Reviewer #3 (Public Review):

The diversity of bacterial species in the human gut microbiome is widely known, but the extensive diversity within each species is far less appreciated. Strains found in individuals on opposite sides of the globe can differ by as little as handfuls of mutations, while strains found in an individual's gut, or in the same household, might have a common ancestor tens of thousands of years ago. What are the evolutionary, ecological, and transmission dynamics that established and maintain this diversity?

The time, T, since the common ancestor of two strains, can be directly inferred by comparing their core genomes and finding the fraction of synonymous (non-amino acid changing) sites at which they differ: dS. With the per-site per-generation mutation rate, μ, and the mean generation times roughly known, this directly yields T (albeit with substantial uncertainty of the generation time.) A traditional way to probe the extent to which selection plays a role is to study pairs of strains and compare the fraction of non-synonymous (amino acid or stop-codon changing) sites, dN, at which the strains differ with their dS. Small dN/dS, as found between distantly related strains, is attributed to purifying selection against deleterious mutations dominating over mutations that have driven adaptive evolution. Large dN/dS as found in laboratory evolution experiments, is caused by beneficial mutations that quickly arise in large bacterial populations, and, with substantial selective advantages, per generation, can rise to high abundance fast enough that very few synonymous mutations arise in the lineages that take over the population.

A number of studies (including by Lieberman's group) have analyzed large numbers of strains of various dominant human gut species and studied how dN/dS varies. Although between closely related strains the variations are large -- often much larger than attributable to just statistical variations -- a systematic trend from dN/dS around unity or larger for close relatives to dN/dS ~ 0.1 for more distant relatives has been found in enough species that it is natural to conjecture a general explanation.<br /> The conventional explanation is that, for close relatives, the effects of selection over the time since they diverged has not yet purged weakly deleterious mutations that arose by chance -- roughly mutations with sT<1 -- while since the common ancestor of more distantly related strains, there is plenty of time for most of those that arose to have been purged.

Torrillo and Lieberman have carried out an in-depth -- sophisticated and quantitative -- analysis of models of some of the evolutionary processes that shape the dependence of dN/dS on dS -- and hence on their divergence time, T. They first review the purifying selection model and show that -- even ignoring its inability to explain dN/dS > 1 for many closely related pairs -- the model has major problems explaining the crossover from dN/dS somewhat less than unity to much smaller values as dS goes through -- on a logarithmic scale -- the 10^-4 range. The first problem, already seen in the infinite-population-size deterministic model, is that a very large fraction of non-synonymous mutations would have to have deleterious s's in the 10^-5 per generation range to fit the data (and a small fraction effectively neutral). As the s's are naturally expected (at least in the absence of quantitative analysis to the contrary) to be spread out over a wide range on a logarithmic scale of s, this seems implausible. But the authors go further and analyze the effects of fluctuations that occur even in the very large populations: ~ >10^12 bacteria per species in one gut, and 10^10 human guts globally. They show that Muller's ratchet -- the gradual accumulation of weakly deleterious mutations that are not purged by selection - leads to a mutational meltdown with the parameters needed to fit the purifying selection model. In particular, with N_e the "effective population size" that roughly parametrizes the magnitude of stochastic birth-death and transition fluctuations, and U the total mutation rate to such deleterious mutations this occurs for U/s > log(sN_e) which they show would obtain with the fitted parameters.

Torrillo and Lieberman promise an alternate model: that there are a modest number of "loci" at which conditionally beneficial mutations can occur that are beneficial in some individual guts (or other environmental conditions) at some times, but deleterious in other (or the same) gut at other times. With the ancestors of a pair of strains having passed through one too many individuals and transmissions, it is possible for a beneficial mutation to occur and rise in the population, only later to be reverted by the beneficial inverse mutation. With tens of loci at which this can occur, they show that this process could explain the drop of dN/dS from short times -- in which very few such mutations have occurred -- to very long times by which most have flipped back and forth so that a random pair of strains will have the same nucleotide at such sites with 50% probability. Their qualitative analysis of a minimally simple model of this process shows that the bacterial populations are plenty big enough for such specific mutations to occur many times in each individual's gut, and with modest beneficials, to takeover. With a few of these conditionally beneficial mutations or reversions occurring during an individuals lifetime, they get a reasonably quantitative agreement with the dN/dS vs dS data with very few parameters. A key assumption of their model is that genetically exact reversion mutations are far more likely to takeover a gut population -- and spread -- than compensatory mutations which have a similar phenotypic-reversion effect: a mutation that is reverted does not show up in dN, while one that is compensated by another shows up as a two-mutation difference after the environment has changed twice.

Strengths:

The quantitative arguments made against the conventional purifying selection model are highly compelling, especially the consideration of multiple aspects that are usually ignored, including -- crucially -- how Muller's ratchet arises and depends on the realistic and needed-to-fit parameters; the effects of bottlenecks in transmission and the possibility that purifying selection mainly occurs then; and complications of the model of a single deleterious s, to include a distribution of selective disadvantages. Generally, the author's approach of focusing on the simplest models with as few as possible parameters (some roughly known), and then adding in various effects one-by-one, is outstanding and, in being used to analyze environmental microbial data, exceptional.

The reversion model the authors propose and study is a simple general one and they again explore carefully various aspects of it -- including dynamics within and between hosts -- and the consequent qualitative and quantitative effects. Again, the quantitive analysis of almost all aspects is exemplary. Although it is hard to make a compelling guess of the number of loci that are subject to alternating selection on the needed time-scales (years to centuries) they make a reasonable argument for a lower bound in terms of the number of known invertible promoters (that can genetically switch gene expression on and off).

Weaknesses:

The primary weakness of this paper is one that the author's are completely open about: the assumption that, collectively, any of possibly-many compensatory mutations that could phenotypically revert an earlier mutation, are less likely to arise and takeover local populations than the exact specific reversion mutation. While detailed analysis of this is, reasonably enough, beyond the scope of the present paper, more discussion of this issue would add substantially to this work. Quantitatively, the problem is that even a modest number of compensatory mutations occurring as the environmental pressures change could lead to enough accumulation of non-synonymous mutations that they could cause dN/dS to stay large -- easily >1 -- to much larger dS than is observed. If, say, the appropriate locus is a gene, the number of combinations of mutations that are better in each environment would play a role in how large dN would saturate to in the steady state (1/2 of n_loci in the author's model). It is possible that clonal interference between compensatory and reversion mutations would result in the mutations with the largest s -- eg, as mentioned, reversion of a stop codon -- being much more likely to take over, and this could limit the typical number of differences between quite well-diverged strains. However, the reversion and subsequent re-reversion would have to both beat out other possible compensatory mutations -- naively less likely. I recommend that a few sentences in the Discussion be added on this important issue along with comments on the more general puzzle -- at least to this reader! -- as to why there appear to be so little adaptive genetic changes in core genomes on time scales of human lifetimes and civilization.

An important feature of gut bacterial evolution that is now being intensely studied is only mentioned in passing at the end of this paper: horizontal transfer and recombination of core genetic material. As this tends to bring in many more mutations overall than occur in regions of a pair of genomes with asexual ancestry, the effects cannot be neglected. To what extent can this give rise to a similar dependence of dN/dS on dS as seen in the data? Of course, such a picture begs the question as to what sets the low dN/dS of segments that are recombined --- often from genetic distances comparable to the diameter of the species.

Reviewer #3 (Public Review):

Ninein is a centrosome protein that has been implicated in microtubule anchorage and centrosome cohesion. Mutations in the human ninein gene have been linked to Seckel syndrome and a rare form of skeletal dysplasia. However, the role of ninein in skeletal development remains unknown. Here, we describe a ninein knockout mouse with advanced endochondral ossification during embryonic development. Although the long bones maintain a regular size, the absence of ninein delays the formation of the bone marrow cavity in the prenatal tibia. Likewise, intramembranous ossification in the skull is more developed, leading to a premature closure of the interfrontal suture. We demonstrate that ninein is strongly expressed in osteoclasts of control mice and that its absence reduces the fusion of precursor cells into syncytial osteoclasts. As a consequence, ninein-deficient osteoclasts have a reduced capacity to resorb bone. At the cellular level, the absence of ninein interferes with<br /> centrosomal microtubule organization, reduces centrosome cohesion, and provokes the loss of centrosome clustering in multinucleated mature osteoclasts. We propose that centrosomal ninein is important for osteoclast fusion, to enable a functional balance between bone-forming osteoblasts and bone-resorbing osteoclasts during skeletal development.

I have run across Jeff Shelton's Analog system (originally via Kickstarter) before. Thanks for the reminder.

There's also a slew of others, especially for folks looking at commercially preprinted cards (though I tend to think they're overpriced compared to blank cards): - The Hipster PDA (Parietal Disgorgement Aid) https://web.archive.org/web/20040906150523/https://merlin.blogs.com/43folders/2004/09/introducing_the.html - Pile of Index Cards (PoIC) https://www.flickr.com/photos/hawkexpress/albums/72157594200490122/ - Levenger https://www.levenger.com/products/triple-decker-pocket-planner?variant=42485422424213 (among others they carry including pocket briefcases) - Notsu https://notsubrand.com/ - Baronfig / Strategist: https://baronfig.com/products/strategist?variant=39787199529043 - Jeff Shelton's Analog system https://ugmonk.com/ - 3x5 Life https://www.3x5life.com/ - Foglietto https://www.nerosnotes.co.uk/collections/foglietto - Jot & Mark https://amzn.to/3Qs26Je

Am I missing any significant or influential examples, particularly branded ones?

Hubnote for 3 x 5" index cards for productivity

Reviewer #3 (Public Review):

Summary:

The goal of this paper is to characterize an anti-diuretic signaling system in insects using Drosophila melanogaster as a model. Specifically, the authors wished to characterize a role of ion transport peptide (ITP) and its isoforms in regulating diverse aspects of physiology and metabolism. The authors combined genetic and comparative genomic approaches with classical physiological techniques and biochemical assays to provide a comprehensive analysis of ITP and its role in regulating fluid balance and metabolic homeostasis in Drosophila. The authors further characterized a previously unrecognized role for Gyc76C as a receptor for ITPa, an amidated isoform of ITP, and in mediating the effects of ITPa on fluid balance and metabolism. The evidence presented in favor of this model is very strong as it combines multiple approaches and employs ideal controls. Taken together, these findings represent an important contribution to the field of insect neuropeptides and neurohormones and have strong relevance for other animals.

Strengths:

Many approaches are used to support their model. Experiments were well-controlled, used appropriate statistical analyses, and were interpreted properly and without exaggeration.

Weaknesses:

No major weaknesses were identified by this reviewer. More evidence to support their model would be gained by using a loss-of-function approach with ITPa, and by providing more direct evidence that Gyc76C is the receptor that mediates the effects of ITPa on fat metabolism. However, these weaknesses do not detract from the overall quality of the evidence presented in this manuscript, which is very strong.

Reviewer #3 (Public Review):

Zhao et al. provide new insights into the mechanism by which a high-fat diet (HFD) induces cardiac arrhythmia employing Drosophila as a model. HFD induces cardiac arrhythmia in both mammals and Drosophila. Both glucagon and its functional equivalent in Drosophila Akh are known to induce arrhythmia. The study demonstrates that Akh mRNA levels are increased by HFD and both Akh and its receptor are necessary for high-fat diet-induced cardiac arrhythmia, elucidating a novel link. Notably, Zhao et al. identify a pair of AKH receptor-expressing neurons located at the posterior of the heart tube. Interestingly, these neurons innervate the heart muscle and form synaptic connections, implying their roles in controlling the heart muscle. The study presented by Zhao et al. is intriguing, and the rigorous characterization of the AKH receptor-expressing neurons would significantly enhance our understanding of the molecular mechanism underlying HFD-induced cardiac arrhythmia.

Many experiments presented in the manuscript are appropriate for supporting the conclusions while additional controls and precise quantifications should help strengthen the authors' augments. The key results obtained by loss of Akh (or AkhR) and genetic elimination of the identified AkhR-expressing cardiac neurons do not reconcile, complicating the overall interpretation.

It is intriguing to see an increase in Akh mRNA levels in HFD-fed animals. This is a key result for linking HFD-induced arrhythmia to Akh. Thus, demonstrating that HFD also increases the Akh protein levels and Akh is secreted more should significantly strengthen the manuscript.

The experiments employing an AkhR null allele nicely demonstrate its requirement for HFD-induced cardiac arrhythmia. Depletion of Akh in Akh-expressing cells recapitulates the consequence of AkhR knockout, supporting that both Akh and its receptor are required for HFD-induced cardiac arrhythmia. Given that RNAi is associated with off-target effects and some RNAi reagents do not work, testing multiple independent RNAi lines is the standard procedure. It is also important to show the on-target effect of the RNAi reagents used in the study.

The most exciting result is the identification of AkhR-expressing neurons located at the posterior part of the heart tube (ACNs). The authors attempted to determine the function of ACNs by expressing rpr with AkhR-GAL4, which would induce cell death in all AkhR-expressing cells, including ACNs. The experiments presented in Figure 6 are not straightforward to interpret. Moreover, the conclusion contradicts the main hypothesis that elevated Akh is the basis of HFD-induced arrhythmia. The results suggest the importance of AkhR-expressing cells for normal heartbeat. However, elimination of Akh or AkhR restores normal rhythm in HFD-fed animals, suggesting that Akh and AkhR are not important for maintaining normal rhythms. If Akh signaling in ACNs is key for HFD-induced arrhythmia, genetic elimination of ACNs should unalter rhythm and rescue the HFD-induced arrhythmia. An important caveat is that the experiments do not test the specific role of ACNs. ACNs should be just a small part of the cells expressing AkhR. The experiments presented in Figure 6 cannot justify the authors' conclusion. Specific manipulation of ACNs will significantly improve the study. Moreover, the main hypothesis suggests that HFD may alter the activity of ACNs in a manner dependent on Akh and AkhR. Testing how HFD changes calcium, possibly by CaLexA (Figure 2) and/or GCaMP, in wild-type and AkhR mutants could be a way to connect ACNs to HFD-induced arrhythmia. Moreover, optogenetic manipulation of ACNs will allow for specific manipulation of ACNs, which is crucial for studying the specific role of ACNs in controlling cardiac rhythms.

Interestingly, expressing rpr with AkhR-GAL4 was insufficient to eliminate both ACNs. It is not clear why it didn't eliminate both ACNs. Given the incomplete penetrance, appropriate quantifications should be helpful. Additionally, the impact on other AhkR-expressing cells should be assessed. Adding more copies of UAS-rpr, AkhR-GAL4, or both may eliminate all ACNs and other AkhR-expressing cells. The authors could also try UAS-hid instead of UAS-rpr.

Reviewer #3 (Public Review):

Summary:

This article addresses an important and interesting question concerning intracellular localization and dynamics of endogenous G proteins. The fate and trafficking of G protein-coupled receptors (GPCRs) have been extensively studied but so far little is known about the trafficking routes of their partner G proteins that are known to dissociate from their respective receptors upon activation of the signaling pathway. The authors utilize modern cell biology tools including genome editing and bystander bioluminescence resonance energy transfer (BRET) to probe intracellular localization of G proteins in various membrane compartments in steady state and also upon receptor activation. Data presented in this manuscript shows that while G proteins are mostly present on the plasma membrane, they can be also detected in endosomal compartments, especially in late endosomes and lysosomes. This distribution, according to data presented in this study, seems not to be affected by receptor activation. These findings will have implications in further studies addressing GPCR signaling mechanisms from intracellular compartments.

Strengths:

The methods used in this study are adequate for the question asked. Especially, the use of genome-edited cells (for the addition of the tag on one of the G proteins) is a great choice to prevent the effects of overexpression. Moreover, the use of bystander BRET allowed authors to probe the intracellular localization of G proteins in a very high-throughput fashion. By combining imaging and BRET authors convincingly show that G proteins are very low abundant on early endosomes (also ER, mitochondria, and medial Golgi), however seem to accumulate on membranes of late endosomal compartments.

Weaknesses:

While the authors provide a novel dataset, many questions regarding G protein trafficking remain open. For example, it is not entirely clear which pathway is utilized to traffic G proteins from the plasma membrane to intracellular compartments. Additionally, future studies should also address the dynamics of G protein trafficking, for example by tracking them over multiple time points.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors report the first evidence of Nav1.5 regulation by a long noncoding RNA, LncRNA-DACH1, and suggest its implication in the reduction in sodium current observed in heart failure. Since no direct interaction is observed between Nav1.5 and the LncRNA, they propose that the regulation is via dystrophin and targeting of Nav1.5 to the plasma membrane.

Strengths:

(1) First evidence of Nav1.5 regulation by a long noncoding RNA.<br /> (2) Implication of LncRNA-DACH1 in heart failure and mechanisms of arrhythmias.<br /> (3) Demonstration of LncRNA-DACH1 binding to dystrophin.<br /> (4) Potential rescuing of dystrophin and Nav1.5 strategy.

Weaknesses:

(1) The fact that the total Nav1.5 protein is reduced by 50% which is similar to the reduction in the membrane reduction questions the main conclusion of the authors implicating dystrophin in the reduced Nav1.5 targeting. The reduction in membrane Nav1.5 could simply be due to the reduction in total protein.

Reviewer #3 (Public Review):

Wang et al. explored the unique biology of the deep-sea mussel Gigantidas platifrons to understand fundamental principles of animal-symbiont relationships. They used single-nucleus RNA sequencing and validation and visualization of many of the important cellular and molecular players that allow these organisms to survive in the deep-sea. They demonstrate that a diversity of cell types that support the structure and function of the gill including bacteriocytes, specialized epithelial cells that host sulfur-oxidizing or methane-oxidizing symbionts as well as a suite of other cell types including supportive cells, ciliary, and smooth muscle cells. By performing experiments of transplanting mussels from one habitat which is rich in methane to methane-limited environments, the authors showed that starved mussels may consume endosymbionts versus in methane-rich environments upregulated genes involved in glutamate synthesis. These data add to the growing body of literature that organisms control their endosymbionts in response to environmental change.

The conclusions of the data are well supported. The authors adapted a technique that would have been technically impossible in their field environment by preserving the tissue and then performing nuclear isolation after the fact. The use of single-nucleus sequencing opens the possibility of new cellular and molecular biology that is not possible to study in the field. Additionally, the in-situ data (both WISH and FISH) are high-quality and easy to interpret. The use of cell-type-specific markers along with a symbiont-specific probe was effective. Finally, the SEM and TEM were used convincingly for specific purposes in the case of showing the cilia that may support water movement.

The one particular area for future exploration surrounds the concept of a proliferative progenitor population within the gills. The authors recover molecular markers for these putative populations and additional future work will uncover if these are indeed proliferative cells contribute to symbiont colonization.

Overall the significance of this work is identifying the relationship between symbionts and bacteriocytes and how these host bacteriocytes modulate their gene expression in response to environmental change. It will be interesting to see how similar or different these data are across animal phyla. For instance, the work of symbiosis in cnidarians may converge on similar principles of there may be independent ways in which organisms have been able to solve these problems.

Reviewer #3 (Public Review):

Summary:

Federer et al. describe the laminar profiles of GABA+ and of PV+ neurons in marmoset V1. They also report on the selectivity and efficiency of expression of a PV-selective enhancer (S5E2). Three further viruses were tested, with a view to characterizing the expression profiles of a GABA-selective enhancer (h56d), but these results are preliminary.

Strengths:

The derivation of cell-type specific enhancers is key for translating the types of circuit analyses that can be performed in mice - which rely on germline modifications for access to cell-type specific manipulation - in higher-order mammals. Federer et al. further validate the utility of S5E2 as a PV-selective enhancer in NHPs.

Additionally, the authors characterize the laminar distribution pattern of GABA+ and PV+ cells in V1. This survey may prove valuable to researchers seeking to understand and manipulate the microcircuitry mediating the excitation-inhibition balance in this region of the marmoset brain.

Weaknesses:

Enhancer/promoter specificity and efficiency cannot be directly compared, because they were packaged in different serotypes of AAV.

The three different serotypes of AAV expressing reporter under the h56D promoter were only tested once each, and all in the same animal. There are many variables that can contribute to the success (or failure) of a viral injection, so observations with an n=1 cannot be considered reliable.

The language used throughout conflates the cell-type specificity conferred by the regulatory elements with that conferred by the serotype of the virus.

Reviewer #3 (Public Review):

This study begins with a chemogenetic screen to discover previously unrecognized regulators of the cell cycle. Using a CRISPR-Cas9 library in HAP1 cells and an assay that scores cell fitness, the authors identify genes that sensitize or desensitize cells to the presence of palbociclib, colchicine, and camptothecin. These three drugs inhibit proliferation through different mechanisms, and with each treatment, expected and unexpected pathways were found to affect drug sensitivity. The authors focus the rest of the experiments and analysis on the polycomb complex PRC2, as the deletion of several of its subunits in the screen conferred palbociclib resistance. The authors find that PRC2, specifically a complex dependent on the MTF2 subunit, methylates histone 3 lysine 27 (H3K27) in promoters of genes associated with various processes including cell-cycle control. Further experiments demonstrate that Cyclin D expression increases upon loss of PRC2 subunits, providing a potential mechanism for palbociclib resistance.

The strengths of the paper are the design and execution of the chemogenetic screen, which provides a wealth of potentially useful information. The data convincingly demonstrate in the HAP1 cell line that the MTF2-PRC2 complex sustains the effects of palbociclib (Figure 4), methylates H3K27 in CpG-rich promoters (Figure 5), and represses Cyclin D expression (Figure 6). These results could be of great interest to those studying cell-cycle control, resistance mechanisms to therapeutic cell-cycle inhibitors, and chromatin regulation and gene expression.

There are several weaknesses that limit the overall quality and potential impact of the study. First, none of the results from the colchicine and camptothecin screens (Figures 1 and 2) are experimentally validated, which lessens the rigor of those data and conclusions. Second, all experiments validating and further exploring results from the palbociclib screen are restricted to the Hap1 cell line, so the reproducibility and generality of the results are not established. While it is reasonable to perform the initial screen to generate hypotheses in the Hap1 line, other cancer and non-transformed lines should be used to test further the validity of conclusions from data in Figures 4-6. Third, conclusions drawn from data in Figures 3D and 4D are not fully supported by the experimental design or results. Finally, there have been other similar chemogenetic screens performed with palbociclib, most notably the study described by Chaikovsky et al. (PMID: 33854239). Results here should be compared and contrasted to other similar studies.

Reviewer #3 (Public Review):

Summary:

The manuscript introduces PAClight1P78A, a novel genetically encoded sensor designed to facilitate the study of class-B1 G protein-coupled receptors (GPCRs), focusing on the human PAC1 receptor. Addressing the significant challenge of investigating these clinically relevant drug targets, the sensor demonstrates a high dynamic range, excellent ligand selectivity, and rapid activation kinetics. It is validated across a variety of experimental contexts including in vitro, ex vivo, and in vivo models in mice and zebrafish, showcasing its utility for high-throughput screening, basic research, and drug development efforts related to GPCR dynamics and pharmacology.

Strengths:

The innovative design of PAClight1P78A successfully bridges a crucial gap in GPCR research by enabling real-time monitoring of receptor activation with high specificity and sensitivity. The extensive validation across multiple models emphasizes the sensor's reliability and versatility, promising significant contributions to both the scientific understanding of GPCR mechanisms and the development of novel therapeutics. Furthermore, by providing the research community with detailed methodologies and access to the necessary viral vectors and plasmids, the authors ensure the sensor's broad applicability and ease of adoption for a wide range of studies focused on GPCR biology and drug targeting.

Weaknesses<br /> To further strengthen the manuscript and validate the efficacy of PAClight1P78A as a selective PACAP sensor, it is crucial to demonstrate the sensor's ability to detect endogenous PACAP release in vivo under physiological conditions. While the current data from artificial PACAP application in mouse brain slices and microinfusion in behaving mice provide foundational insights into the sensor's functionality, these approaches predominantly simulate conditions with potentially higher concentrations of PACAP than naturally occurring levels.

Although the sensor's specificity for the PAC1 receptor and its primary ligand is a pivotal achievement, exploring its potential application to other GPCRs within the class-B1 family or broader categories could enhance the manuscript's impact, suggesting ways to adapt this technology for a wider array of receptor studies. Additionally, while the sensor's performance is convincingly demonstrated in short-term experiments, insights into its long-term stability and reusability in more prolonged or repeated measures scenarios would be valuable for researchers interested in chronic studies or longitudinal behavioral analyses. Addressing these aspects could broaden the understanding of the sensor's practical utility over extended research timelines.

Furthermore, the current in vivo experiments involving microinfusion of PACAP near sensor-expressing areas in behaving mice are based on a relatively small sample size (n=2), which might limit the generalizability of the findings. Increasing the number of subjects in these experimental groups would enhance the statistical power of the results and provide a more robust assessment of the sensor's in vivo functionality. Expanding the sample size will not only validate the findings but also address potential variability within the population, thereby reinforcing the conclusions drawn from these crucial experiments.

Reviewer #3 (Public Review):

Summary:

In this study, the authors probe the connections between clustering of the Met4/32 transcription factors (TFs), clustering of their regulatory targets, and transcriptional regulation. While there is an increasing number of studies on TF clustering in vitro and in vivo, there is an important need to probe whether clustering plays a functional role in gene expression. Another important question is whether TF clustering leads to the clustering of relevant gene targets in vivo. Here the authors provide several lines of evidence to make a compelling case that Met4/32 and their target genes cluster and that this leads to an increase in transcription of these genes in the induced state. First, they found that, in the induced state, Met4/32 forms co-localized puncta in vivo. This is supported by in vitro studies showing that these TFs can form condensates in vitro with Med32 being the driver of these condensates. They found that two target genes, MET6 and MET13 have a higher probability of being co-localized with Met4 puncta compared with non-target loci. Using a targeted DNA methylation assay, they found that MET13 and MET6 show Met4-dependent long-range interactions with other Met4-regulated loci, consistent with the clustering of at least some target genes under induced conditions. Finally, by inserting a Met4-regulated reporter gene at variable distances from MET6, they provide evidence that insertion near this gene is a modest hotspot for activity.

Weaknesses:

(1) Please provide more information on the assay for puncta formation (Figure 1). It's unclear to me from the description provided how this assay was able to quantitate the number of puncta in cells.

2) How does the number of puncta in cells correspond with the number of Met-regulated genes? What are the implications of this calculation?

3) A control for chromosomal insertion of the Met-regulated reporter was a GAL4 promoter derivative reporter. However, this control promoter seems 5-10 fold more active than the Met-regulated promoter (Figure 6). It's possible that the high activity from the control promoter overcomes some other limiting step such that chromosomal location isn't important. It would be ideal if the authors used a promoter with comparable activity to the Met-reporter as a control.

(4) It seems like transcription from a very large number of genes is altered in the Met4 IDR mutant (Figure 7F). Why is this and could this variability affect the conclusions from this experiment?

Reviewer #3 (Public Review):

The manuscript by Ruan et al. addresses an important issue in Panx1 research, i.e. the activation of the channel formed by Panx1 via protein phosphorylation. If the authors' conclusions are correct, the previous claims for Panx1 phosphorylation on the basis of the commercial anti-phospho-Panx1 antibodies would be in question.

This is a very detailed and comprehensive analysis making use of state-of-the-art techniques, including mass spectrometry and phos-tag gel electrophoresis.

In general, the study is well-controlled as relating to negative controls.

The value of this manuscript is, that it could spawn new, more function-oriented studies on the activation of Panx1 channels.

The weaknesses identified previously are reproduced below:

Weaknesses:

Although the manuscript addresses an important issue, the activation of the ATP-release channel Panx1 by protein phosphorylation, the data provided do not support the firm conclusion that such activation does not exist. The failure to reproduce published data obtained with commercial anti-phospho Panx1 antibodies can only be of limited interest for a subfield.

(1) The title claiming that "Panx1 is NOT phosphorylated..." is not justified by the failure to reproduce previously published data obtained with these antibodies. If, as claimed, the antibodies do not recognize Panx1, their failure cannot be used to exclude tyrosine phosphorylation of the Panx1 protein. There is no positive control for the antibodies.

(2) The authors claim that exogenous SRC expression does not phosphorylate Y198. DeLalio et al. 2019 show that Panx1 is constitutively phosphorylated at Y198, so an effect of exogenous SRC expression is not necessarily expected.

(3) The authors argue that the GFP tag of Panx1at the COOH terminus does not interfere with folding since the COOH modified (thrombin cleavage site) Panx1 folds properly, forming an amorphous glob in the cryo-EM structure. However, they do not show that the COOH-modified Panx1 folds properly. It may not, because functional data strongly suggest that the terminal cysteine dives deep into the pore. For example, the terminal cysteine, C426, can form a disulfide bond with an engineered cysteine at position F54 (Sandilos et al. 2012).

(4) The authors dismiss the additional arguments for tyrosine phosphorylation of Panx1 given by the various previous studies on Panx1 phosphorylation. These studies did not, as implied, solely rely on the commercial anti-phospho-Panx1 antibodies, but also presented a wealth of independent supporting data. Contrary to the authors' assertion, in the previous papers the pY198 and pY308 antibodies recognized two protein bands in the size range of glycosylated and partial glycosylated Panx1.

(5) A phosphorylation step triggering channel activity of Panx1 would be expected to occur exclusively on proteins embedded in the plasma membrane. The membrane-bound fraction is small in relation to the total protein, which is particularly true for exogenously expressed proteins. Thus, any phosphorylated protein may escape detection when total protein is analyzed. Furthermore, to be of functional consequence, only a small fraction of the channels present in the plasma membrane need to be in the open state. Consequently, only a fraction of the Panx1 protein in the plasma membrane may need to be phosphorylated. Even the high resolution of mass spectroscopy may not be sufficient to detect phosphorylated Panx1 in the absence of enrichment processes.

(6) In the electrophysiology experiments described in Figure 7, there is no evidence that the GFP-tagged Panx1 is in the plasma membrane. Instead, the image in Figure 7a shows prominent fluorescence in the cytoplasm. In addition, there is no evidence that the CBX-sensitive currents in 7b are mediated by Panx1-GFP and are not endogenous Panx1. Previous literature suggests that the hPanx1 protein needs to be cleaved (Chiu et al. 2014) or mutated at the amino terminus (Michalski et al 2018) to see voltage-activated currents, so it is not clear that the currents represent hPANX1 voltage-activated currents.

Note from the editors: The authors provided a rebuttal to the latest review, but no additional data, so we encourage readers to read the concerns and the author responses.

Reviewer #3 (Public Review):

Summary and Strengths:

The manuscript by Lewis et al, investigates whether myosin ATP activity may differ between states of hibernation and activity in both large and small mammals. The study interrogates (primarily) permeabilized muscle strips or myofibrils using several state-of-the-art assays, including the mant-ATP assay to investigate ATP utilization of myosin, X-ray diffraction of muscles, proteomics studies, metabolic tests, and computational simulations. The overall data suggests that ATP utilization of myosin during hibernation is different than in active conditions.

A clear strength of this study is the use of multiple animals that utilize two different states of hibernation or torpor. Two large animal hibernators (Eurasian Brown Bear, American Black Bear) represent large animal hibernators that typically undergo a prolonged hibernation. Two small animal hibernators (Garden Dormouse, 13 Lined Ground Squirrel) undergo torpor with more substantial reductions in heart rate and body temperature, but whose torpor bouts are interrupted by short arousals that bring the animals back to near-summer like metabolic conditions.

Especially interesting, the investigators analyze the impact that body temperature may have on myosin ATP utilization by performing assays at two different temperatures (8 and 20 degrees C, in 13 Lined Ground Squirrels).

The multiple assays utilized provide a more comprehensive set of methods with which to test their hypothesis that muscle myosins change their metabolic efficiency during hibernation.

Suggestions and potential Weaknesses:

The following highlight comments from the first Public Review that this reviewer acknowledges authors may not be able to address in the current study but may merit carrying to the revised article of record.

(1) Statistical Analysis<br /> The revised manuscript addresses the substantial issues. The two remaining questions may be noted for future experimental design(s): 1.c. That myosin isoforms may be considered a main effect and 1.e. The importance of biological vs statistical significance, especially for the mant-ATP chase data from the American Black Bear, where there appear to be shifts between the summer and winter data.

(2). Consistency of DRX/SRX data.<br /> The responses to the first Public Review on the prior version of this manuscript highlight that a potential disconnect between the mant-ATP-predicted SRX:DRX proportions and x-ray diffraction studies measuring the position of the myosin heads (Mohran et al PMID 38103642) may be outside of the scope of the current manuscript. The reviewer accepts that a substantial discussion is outside of this article, but considers a brief mention possible differences between ATP kinetics and structural movements of value.

Overall, the manuscript represents a valuable data set comparing myosin properties of skeletal muscles multiple species exhibiting different forms of hibernation/torpor.

Reviewer #3 (Public Review):

This is interesting biology. Vitamin B6 deficiency has been linked to cognitive impairment. It is not clear whether supplements are effective in restoring functional B6 levels. Vitamin B6 is composed of pyridoxal compounds and their phosphorylated forms, with pyridoxal 5-phosphate (PLP) being of particular importance. The levels of PLP are determined by the balance between pyridoxal kinase and phosphatase activities. The authors are testing the hypothesis that inhibition of pyridoxal phosphatase (PDXP) would arrest the age-dependent decline in PLP, offering an alternative therapeutic strategy to supplements. Published data illustrating that ablation of the Pdxp gene in mice led to increases in PLP levels and improvement in learning and memory trials are consistent with this hypothesis.

In this report, the authors conduct a screen of a library of ~40k small molecules and identify 7,8-dihydroxyflavone (DHF) as a candidate PDXP inhibitor. They present an initial characterization of this micromolar inhibitor, including a co-crystal structure of PDXP and 7,8-DHF. In addition, they demonstrate that treatment of cells with 7,8 DHP increases PLP levels. Overall, this study provides further validation of PDXP as a therapeutic target for the treatment of disorders associated with vitamin B6 deficiency and provides proof-of-concept for inhibition of the target with small-molecule drug candidates.

Strengths include the biological context, the focus on an interesting and under-studied class of protein phosphatases that includes several potential therapeutic targets, and the identification of a small molecule inhibitor that provides proof-of-concept for a new therapeutic strategy. Overall, the study has the potential to be an important development for the phosphatase field in general.

Weaknesses include the fact that the compound is very much an early-stage screening hit. It is an inhibitor with micromolar potency for which mechanisms of action other than inhibition of PDXP have been reported. Extensive further development will be required to demonstrate convincingly the extent to which its effects in cells are due to on-target inhibition of PDXP.

Reviewer #3 (Public Review):

Summary:

The manuscript by Yeo et al. investigates the intracellular trafficking of Botulinum neurotoxin A (BoNT/A), a potent toxin used in clinical and cosmetic applications. Contrary to the prevailing understanding of BoNT/A translocation into the cytosol, the study suggests a retrograde migration from the synapse to the soma-localized Golgi in neurons. Using a genome-wide siRNA screen in genetically engineered neurons, the researchers identify over three hundred genes involved in this process. The study employs organelle-specific split-mNG complementation, revealing that BoNT/A traffics through the Golgi in a retromer-dependent manner before moving to the endoplasmic reticulum (ER). The Sec61 complex is implicated in the retro-translocation of BoNT/A from the ER to the cytosol. Overall, the research challenges the conventional model of BoNT/A translocation, uncovering a complex route from synapse to cytosol for efficient intoxication. The findings are based on a comprehensive approach, including the introduction of a fluorescent reporter for BoNT/A catalytic activity and genetic manipulations in neuronal cell lines. The conclusions highlight the importance of retrograde trafficking and the involvement of specific genes and cellular processes in BoNT/A intoxication.

Strengths:

The major part of the experiments are convincing. They are well-controlled and the interpretation of their results is balanced and sensitive.

Weaknesses:

To my opinion, the main weakness of the paper is that all experiments are performed using a single cellular system (RenVM neurons), as stated in the title. It is therefore unclear at the moment to what extent the findings in this paper can be generalized to other neuronal cell models / in vivo situation.

Reviewer #3 (Public Review):

Summary:

This paper presents novel and innovative force measurements of the biophysics of gliding cyanobacteria filaments. These measurements allow for estimates of the resistive force between the cell and substrate and provide potential insight into the motility mechanism of these cells, which remains unknown.

Strengths:

The authors used well-designed microfabricated devices to measure the bending modulus of these cells and to determine the critical length at which the cells buckle. I especially appreciated the way the authors constructed an array of pillars and used it to do 3-point bending measurements and the arrangement the authors used to direct cells into a V-shaped corner in order to examine at what length the cells buckled at. By examining the gliding speed of the cells before buckling events, the authors were able to determine how strongly the buckling length depends on the gliding speed, which could be an indicator of how the force exerted by the cells depends on cell length; however, the authors did not comment on this directly.

Weaknesses:

There are no major weaknesses in the paper.

Reviewer #3 (Public Review):

In this study, the authors utilized mass spectrometry-based quantification of polar metabolites and lipids in normal and cancerous tissue interstitial fluid and plasma. This showed that nutrient availability in tumor interstitial fluid was similar to that of interstitial fluid in adjacent normal kidney tissue, but that nutrients found in both interstitial fluid compartments were different from those found in plasma. This suggests that the nutrients in kidney tissue differ from those found in blood and that nutrients found in kidney tumors are largely dictated by factors shared with normal kidney tissue. Those data could be useful as a resource to support further study and modeling of the local environment of RCC and normal kidney physiology.

Reviewer #3 (Public Review):

Summary:

Secondary metabolites are produced by numerous microorganisms and have important ecological functions. A major problem is that neither the function of a secondary metabolite enzyme nor the resulting metabolite can be precisely predicted from gene sequence data.

In the current paper, the authors addressed this highly relevant question.

The authors developed a bioinformatic pipeline to reconstruct the complete secondary metabolism pathway of pyoverdines, a class of iron-scavenging siderophores produced by Pseudomonas spp. These secondary metabolites are biosynthesized by a series of non-ribosomal peptide synthetases and require a specific receptor (FpvA) for uptake. The authors combined knowledge-guided learning with phylogeny-based methods to predict with high accuracy encoding NRPSs, substrate specificity of A domains, pyoverdine derivatives, and receptors. After validation, the authors tested their pipeline with sequence data from 1664 phylogenetically distinct Pseudomonas strains and were able to determine 18,292 enzymatic A domains involved in pyoverdine synthesis, reliably predicted 97.8% of their substrates, identified 188 different pyoverdine molecule structures and 4547 FpvA receptor variants belonging to 94 distinct groups. All the results and predictions were clearly superior to predictions that are based on antiSMASH. Novel pyoverdine structures were elucidated experimentally by UHPLC-HR-MS/MS.

To assess the extendibility of the pipeline, the authors chose Burkholderiales as a test case which led to the results that the pipeline consistently maintains high prediction accuracy within Burkholderiales of 83% which was higher than for antiSMASH (67%).

Together, the authors concluded that supervised learning based on a few known compounds produced by species from the same genus probably outperforms generalized prediction algorithms trained on many products from a diverse set of microbes for NRPS substrate predictions. As a result, they also show that both pyoverdine and receptor diversity have been vastly underestimated.

Strengths:

The authors developed a very useful bioinformatic pipeline with high accuracy for secondary metabolites, at least for pyoverdines. The pipelines have several advantages compared to existing pipelines like the extensively used antiSMASH program, e.g. it can be applied to draft genomes, shows reduced erroneous gene predictions, etc. The accuracy was impressively demonstrated by the discovery of novel pyoverdines whose structures were experimentally substantiated by UHPLC-HR-MS/MS.

The manuscript is very well written, and the data and the description of the generation of pipelines are easy to follow.

Weaknesses:

The only major comment I have is the uncertainty of whether the pipeline can be applied to more complex non-ribosomal peptides. In the current study, the authors only applied their pipeline to a very narrow field, i.e., pyoverdines of Pseudomonas and Burkholderia strains.

Reviewer #3 (Public Review):

The study presents strong evidence for allosteric activation of plant receptor kinases, which enhances our understanding of the non-catalytic mechanisms employed by this large family of receptors.

Plant receptor kinases (RKs) play a critical role in transducing extracellular signals. The activation of RKs involves homo- or heterodimerization of the RKs, and it is believed that mutual phosphorylation of their intracellular kinase domains initiates downstream signaling. However, this model faces a challenge in cases where the kinase domain exhibits pseudokinase characteristics. In their recent study, Mühlenbeck et al. reveal the non-catalytic activation mechanisms of the EFR-BAK1 complex in plant receptor kinase signaling. Specifically, they aimed to determine that the EFR kinase domain activates BAK1 not through its kinase activity, but rather by utilizing a "conformational toggle" mechanism to enter an active-like state, enabling allosteric trans-activation of BAK1. The study sought to elucidate the structural elements and mutations of EFR that affect this conformational switch, as well as explore the implications for immune signaling in plants. To investigate the activation mechanisms of the EFR-BAK1 complex, the research team employed a combination of mutational analysis, structural studies, and hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. For instance, through HDX-MS analysis, Mühlenbeck et al. discovered that the EFR (Y836F) mutation impairs the accessibility of the active-like conformation. On the other hand, they identified the EFR (F761H) mutation as a potent intragenic suppressor capable of stabilizing the active-like conformation, highlighting the pivotal role of allosteric regulation in BAK1 kinase activation. The data obtained from this methodology strengthens their major conclusion. Moreover, the researchers propose that the allosteric activation mechanism may extend beyond the EFR-BAK1 complex, as it may also be partially conserved in the Arabidopsis LRR-RK XIIa kinases. This suggests a broader role for non-catalytic mechanisms in plant RK signaling.

The allosteric activation mechanism was demonstrated for receptor tyrosine kinases (RTKs) many years ago. A similar mechanism has been suggested for the activation of plant RKs, but experimental evidence for this conclusion is lacking. Data in this study represent a significant advancement in our understanding of non-catalytic mechanisms in plant RK signaling. By shedding light on the allosteric regulation of BAK1, the study provides a new paradigm for future research in this area.

Reviewer #3 (Public Review):

This paper compares the synaptic and membrane properties of two main subtypes of interneurons (PV+, SST+) in the auditory cortex of control mice vs mutants with Syngap1 haploinsufficiency. The authors find differences at both levels, although predominantly in PV+ cells. These results suggest that altered PV-interneuron functions in the auditory cortex may contribute to the network dysfunction observed in Syngap1 haploinsufficiency-related intellectual disability. The subject of the work is interesting, and most of the approach is direct and quantitative, which are major strengths. There are also some weaknesses that reduce its impact for a broader field.

(1) The choice of mice with conditional (rather than global) haploinsufficiency makes the link between the findings and Syngap1 relatively easy to interpret, which is a strength. However, it also remains unclear whether an entire network with the same mutation at a global level (affecting also excitatory neurons) would react similarly.

(2) There are some (apparent?) inconsistencies between the text and the figures. Although the authors appear to have used a sophisticated statistical analysis, some datasets in the illustrations do not seem to match the statistical results. For example, neither Fig 1g nor Fig 3f (eNMDA) reach significance despite large differences. Also, the legend to Fig 9 indicates the presence of "a significant decrease in AP half-width from cHet in absence or presence of a-DTX", but the bar graph does not seem to show that.

(3) The authors mention that the lack of differences in synaptic current kinetics is evidence against a change in subunit composition. However, in some Figures, for example, 3a, the kinetics of the recorded currents appear dramatically different. It would be important to know and compare the values of the series resistance between control and mutant animals.

(4) A significant unexplained variability is present in several datasets. For example, the AP threshold for PV+ includes points between -50-40 mV, but also values at around -20/-15 mV, which seems too depolarized to generate healthy APs (Fig 5c, Fig7c).

(5) I am unclear as to how the authors quantified colocalization between VGluts and PSD95 at the low magnification shown in Supplementary Figure 2.

(6) The authors claim that "cHet SST+ cells showed no significant changes in active and passive membrane properties", but this claim would seem to be directly refused by the data of Fig 8f. In the absence of changes in either active or passive membrane properties shouldn't the current/#AP plot remain unchanged?

(7) The plots used for the determination of AP threshold (Figs 5c, 7c, and 7h) suggest that the frequency of acquisition of current-clamp signals may not have been sufficient, this value is not included in the Methods section.

Reviewer #3 (Public Review):

Building on their previous work that defined four major subgroups, or clades, of V1 interneurons largely by their transcriptional signatures, they do meticulous yet comprehensive analysis of the birth timing of V1 interneurons by clade, and even intra-clade, subtypes. This analysis establishes new relationships between the molecular identity, settling position, and birth time with extraordinary precision.

These relationships are then explored from the lens of synaptic connectivity. Focusing on the FoxP2 clade, they show tight spatial correspondence between V1 and motor neuron position, and through detailed synaptic analysis, find the FoxP2 V1 clade, as compared to Renshaw cells and other V1s, are the major contributors to V1-to-limb motor neuron connectivity. Finally, by analyzing sensory-to-V1 connectivity too, they show that the FoxP2 clade exhibits Ia-reciprocal interneuron-like convergence of proprioceptive and Renshaw cell synapses.

Taking the development and connectivity analysis together, their work substantially advances our understanding of spinal interneurons and yields fundamental basic information about how cell type heterogeneity corresponds across developmental, molecular and anatomical features.

An additional strength of this study is that they generate new genetic tools for labeling interneuron subpopulations, and provide insider knowledge into antibody, genetic and viral labeling that often get tucked under the rug, providing a very useful resource for further studies.

My only criticism is that some of the main messages of the paper are buried in technical details. Better separation of the main conclusions of the paper, which should be kept in the main figures and text, and technical details/experimental nuances, which are essential but should be moved to the supplement, is critical. This will also correct the other issue with the text at present, which is that it is too long.

Reviewer #3 (Public Review):

Summary:

This study explored how the motor system adapts to new environments by modifying redundant body movements. Using a novel bimanual stick manipulation task, participants manipulated a virtual stick to reach targets, focusing on how tip-movement direction perturbations affected both tip movement and stick-tilt adaptation. The findings indicated a consistent strategy among participants who flexibly adjusted the tilt angle of the stick in response to errors. The adaptation patterns are influenced by physical space relationships, guiding the motor system's choice of movement patterns. Overall, this study highlights the adaptability of the motor system through changes in redundant body movement patterns.

Strengths:

This paper introduces a novel bimanual stick manipulation task to investigate how the motor system adapts to novel environments by altering the movement patterns of our redundant body.

Weaknesses:

The generalizability of the findings is quite limited. It would have been interesting to see if the same relationships were held for different stick lengths (i.e., the hands positioned at different start locations along the virtual stick) or when reaching targets to the left and right of a start position, not just at varying angles along one side. Alternatively, this study would have benefited from a more thorough investigation of the existing literature on redundant systems instead of primarily focusing on the lack of redundancy in endpoint-reaching tasks. Although the novel task expands the use of endpoint robots in motor control studies, the utility of this task for exploring motor control and learning may be limited.

Reviewer #3 (Public Review):

Summary:

Bennion et al. investigate how semantic relatedness proactively benefits the learning of new word pairs. The authors draw predictions from Osgood (1949), which posits that the degree of proactive interference (PI) and proactive facilitation (PF) of previously learned items on to-be-learned items depends on the semantic relationships between the old and new information. In the current study, participants learn a set of word pairs ("supplemental pairs"), followed by a second set of pairs ("base pairs"), in which the cue, target, or both words are changed, or the pair is identical. Pairs were drawn from either a narrower or wider stimulus set and were tested after either a 5-minute or 48-hour delay. The results show that semantic relatedness overwhelmingly produces PF and greater memory interdependence between base and supplemental pairs, except in the case of unrelated pairs in a wider stimulus set after a short delay, which produced PI. In their final analyses, the authors compare their current results to previous work from their group studying the analogous retroactive effects of semantic relatedness on memory. These comparisons show generally similar, if slightly weaker, patterns of results. The authors interpret their results in the framework of recursive reminders (Hintzman, 2011), which posits that the semantic relationships between new and old word pairs promote reminders of the old information during the learning of the new to-be-learned information. These reminders help to integrate the old and new information and result in additional retrieval practice opportunities that in turn improve later recall.

Strengths:

Overall, I thought that the analyses were thorough and well-thought-out and the results were incredibly well-situated in the literature. In particular, I found that the large sample size, inclusion of a wide range of semantic relatedness across the two stimulus sets, variable delays, and the ability to directly compare the current results to their prior results on the retroactive effects of semantic relatedness were particular strengths of the authors' approach and make this an impressive contribution to the existing literature. I thought that their interpretations and conclusions were mostly reasonable and included appropriate caveats (where applicable).

Weaknesses:

Although I found that the paper was very strong overall, I have three main questions and concerns about the analyses.

My first concern lies in the use of the narrow versus wider stimulus sets. I understand why the initial narrow stimulus set was defined using associative similarity (especially in the context of their previous paper on the retroactive effects of semantic similarity), and I also understand their rationale for including an additional wider stimulus set. What I am less clear on, however, is the theoretical justification for separating the datasets. The authors include a section combining them and show in a control analysis that there were no directional effects in the narrow stimulus set. The authors seem to imply in the Discussion that they believe there are global effects of the lower average relatedness on differing patterns of PI vs PF across stimulus sets (lines 549-553), but I wonder if an alternative explanation for some of their conflicting results could be that PI only occurs with pairs of low semantic relatedness between the supplemental and base pair and that because the narrower stimulus set does not include the truly semantically unrelated pairs, there was no evidence of PI.

My next concern comes from the additive change in both measures (change in Cue + change in Target). This measure is simply a measure of overall change, in which a pair where the cue changes a great deal but the target doesn't change is treated equivalently to a pair where the target changes a lot, but the cue does not change at all, which in turn are treated equivalently to a pair where the cue and target both change moderate amounts. Given that the authors speculate that there are different processes occurring with the changes in cue and target and the lack of relationship between cue+target relatedness and memorability, it might be important to tease apart the relative impact of the changes to the different aspects of the pair.

Finally, it is unclear to me whether there was any online spell-checking that occurred during the free recall in the learning phase. If there wasn't, I could imagine a case where words might have accidentally received additional retrieval opportunities during learning - take for example, a case where a participant misspelled "razor" as "razer." In this example, they likely still successfully learned the word pair but if there was no spell-checking that occurred during the learning phase, this would not be considered correct, and the participant would have had an additional learning opportunity for that pair.

Reviewer #3 (Public Review):

Summary:

This work investigates the role of cellular senescence in the progression of Periodontitis using a combination of in vivo and in vitro mouse modelling experiments, human periodontitis samples, and transcriptomic analyses.

The authors propose that gum fibroblasts from either patient periodontitis samples or a mouse model of periodontitis can enter a state of cellular senescence (Figure 1). Treatment of their periodontitis mouse model with the compound Metformin attenuated this senescent phenotype and mildly reduced symptom severity. Therefore providing a potential mechanistic link between the senescent state and disease progression (Figure 2).

Leveraging analysis of published single-cell RNA-sequencing datasets of human healthy and periodontitis gum samples, the authors identify CD81+ gum fibroblasts as the cell type with the greatest enrichment of senescence-associated gene expression (Figures 3 and 4) as well as possessing metabolic alterations (Figure 5). Finally, the authors propose that these senescent gum fibroblasts are able to recruit neutrophils through C3 signalling, generating a sustained inflammatory environment that promotes disease progression (Figure 6).

The conclusions of this research are mostly well supported by that data. However, the characterisation of the senescent state and its causal involvement in disease progression could be further improved.

Strengths:

(1) The authors' use of both human and mouse samples provides important translational relevance to their research by finding analogous populations of putatively senescent fibroblasts in both systems.

(2) The use of single-cell RNA-sequencing datasets derived from patient control and periodontitis samples provides a powerful system for interrogating specific cell types. Such an analysis allowed for the characterisation of fibroblast heterogeneity revealing the unique CD81-expressing subset as having the greatest senescent characteristics. Importantly, this result was validated by immunofluorescence in both mouse and human periodontitis systems.

Weaknesses:

(1) The assessment of cellular senescence induction during periodontitis is rather superficial, relying on p16 and p21 Immunohistochemical staining and geneset enrichment analysis (Figure 1). This could be bolstered by their in vitro human fibroblast culture system utilising LPS stimulation. Specifically, their assessment could be more robust by including further markers of senescence such as (i) expression of DNA-damage markers, (ii) evidence of proliferative arrest, and (iii) assessment of an induced secretory phenotype. While a SASP signature was defined in Figure 5A, this was derived from a published single-cell RNA-sequencing dataset. Finding an analogous SASP signature in their human fibroblast cultures/bulk RNA-sequencing comparison of mouse normal-versus-periodontitis tissue would provide more compelling evidence for senescence induction.

(2) While Metformin treatment has an existing basis in the literature as a therapeutic strategy for treating periodontitis, the authors of the current study provide novelty by proposing that Metformin acts by reducing the senescent cell burden during periodontitis. While Metformin treatment is able to significantly reduce the severity of bone damage in ligation-induced periodontitis, the effect is quite mild and the evidence presented does not compellingly show an effect on the putatively senescent p16+ and p21+ cell populations in the gum (Figures 2E and F). Moreover, while the authors show that Metformin treatment is able to attenuate senescence by reducing the expression of senescence-associated Beta-galactosidase (Supplementary Figure 2E), this raises several questions. Namely, (i) Does Metformin prevent the acquisition of a senescent state or (ii) is it acting as a senolytic by actively killing the senescent fibroblasts? It would be important to address these questions to better assess whether Metformin treatment is efficacious only prophylactically, or whether it can have an effect during disease progression. Furthermore, experimental testing if other, widely utilised, senolytics strategies (i.e Navitoclax, Dasatinib+Quercetin, Fisetin etc...) or testing if a p16-/- genetic background is able to attenuate senescence and produce similar protective response would provide more compelling evidence to support their conclusion that cellular senescence is having a causal role in disease progression.

(3) The authors' metabolic profiling of their senescent gum fibroblasts, through interrogation of the transcriptomic datasets, reveals an upregulated synthesis of arachidonic acid. Through this they propose that it can be converted into prostaglandins and leukotrienes, by COXs expressed by the fibroblasts, fuelling tissue inflammation. However, this mechanism promoting inflammation is speculative and lacks experimental demonstration. To support this mechanism it would be important to show (i) increased prostaglandin/leukotrienes expression in periodontitis (relative to healthy control) and (ii) the ability to reduce this by attenuating the senescent phenotype (either by Metformin or other senolytics strategies).

for - sleep hygiene - 3 - temperature

advice - sleep hygiene - 3 - temperature - sleep cool - 18.5 Deg C is ideal

Reviewer #3 (Public Review):

Summary:

This paper assesses the size and clearance kinetics of proviral HIV DNA (intact and total) in women in South Africa with clade C virus. who started ART at different time points of infection (very early vs late).

Strengths:

The cohort is excellent. The paper is easy to read. The methodology is appropriate. Some conclusions, particularly the differing kinetics of total HIV DNA despite a similar amount of virus in early vs late treated women are novel and thought-provoking. I really enjoyed reading this paper!

Weaknesses:

There are several areas in the paper that could be explicated a bit more accurately with more detailed references to past work.

(1) The word reservoir should not be used to describe proviral DNA soon after ART initiation. It is generally agreed upon that there is still HIV DNA from actively infected cells (phase 1 & 2 decay of RNA) during the first 6-12 months of ART. Only after a full year of uninterrupted ART is it really safe to label intact proviral HIV DNA as an approximation of the reservoir. This should be amended throughout.

(2) All raw, individualized data should be made available for modelers and statisticians. It would be very nice to see the RNA and DNA data presented in a supplementary figure by an individual to get a better grasp of intra-host kinetics.

(3) The legend of Supplementary Figure 2 should list when samples were taken.

Reviewer #3 (Public Review):

Summary:

The paper by Gao et al. describes that capsaicin (CAP) might act as a novel NRF2 agonist capable of suppressing ethanol (EtOH)-induced oxidative damage in the gastric mucosa by disrupting the KEAP1-NRF2 interaction. Initially, the authors established and validated a cell model for EtOH-induced oxidative stress which they used to experiment with different CAP concentrations and to determine changes in a variety of parameters such as cell morphology, ROS production, status of redox balance to mitochondrial function, amongst others.

The proposed mechanism by which CAP activates NRF2 and mitigates oxidative stress is thought to be via non-covalent binding to the Kelch domain of KEAP1. A variety of assays such as BLI, CETSA, Pull-down, Co-IP, and HDX-MS were employed to investigate the KEAP1 binding behavior of CAP both in vitro and in GES1 cells. Consequently, the authors developed in vivo nanoparticles harboring CAP and tested those in a rat model. They found that pretreatment with the CAP-nanoparticles led to significant upregulation of NRF2 and subsequent effects on pro- (suppression of IL-1β, TNF-α, IL-6, and CXCL1) and anti-inflammatory (activation of IL-10) cytokines pointing to a resolved state of inflammation and oxidative stress.

Strengths:

The work comprises a comprehensive approach with a variety of in vitro assays as well as cell culture experiments to investigate CAP binding behaviour to KEAP1. In addition, the authors employ in vivo validation by establishing an ethanol-induced acute gastric mucosal damage rat model and providing evidence of the potential therapeutic effect of CAP.

The study further provides novel insights into the mode of CAP action by elucidating the mechanism by which CAP promotes NRF2 expression and downstream antioxidant target gene activation.

The design of IR-Dye800 modified albumin-coated CAP nanoparticles for enhanced drug solubility and delivery efficiency demonstrates a valuable practical application of the research findings.

In summary, the study's findings suggest that CAP could be a safe and novel NRF2 agonist with implications for the development of lead drugs for oxidative stress-related diseases. Collectively, the data support the significance and potential impact of CAP as a therapeutic agent for oxidative stress-related conditions.

Weaknesses:

While the study provides valuable insights into the molecular mechanisms and in vivo effects of CAP, further clinical studies are needed to validate its efficacy and safety in human subjects. The study primarily focuses on the acute effects of CAP on ethanol-induced gastric mucosa damage. Long-term studies are necessary to assess the sustained therapeutic effects and potential side effects of CAP treatment.

Furthermore, the study primarily focuses on the interaction between CAP and the KEAP1-NRF2 axis in the context of ethanol-induced gastric mucosa damage. It may be beneficial to explore the broader effects of CAP on other pathways or conditions related to oxidative stress. CAP has been known for its interaction with the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel and subsequent NRF2 signaling pathway activation. Those receptors are also expressed within the gastric mucosa and could potentially cross-react with CAP leading to the observed outcome. Including experiments to investigate this route of activation could strengthen the present study.

While the design of CAP nanoparticles is innovative, further research is needed to optimize the nanoparticle formulation for enhanced efficacy and targeted delivery to specific tissues.

Addressing these weaknesses through additional research and clinical trials can strengthen the validity and applicability of CAP as a therapeutic agent for oxidative stress-related conditions.

Reviewer #3 (Public Review):

Summary:

The authors. sought to quantify the influence of the gut microbiome on metabolite cycling in a Drosophila model with extensive metabolomic profiling over a 24-hour period. The major strength of the work is the production of a large dataset of metabolites that can be the basis for hypothesis generation for more specific experiments. There are several weaknesses that make the conclusions difficult to evaluate. Additional experiments to quantify food intake over time will be required to determine the direct role of the microbiome in metabolite cycling.

Strengths:

An extensive metabolomic dataset was collected with treatments designed to determine the influence of the gut microbiome on metabolite circadian cycling.

Weaknesses:

(1) The major strength of this study is the extensive metabolomic data, but as far as I can tell, the raw data is not made publicly available to the community. The presentation of highly processed data in the figures further underscores the need to provide the raw data (see comment 3).

(2) Feeding times heavily influence the metabolome. The authors use timed feeding to constrain when flies can eat, but there is no measurement of how much they ate and when. That needs to be addressed.

Since food is the major source of metabolites, the timing of feeding needs to be measured for each of the treatment groups. In the previous paper (Zhang et al 2023 PNAS), the feeding activity of groups of 4 male flies was measured for the wildtype flies. That is not sufficient to determine to what extent feeding controls the metabolic profile of the flies. Additionally, timed feeding opportunities do not equate to the precise time of feeding. They may also result in dietary restriction, leading to the loss of stress resistance in the TF flies. The authors need to measure food consumption over time in the exact conditions under which transcriptomic and metabolomic cycling are measured. I suggest using the EX-Q assay as it is much less effort than the CAFE assay and can be more easily adapted to the rearing conditions of the experiments.

(3) The data on the cycling of metabolites is presented in a heavily analyzed form, making it difficult to evaluate the validity of the findings, particularly when a lack of cycling is detected. The normalization to calculate the change in cycling due to particular treatments is particularly unclear and makes me question whether it is affecting the conclusions. More presentation of the raw data to show when cycling is occurring versus not would help address this concern, as would a more thorough explanation of how the normalization is calculated - the brief description in the methods is not sufficient.

For instance, the authors state that "timed feeding had less effect on flies containing a microbiome relative to sterile flies." One alternative interpretation of that result is that both treatments are cycling but that the normalization of one treatment to the other removes the apparent effect. This concern should be straightforward to address by showing the raw data for individual metabolites rather than the group.

Reviewer #3 (Public Review):

This study was focused on the conserved mechanisms across the Transmembrane Channel/Scramblase superfamily, which includes members of the TMEM16, TMEM63/OSCA, and TMC families. The authors show that the introduction of lysine residues at the TM4-TM6 interface can disrupt gating and confer scramblase activity to non-scramblase proteins. Specifically, they show this to be true for conserved TM4 residues across TMEM16F, TMEM16A, OSCA1.2, and TMEM63A proteins. This breadth of data is a major strength of the paper and provides strong evidence for an underlying linked mechanism for ion conduction and phospholipid transport. Overall, the confocal imaging experiments, patch clamping experiments, and data analysis are performed well.

However, there are several concerns regarding the scope of experiments supporting some claims in the paper. Although the authors propose that the TM4/TM6 interface is critical to ion conduction and phospholipid scramblase activity, in each case, there is very narrow evidence of support consisting of 1-3 lysine substitutions at specific residues on TM4. Given that the authors postulate that the introduction of a positive charge via the lysine side chain is essential to the constitutive activity of these proteins, additional mutation controls for side chain size (e.g. glutamine/methionine) or negative charge (e.g. glutamic acid), or a different positive charge (i.e. arginine) would have strengthened their argument. To more comprehensively understand the TM4/TM6 interface, mutations at locations one turn above and one turn below could be studied until there is no phenotype. In addition, the equivalent mutations on the TM6 side should be explored to rule out the effects of conformational changes that arise from mutating TM4 and to increase the strength of evidence for the importance of side-chain interactions at the TM6 interface. The experiments for OSCA1.2 osmolarity effects on gating and scramblase in Figure 4 could be improved by adding different levels of osmolarity in addition to time in the hypotonic solution.

Reviewer #3 (Public Review):

Summary:

The manuscript by Wang and colleagues describes single molecule localization microscopy to quantify the distribution and organization of Nipah virus F expressed on cells and on virus-like particles. Notably the crystal structure of F indicated hexameric assemblies of F trimers. The authors propose that F clustering favors membrane fusion.

Strengths:

The manuscript provides solid data on imaging of F clustering with the main findings of:<br /> - F clusters are independent of expression levels<br /> - Proteolytic cleavage does not affect F clustering<br /> - Mutations that have been reported to affect the hexamer interface reduce clustering on cells and its distribution on VLPs<br /> - - F nanoclusters are stabilized by AP

Weaknesses:

The relationship between F clustering and fusion is per se interesting, but looking at F clusters on the plasma membrane does not exclude that F clustering occurs for budding. Many viral glycoproteins cluster at the plasma membrane to generate micro domains for budding. This does not exclude that these clusters include hexamer assemblies or clustering requires hexamer assemblies.<br /> Assuming that the clusters are important for entry, hexameric clusters are not unique to Nipah virus F. Similar hexameric clusters have been described for the HEF on influenza virus C particles (Halldorsson et al 2021) and env organization on Foamy virus particles (Effantin et al 2016), both with specific interactions between trimers. What is the organization of F on Nipah virus particles? If F requires to be hexameric for entry, this should be easily imaged by EM on infectious or inactivated virus particles.<br /> AP stabilization of the F clusters is curious if the clusters are solely required for entry? Virus entry does not recruit the clathrin machinery. Is it possible that F clusters are endocytosed in the absence of budding?

Other points:<br /> Fig. 3: Some of the V108D and L53D clusters look similar in size than wt clusters. It seems that the interaction is important but not absolutely essential? Would a double mutant abrogate clustering completely?<br /> Fig. 4: The distribution of F on VLPs should be confirmed by cryoEM analyses. This would also confirm the symmetry of the clusters.

The manuscript by Chernomordik et al. JBC 2004 showed that influenza HA outside the direct contact zone affects fusion, which could be further elaborated in the context of F clusters and the fusion mechanism.

Reviewer #3 (Public Review):

Summary:

In this work, Link and colleagues have investigated the localization and function of the actomyosin system in the parasite Trypanosoma brucei, which represents a highly divergent and streamlined version of this important cytoskeletal pathway. Using a variety of cutting-edge methods, the authors have shown that the T. brucei Myo1 homolog is a dynamic motor that can translocate actin, suggesting that it may not function as a more passive crosslinker. Using expansion microscopy, iEM, and CLEM, the authors show that MyoI localizes to the endosomal pathway, specifically the portion tasked with internalizing and targeting cargo for degradation, not the recycling endosomes. The glycosomes also appear to be associated with MyoI, which was previously not known. An actin chromobody was employed to determine the localization of filamentous actin in cells, which was correlated with the localization of Myo1. Interestingly, the pool of actomyosin was not always closely associated with the flagellar pocket region, suggesting that portions of the endolysomal system may remain at a distance from the sole site of parasite endocytosis. Lastly, the authors used actin-perturbing drugs to show that disrupting actin causes a collapse of the endosomal system in T. brucei, which they have shown recently does not comprise distinct compartments but instead a single continuous membrane system with subdomains containing distinct Rab markers.

Strengths:

Overall, the quality of the work is extremely high. It contains a wide variety of methods, including biochemistry, biophysics, and advanced microscopy that are all well-deployed to answer the central question. The data is also well-quantitated to provide additional rigor to the results. The main premise, that actomyosin is essential for the overall structure of the T. brucei endocytic system, is well supported and is of general interest, considering how uniquely configured this pathway is in this divergent eukaryote and how important it is to the elevated rates of endocytosis that are necessary for this parasite to inhabit its host.

Weaknesses:

(1) Did the authors observe any negative effects on parasite growth or phenotypes like BigEye upon expression of the actin chromobody?

(2) The Garcia-Salcedo EMBO paper cited included the production of anti-actin polyclonal antibodies that appeared to work quite well. The localization pattern produced by the anti-actin polyclonals looks similar to the chromobody, with perhaps a slightly larger labeling profile that could be due to differences in imaging conditions. I feel that the anti-actin antibody labeling should be expressly mentioned in this manuscript, and perhaps could reflect differences in the F-actin vs total actin pool within cells.

(3) The authors showed that disruption of F-actin with LatA leads to disruption of the endomembrane system, which suggests that the unique configuration of this compartment in T. brucei relies on actin dynamics. What happens under conditions where endocytosis and endocyctic traffic is blocked, such as 4 C? Are there changes to the localization of the actomyosin components?

(4) Along these lines, the authors suggest that their LatA treatments were able to disrupt the endosomal pathway without disrupting clathrin-mediated endocytosis at the flagellar pocket. Do they believe that actin is dispensable in this process? That seems like an important point that should be stated clearly or put in greater context.

Reviewer #3 (Public Review):

Summary:

This paper used RNAseq, ATACseq, and Hi-C to assess gene expression, chromatin accessibility, and chromatin physical associations for native CD4+ T cells as they respond to stimulation through TCR and CD28. With these data in hand, the author identified 423 GWAS signals to their respective target genes, where most of these were not in the proximal promoter, but rather distal enhancers. The IL-2 gene was used as an example to identify new distal cis-regulatory regions required for optimal IL-2 gene transcription. These distal elements interact with the proximal IL2 promoter region. When the distal enhancer contained an autoimmune SNP, it affected IL-2 gene transcription. The authors also identified genetic risk variants that were associated with genes upon activation. Some of these regulate proliferation and cytokine production, but others are novel.

Strengths:

This paper provides a wealth of data related to gene expression after CD4 T cells are activated through the TCR and CD28. An important strength of this paper is that these data were intensively analyzed to uncover autoimmune disease SNPs in cis-acting regions. Many of these could be assigned to likely target genes even though they often are in distal enhancers. These findings help to provide a better understanding concerning the mechanism by which GWAS risk elements impact gene expression.

Another strength of this study was the proof-of-principle studies examining the IL-2 gene. Not only were new cis-acting enhancers discovered, but they were functionally shown to be important in regulating IL-2 expression, including susceptibility to colitis. Their importance was also established with respect to such distal enhancers harboring disease-relevant SNPs, which were shown to affect IL-2 transcription.

The data from this study were also mined against past CRISPR screens that identified genes that control aspects of CD4 T cell activation. From these comparisons, novel genes were identified that function during T cell activation.

Weaknesses:

A weakness of this study is that few individuals were analyzed, i.e., RNAseq and ATACseq (n=3) and HiC (n=2). Thus, the authors may have underestimated potentially relevant risk associations by their chromatin capture-based methodology. This might account for the low overlap of their data with the eQTL-based approach or the HIEI truth set.

Impact:

This study indicates that defining distal chromatin interacting regions helps to identify distal genetic elements, including relevant variants, that contribute to gene activation.

Reviewer #3 (Public Review):

Summary:

In their manuscript "Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans", Lenka et al present data that could suggest an "in trans" model of Parkin ubiquitination activity. Parkin is an intensely studied E3 ligase implicated in mitophagy, whereby missense mutations to the PARK2 gene are known to cause autosomal recessive juvenile parkinsonism. From a mechanistic point of view, Parkin is extremely complex. Its activity is tightly controlled by several modes of auto-inhibition that must be released by queues of mitochondrial damage. While the general overview of Parkin activation has been mapped out in recent years, several details have remained murky. In particular, whether Parkin dimerizes as part of its feed-forward signaling mechanism, and whether said dimerization can facilitate ligase activation, has remained unclear. Here, Lenka et al. use various truncation mutants of Parkin in an attempt to understand the likelihood of dimerization (in support of an "in trans" model for catalysis).

Strengths:

The results are bolstered by several distinct approaches including analytical SEC with cleavable Parkin constructs, ITC interaction studies, ubiquitination assays, protein crystallography, and cellular localization studies.

Weaknesses:

As presented, however, the storyline is very confusing to follow and several lines of experimentation felt like distractions from the primary message. Furthermore, many experiments could only indirectly support the author's conclusions, and therefore the final picture of what new features can be firmly added to the model of Parkin activation and function is unclear.

Major concerns:

(1) This manuscript solves numerous crystal structures of various Parkin components to help support their idea of in trans transfer. The way these structures are presented more resemble models and it is unclear from the figures that these are new complexes solved in this work, and what new insights can be gleaned from them.

(2) There are no experiments that definitively show the in trans activation of Parkin. The binding experiments and size exclusion chromatography are a good start, but the way these experiments are performed, they'd be better suited as support for a stronger experiment showing Parkin dimerization. In addition, the rationale for an in trans activation model is not convincingly explained until the concept of Parkin isoforms is introduced in the Discussion. The authors should consider expanding this concept into other parts of the manuscript.

2a. For the in trans activation experiment using wt Parkin and pParkin (T270R/C431A) (Figure 3D), there needs to be a large excess of pParkin to stimulate the catalytic activity of wt Parkin. This experiment has low cellular relevance as these point mutations are unlikely to occur together to create this nonfunctional pParkin protein. In the case of pParkin activating wt Parkin (regardless of artificial point mutations inserted to study specifically the in trans activation), if there needs to be much more pParkin around to fully activate wt Parkin, isn't it just more likely that the pParkin would activate in cis?

2ai. Another underlying issue with this experiment is that the authors do not consider the possibility that the increased activity observed is a result of increased "substrate" for auto-ubiquitination, as opposed to any role in catalytic activation. Have the authors considered looking at Miro as a substrate in order to control for this?

2b. The authors mention a "higher net concentration" of the "fused domains" with RING0, and use this to justify artificially cleaving the Ubl or RING2 domains from the Parkin core. This fact should be moot. In cells, it is expected there will only be a 1:1 ratio of the Parkin core with the Ubl or RING2 domains. To date, there is no evidence suggesting multiple pUbls or multiple RING2s can bind the RING0 binding site. In fact, the authors here even show that either the RING2 or pUbl needs to be displaced to permit the binding of the other domain. That being said, there would be no "higher net concentration" because there would always be the same molar equivalents of Ubl, RING2, and the Parkin core.

2c. A larger issue remaining in terms of Parkin activation is the lack of clarity surrounding the role of the linker (77-140); particularly whether its primary role is to tether the Ubl to the cis Parkin molecule versus a role in permitting distal interactions to a trans molecule. The way the authors have conducted the experiments presented in Figure 2 limits the possible interactions that the activated pUbl could have by (a) ablating the binding site in the cis molecule with the K211N mutation; (b) further blocking the binding site in the cis molecule by keeping the RING2 domain intact. These restrictions to the cis parkin molecule effectively force the pUbl to bind in trans. A competition experiment to demonstrate the likelihood of cis or trans activation in direct comparison with each other would provide stronger evidence for trans activation.

(3) A major limitation of this study is that the authors interpret structural flexibility from experiments that do not report directly on flexibility. The analytical SEC experiments report on binding affinity and more specifically off-rates. By removing the interdomain linkages, the accompanying on-rate would be drastically impacted, and thus the observations are disconnected from a native scenario. Likewise, observations from protein crystallography can be consistent with flexibility, but certainly should not be directly interpreted in this manner. Rigorous determination of linker and/or domain flexibility would require alternative methods that measure this directly.

(4) The analysis of the ACT element comes across as incomplete. The authors make a point of a competing interaction with Lys48 of the Ubl domain, but the significance of this is unclear. It is possible that this observation could be an overinterpretation of the crystal structures. Additionally, the rationale for why the ACT element should or shouldn't contribute to in trans activation of different Parkin constructs is not clear. Lastly, the conclusion that this work explains the evolutionary nature of this element in chordates is highly overstated.

(5) The analysis of the REP linker element also seems incomplete. The authors identify contacts to a neighboring pUb molecule in their crystal structure, but the connection between this interface (which could be a crystallization artifact) and their biochemical activity data is not straightforward. The analysis of flexibility within this region using crystallographic and AlphaFold modeling observations is very indirect. The authors also draw parallels with linker regions in other RBR ligases that are involved in recognizing the E2-loaded Ub. Firstly, it is not clear from the text or figures whether the "conserved" hydrophobic within the linker region is involved in these alternative Ub interfaces. And secondly, the authors appear to jump to the conclusion that the Parkin linker region also binds an E2-loaded Ub, even though their original observation from the crystal structure seems inconsistent with this. The entire analysis feels very preliminary and also comes across as tangential to the primary storyline of in trans Parkin activation.

Reviewer #3 (Public Review):

Summary:

The authors combine classical theories of phase separation and self-assembly to establish a framework for explaining the coupling between the two phenomena in the context of protein assemblies and condensates. By starting from a mean-field free energy for monomers and assemblies immersed in solvent and imposing conditions of equilibrium, the authors derive phase diagrams indicating how assemblies partition into different condensed phases as temperature and the total volume fraction of proteins are varied. They find that phase separation can promote assembly within the protein-rich phase, providing a potential mechanism for spatial control of assembly. They extend their theory to account for the possibility of gelation. They also create a theory for the kinetics of self-assembly within phase separated systems, predicting how assembly size distributions change with time within the different phases as well as how the volumes of the different phases change with time.

Strengths:

The theoretical framework that the authors present is an interesting marriage of classic theories of phase separation and self-assembly. Its simplicity should make it a powerful general tool for understanding the thermodynamics of assembly coupled to phase separation, and it should provide a useful framework for analyzing experiments on assembly within biomolecular condensates.

The key advance over previous work is that the authors now account for how self-assembly can change the boundaries of the phase diagram.

A second interesting point is the explicit theoretical consideration for the possibility that gelation (i.e. self-assembly into a macroscopic aggregate) could account for widely observed solidification of condensates. While this concept has been broadly discussed, to date I have yet to see a rigorous theoretical analysis of the possibility.

The kinetic theory in sections 5 and 6 is also interesting as it extends on previous work by considering the kinetics of phase separation as well as those of self-assembly.

Weaknesses:

A key point the authors make about their theory is that it allows, as opposed to previous research, to study non-dilute limits. It is true that they consider gelation when the 3D assemblies become macroscopic. However, dilute solution theory assumptions seem to be embedded in many aspects of their theory, and it is not always clear where else the non-dilute limits are considered. Is it in the inter-species interaction \chi_{ij}? Why then do they never explore cases for which \chi_{ij} is nonzero in their analysis?

The connection between this theory and biological systems is described in the introduction but lost along the main text. It would be very helpful to point out, for instance, that the presence of phase separation might induce aggregation of proteins. This point is described formally at the end of Section 3, but a more qualitative connection to biological systems would be very useful here.

Building on the previous point, it would be helpful to give an intuitive sense of where the equations derived in the Appendices and presented in the main text come from and to spell out clear physical interpretations of the results. For example, it would be helpful to point out that Eq. 4 is a form of the law of mass action, familiar from introductory chemistry.

It would be useful to better explain how the current work extends on existing previous work from these authors as well as others. Along these lines, closely related work by W. Jacobs and B. Rogers [O. Hedge et al. 2023, https://arxiv.org/abs/2301.06134; T. Li et al. 2023, https://arxiv.org/abs/2306.13198] should be cited in the introduction.

The results discussed in the first paragraph of Section 3 on assembly size distributions in a homogeneous system are well-known from classic theories of self-assembly. This should be acknowledged and appropriate references should be added; see for instance Rev. Mod. Phys. 93, 025008 and Statistical Thermodynamics Of Surfaces, Interfaces, And Membranes by Sam Safran.

Equation 14 for the kinetic of volume fractions is given with a reference to Bauermann et al 2022, but it should be accompanied by a better intuitive interpretation of its terms in the main text. In particular, how should one understand the third term in this equation? Why does the change in volume impact the change of volume fraction in this way?

The discussion in the last paragraph of Section 6 should be clarified. How can the total amount of protein in both phases decrease? This would necessarily violate either mass or volume conservation. Also, the discussion of why the volume is non-monotonic in time is not clear.

Reviewer #3 (Public Review):

In this manuscript, the authors detail improvements in the core CTFFIND (CTFFIND5 as implemented in cisTEM) algorithm that better estimates CTF parameters from titled micrographs and those that exhibit signal attenuation due to ice thickness. These improvements typically yield more accurate CTF values that better represent the data. Although some of the improvements result in slower calculations per micrograph, these can be easily overcome through parallelization.

There are some concerns outlined below that would benefit from further evaluation by the authors.

For the examples shown in Figure 3b, given the small differences in estimated defocus1 and 2, what type of improvements would be expected in the reconstructed tomograms? Do such improvements in estimates manifest in better tilt-series reconstruction?

Similarly, the data shown in Figure 3C shows minimal improvements in the CTF resolution estimate (e.g., 4.3 versus 4.2 Å), but exhibited several hundred Å difference in defocus values. How do such differences impact downstream processing? Is such a difference overcame by per-particle (local) CTF refinements (like the authors mention in the discussion, see below)?

At which point does the thickness of the specimen preclude the ice thickness modulation to be included for "accurate" estimate? 500Å? 1000Å? 2000Å? Based on the data shown in Figure 3B, as high as 969 Å thick specimens benefit moderately (4.6 versus 3.4 Å fit estimate), but perhaps not significantly, from the ice thickness estimation. Considering the increased computational time for ice thickness estimation, such an estimate of when to incorporate for single-particle workflows would be beneficial.

It would seem that this statement could be evaluated herein: "the analysis of images of purified samples recorded at lower acceleration voltages, e.g., 100 keV (McMullan et al., 2023), may also benefit since thickness-dependent CTF modulations will appear at lower resolution with longer electron wavelengths". There are numerous examples of 300kV, 200kV, and 100kV EMPIAR datasets to be compared and recommendations would be welcomed.

Although logical, this statement is not supported by the data presented in this manuscript: "The improvements of CTFFIND5 will provide better starting values for this refinement, yielding better overall CTF estimation and recovery of high-resolution information during 3D reconstruction."

Moreso, the lack of single-particle data evaluation does present a concern. Naively, these improvements would benefit all cryoEM data, regardless of modality.

Reviewer #3 (Public Review):

Summary:

The authors conducted a time-resolved EEG decoding study where they presented sequences of dot locations (4 locations onscreen) or single elements of those sequences, presented at the correct temporal epoch for if they had been presented in the full sequence. They were interested in examining whether presenting single items would activate representations of the anticipated following events that were never presented. Stimuli were presented for 100 ms and separated by 200 ms ISIs. They also had pattern estimation blocks with 600 ms ISIs. They found indeed, that anticipated events could be decoded at their correct moment in time, although future anticipated elements could not.

The decoding of presented dots was fairly confined to the diagonal of the decoding matrix (training time x testing time), suggesting little temporal generalisation. This was in contrast with successor representations which were temporally more diffuse. The subsequent successor could be decoded but not future successors.

Strengths:

I liked this paper. The design was simple and clean and the implications of the findings are clear. The authors achieved their aims with this design, with the results supporting the conclusions. The findings will be of interest to a range of researchers studying learning and perception mechanisms, as well as the more generic role of prediction in the brain.

Weaknesses:

The sample size is fairly low for an EEG study. The authors justify it according to a previous Hogendoorn study, but not according to effect sizes in that study and particular power values.

For understandable reasons, the long ISI blocks were presented before the main test blocks (I would have made the same decision) but there is the risk that participants then come to expect stimuli at larger temporal separations in the main blocks. I do wonder whether this is part of the reason for the greater temporal generalisation for anticipated event representations.

Additional context:

My memory of Ekman et al. 2017 is that single events (presented at position 1) elicited predictive activation of anticipated future events, but that there was a temporal compression. The present study appears to show no temporal compression but that the representations are activated at the correct moment in time. This seems like a potentially interesting difference and one with mechanistic implications for the field.

Reviewer #3 (Public Review):

Summary:

Huang et al. investigated the phenotype of Bend2 mutant mice which expressed a truncated isoform. This mutant male showed increasing apoptosis due to unrepaired double-strand breaks. However, this mutant male has fertility, and this enabled them to analyze Bend2 function in females. They revealed that Bend2 mutation in females showed decreasing follicle numbers which leads to loss of ovarian reserve.

Strengths:

Since their Bend2 mutant males were fertile, they were able to analyze the function of Bend2 in females and they revealed that loss of Bend2 causes less follicle formation.

Weaknesses:

Why the phenotype of their mutant male is different from previous work (Ma et al.) is not clear enough although they discuss it.

Reviewer #3 (Public Review):

Summary:

In this work, MacFarland et.al. show that difference in the time of contact between axons of LC4 and LPLC2 visual projection neurons (VPNs) in the optic glomeruli and dendrites of large descending neuron, the giant fiber (GF) shapes the differential connectivity between these neurons.

Strengths:

The authors analyzed the development of a well-known circuit between GF dendrites and LC4 andLPLC2 axons using different approaches. Additionally, they developed an ex-vivo patch clamping technique to show, together with correlative RNA-sequencing data, that contact site restriction is not dependent on neuronal activity. Based on this study, the connectivity pattern between GF and the adjacent different sets of VPNs now provides a very interesting model to investigate developmental programs that lead to synaptic specificity.

Weaknesses:

Following are the concerns that significantly impact the veracity of conclusions drawn based on the data provided.

(1) All the data related to the activity of VPNs and GF and how this activity is related to the connectivity and/or maintaining and stabilizing this connectivity is correlative. The expression profiles of synaptic molecules (only at RNA level) over time or the appearance of pre and post synaptic proteins or the spontaneous spike patterns in GF do not show the role of activity in synapse specificity program. Synaptic molecules have been previously shown to be present at presynaptic sites without being involved in activity (Chen et al., 2014, Jin et al., 2018). To show whether activity is indeed not required for connectivity for either of the cell types (LC4 and LPLC2), they should silence each and also both cell types as early as possible (with the LC4 driver that does not ablate them) and then quantify the contacts with GF. In the same vein, the authors should knock down components of the synaptic machinery as early as possible to show directly the effect on 1) contact formation and 2) contact stabilization. For example, authors state in the lines 267-269 "VPN cholinergic machinery arrives too late to contribute to the initial targeting and localization of VPN axons on GF dendrites. Cholinergic activity instead is likely to participate in VPN and GF synapse refinement and stabilization." This statement would only be valid if the authors knock down the cholinergic machinery and find the contact numbers unchanged in the early stages but significantly different in later stages in comparison to the controls. Furthermore, authors only show increase in the VAChT and ChAT in the presynaptic cells but do not show if the cholinergic receptor AChRs are even expressed in GF cells or at what point they are expressed. Without these receptor expression, cholinergic system might not even be involved in the process. Also, there might be other neurotransmitter systems involved. Authors should at least check if other neurotransmitter systems are expressed in these cells, both pre-and post-synaptic.<br /> Line 371-374: "In the later stages of development, the frequency of synaptic events increase as gap junction proteins are downregulated and cholinergic presynaptic machinery is upregulated to enhance and stabilize synapses with intended synaptic partners while refining unintended contacts". The authors did not show the activity they observed in GF is due to the contacts they make with LC4s and LPLC2s. The functionality of these contacts can be shown by silencing the LC4s and LPLC2s and then doing the patch clamping in GF to see a decrease in the activity. Further, the authors did not show that the reduction in contacts are only by refining "unintended" contacts. There is no evidence that can support this statement.

(2) In the LC4 ablation experiments, authors claim that LC4_4 split Gal4 line is expressed around 18APF, prior to GF LC4 initial contact (Line 387). However, authors do not show the time point of first contact between GF dendrites and LC4 cells. In Fig. 2 the first time point shown is at P36, where there is already significant overlap between GF dendrites and LC4 axons. Authors should show the very first time point where they see any, even if minimal, overlap and/or contact between GFs and LC4s. Once the LC4s are ablated, is the increase in the colocalization between GF and LPLC2 due to LPLC2s increasing their contact numbers or due to them not decreasing the maximum contact numbers that the authors observed at P72 (Fig 2G)? In other words, once the LC4s are ablated, what would the new graph for temporal contact numbers for LPLC2 look like and how it would compare to Fig2G?

(3) If the developmental stages for different lines match, that would be more helpful for comparison. Also, as the authors analyzed expression every 12 hours from 0APF, the panel should also contain earlier time points (e.g. P0, P12) for all lines. This is critical to understand at what point the axons of LC4, LPLC2 and LPLC1 reach their position. From the scale bar in Supp Fig.4, it seems LC4 axons have already reached final position at P24 and there is no extension between P24 and P60. Do the authors know at what point LC4 axons start extending and reach the final position? If the LC4 and LPLC2 arbors are already separated medio-laterally even before GF dendrites extend towards them, it would explain why GF dendrites extending from medial region of the brain would encounter LC4 axons first and LPLC2 axons later, just based on their localization in space.<br /> Further to this point, the authors show in the section two of the paper that it is the GF dendrites that extend, elaborate and refine during the phase the authors analyzed and the authors do not show any morphological change in the axons of the VPNs. Therefore, the title of the paper is 'axon arrival times and physical occupancy establish visual projection neuron integration on developing dendrites in the Drosophila optic glomeruli' is slightly misguided.

(4) In the absence of LC4s, does the LPLC1 and GF colocalization increase or do they still stay disconnected?

(5) Does the absence of LC4s have any effect on GF arbor complexity? Does the graph in Fig 2B and C change? Can the increase in colocalization between LPLC2 and GF be at least partially due to the expansion of GF dendritic volume?

(6) Why is there a segregation in the medial-lateral axis but not in the dorso-ventral axis? Wouldn't the same segregation mechanism be in play in both axes? Also, the authors should clarify if this reduction in dorsal-ventral distribution is because dorso-ventral expansion of GF dendrites beyond the LC4 and LPLC2 axons? Theoretically that would seem to make the LC4s move more ventrally and LPLC2 move more dorsally in comparison to the total arbor.

(7) Why the LPLC2 medial connections are regarded as "mistargeting" in the heading of Supplemental Figure 1? Both in EM data and in some of the confocal datasets, these connections are observed. What is the criteria to label a connection "mistargeting" if it is observed, albeit occasionally, both in EM and confocal datasets?

(8) In Line 126-127, authors state that "we sought to determine how the precise VPN localization along GF dendrites arises across development". However, based in EM and microscopic data, there is considerable variability in the contact numbers and distribution. With such variability present, how can the localization be termed "precise"? Authors should clarify.

Reviewer #3 (Public Review):

Summary:

In the present manuscript, the authors use a few minutes of voltage imaging of CA1 pyramidal cells in head-fixed mice running on a track while local field potentials (LFPs) are recorded. The authors suggest that synchronous ensembles of neurons are differentially associated with different types of LFP patterns, theta and ripples. The experiments are flawed in that the LFP is not "local" but rather collected in the other side of the brain, and the investigation is flawed due to multiple problems with the point process analyses. The synchrony terminology refers to dozens of milliseconds as opposed to the millisecond timescale referred to in prior work, and the interpretations do not take into account theta phase locking as a simple alternative explanation.

Weaknesses:

The two main messages of the manuscript indicated in the title are not supported by the data. The title gives two messages that relate to CA1 pyramidal neurons in behaving head-fixed mice: (1) synchronous ensembles are associated with theta (2) synchronous ensembles are not associated with ripples.

There are two main methodological problems with the work: (1) experimentally, the theta and ripple signals were recorded using electrophysiology from the opposite hemisphere to the one in which the spiking was monitored. However, both signals exhibit profound differences as a function of location: theta phase changes with the precise location along the proximo-distal and dorso-ventral axes, and importantly, even reverses with depth. And ripples are often a local phenomenon - independent ripples occur within a fraction of a millimeter within the same hemisphere, let alone different hemispheres. Ripples are very sensitive to the precise depth - 100 micrometers up or down, and only a positive deflection/sharp wave is evident. (2) The analysis of the point process data (spike trains) is entirely flawed. There are many technical issues: complex spikes ("bursts") are not accounted for; differences in spike counts between the various conditions ("locomotion" and "immobility") are not accounted for; the pooling of multiple CCGs assumes independence, whereas even conditional independence cannot be assumed; etc.

Beyond those methodological issues, there are two main interpretational problems: (1) the "synchronous ensembles" may be completely consistent with phase locking to the intracellular theta (as even shown by the authors themselves in some of the supplementary figures). (2) The definition of "synchrony" in the present work is very loose and refers to timescales of 20-30 ms. In previous literature that relates to synchrony of point processes, the timescales discussed are 1-2 ms, and longer timescales are referred to as the "baseline" which is actually removed (using smoothing, jittering, etc.).

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

(1) A full characterization of the effect of sUA in other NAD-consuming and synthesizing enzymes is needed to validate the statement that the mechanism of regulation of NAD by sUA is mediated by CD38, The CD38 KO may not serve as the ideal control since it may saturate NAD levels already. Analysis of multiple tissues is needed.

(2) The physiological role of sUA as an endogenous inhibitor of CD38 needs stronger validation (sUA deficient model?).

(3) Flux studies would also be necessary to make the conclusion stronger.

Reviewer #3 (Public Review):

Summary:

Suzuki-Okutani and collogues reported a new live-attenuated SARS-CoV-2 vaccine (BK2102) containing multiple deletion/substitution mutations. They show that the vaccine candidate is highly attenuated and demonstrates a great safety profile in multiple animal models (hamsters and Tg-Mice). Importantly, their data show that single intranasal immunization with BK2102 leads to strong protection of hamsters against D614G and BA.5 challenge in both lungs and URT (nasal wash). Both humoral and cellular responses were induced, and neutralization activity remained for >360 after a single inoculation.

Strengths:

The manuscript describes a comprehensive study that evaluates the safety, immunogenicity, and efficacy of a new live-attenuated vaccine. Strengths of the study include (1) strong protection against immune evasive variant BA.5 in both lungs and NW; (2) durability of immunity for >360 days; (3) confirmation of URT protection through a transmission experiment.

While first-generation COVID-19 vaccines have achieved much success, new vaccines that provide mucosal and durable protection remain needed. Thus, the study is significant.

Weaknesses:

Lack of a more detailed discussion of this new vaccine approach in the context of reported live-attenuated SARS-CoV-2 vaccines in terms of its advantages and/or weaknesses.

Antibody endpoint titers could be presented.

Lack of elaboration on immune mechanisms of protection at the upper respiratory tract (URT) against an immune evasive variant in the absence of detectable neutralizing antibodies.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

This configuration eliminates the need for transferring gels to other dishes or wells, thereby enhancing the throughput and reproducibility of parallel expansion microscopy. This methodological uniqueness indicates the applicability of HiExM in detecting subtle cellular changes on a large scale.

Weaknesses:

To demonstrate the potential utility of HiExM in cell phenotyping, drug studies, and toxicology investigations, the authors treated hiPS-derived cardiomyocytes with a low dose of doxycycline (dox) and quantitatively assessed changes in nuclear morphology. However, this reviewer is not fully convinced of the validity of this specific application. Furthermore, some data about the effect of expansion require reconsideration.

Reviewer #3 (Public Review):

Summary:

infectious bursal disease virus (IBDV) is a birnavirus and an important avian pathogen. Interestingly, IBDV appears to be a unique dsRNA virus that uses early endosomes for RNA replication that is more common for +ssRNA viruses such as for example SARS-CoV-2.

This work builds on previous studies showing that IBDV VP3 interacts with PIP3 during virus replication. The authors provide further biophysical evidence for the interaction and map the interacting domain on VP3.

Strengths:

Detailed characterization of the interaction between VP3 and PIP3 identified R200D mutation as critical for the interaction. Cryo-EM data show that VP3 leads to membrane deformation.

Weaknesses:

The work does not directly show that the identified R200 residues are directly involved in VP3-early endosome recruitment during infection. The majority of work is done with transfected VP3 protein (or in vitro) and not in virus-infected cells.

Additional controls such as the use of PIP3 antagonizing drugs in infected cells together with a colocalization study of VP3 with early endosomes would strengthen the study.

In addition, it would be advisable to include a control for cryo-EM using liposomes that do not contain PIP3 but are incubated with HIS-VP3-FL. This would allow ruling out any unspecific binding that might not be detected on WB.

The authors also do not propose how their findings could be translated into drug development that could be applied to protect poultry during an outbreak. The title of the manuscript is broad and would improve with rewording so that it captures what the authors achieved.

Reviewer #3 (Public Review):

In this study, Zhang and colleagues proposed an ELMo-based embedding model (catELMo) for TCRβ CDR3 amino acid sequences. They showed the effectiveness of catELMo in both supervised TCR binding prediction and unsupervised clustering, surpassing existing methods in accuracy and reducing annotation costs. The study provides insights on the effect of model architectures to TCR specificity prediction and clustering tasks.

The authors have addressed our prior critiques of the manuscript.

Reviewer #3 (Public Review):

This paper studies chromatic coding in mouse primary visual cortex. Calcium responses of a large collection of cells are measured in response to a simple spot stimulus. These responses are used to estimate chromatic tuning properties - specifically sensitivity to UV and green stimuli presented in a large central spot or a larger still surrounding region. Cells are divided based on their responses to these stimuli into luminance or chromatic sensitive groups. The results are interesting and many aspects of the experiments and conclusions are well done; several technical concerns, however, limit the support for several main conclusions,

Limitations of stimulus choice<br /> The paper relies on responses to a large (37.5 degree diameter) modulated spot and surround region. This spot is considerably larger than the receptive fields of both V1 cells and retinal ganglion cells (it is twice the area of the average V1 receptive field). As a result, the spot itself is very likely to strongly activate both center and surround mechanisms, and responses of cells are likely to depend on where the receptive fields are located within the spot (and, e.g., how much of the true neural surround samples the center spot vs the surround region). Most importantly, the surrounds of most of the recorded cells will be strongly activated by the central spot. This brings into question statements in the paper about selective activation of center and surround (e.g. page 2, right column). This in turn raises questions about several subsequent analyses that rely on selective center and surround activation.

Comparison with retina<br /> A key conclusion of the paper is that the chromatic tuning in V1 is not inherited from retinal ganglion cells. This conclusion comes from comparing chromatic tuning in a previously-collected data set from retina with the present results. But the retina recordings were made using a considerably smaller spot, and hence it is not clear that the comparison made in the paper is accurate. For example, the stimulus used for the V1 experiments almost certainly strongly stimulates both center and surround of retinal ganglion cells. The text focuses on color opponency in the receptive field centers of retinal ganglion cells, but center-surround opponency seems at least as relevant for such large spots. This issue needs to be described more clearly and earlier in the paper.

Limitations associated with ETA analysis<br /> One of the reviewers in the previous round of reviews raised the concern that the ETA analysis may not accurately capture responses of cells with nonlinear receptive field properties such as On/Off cells. This possibility and whether it is a concern should be discussed.

Discrimination performance poor<br /> Discriminability of color or luminance is used as a measure of population coding. The discrimination performance appears to be quite poor - with 500-1000 neurons needed to reliably distinguish light from dark or green from UV. Intuitively I would expect that a single cell would provide such discrimination. Is this intuition wrong? If not, how do we interpret the discrimination analyses?

Reviewer #3 (Public Review):

Summary:

The authors conducted a human fMRI study investigating the omission of expected electrical shocks with varying probabilities. Participants were informed of the probability of shock and shock intensity trial-by-trial. The time point corresponding to the absence of the expected shock (with varying probability) was framed as a prediction error producing the cognitive state of relief/pleasure for the participant. fMRI activity in the VTA/SN and ventral putamen corresponded to the surprising omission of a high probability shock. Participants' subjective relief at having not been shocked correlated with activity in brain regions typically associated with reward-prediction errors. The overall conclusion of the manuscript was that the absence of an expected aversive outcome in human fMRI looks like a reward-prediction error seen in other studies that use positive outcomes.

Strengths:

Overall, I found this to be a well-written human neuroimaging study investigating an often overlooked question on the role of aversive prediction errors, and how they may differ from reward-related prediction errors. The paper is well-written and the fMRI methods seem mostly rigorous and solid.

Comments on revised version:

The authors were extremely responsive to the comments and provided a comprehensive rebuttal letter with a lot of detail to address the comments. The authors clarified their methodology, and rationale for their task design, which required some more explanation (at least for me) to understand. Some of the design elements were not clear to me in the original paper.

The initial framing for their study is still in the domain of learning. The paper starts off with a description of extinction as the prime example of when threat is omitted. This could lead a reader to think the paper would speak to the role of prediction errors in extinction learning processes. But this is not their goal, as they emphasize repeatedly in their rebuttal letter. The revision also now details how using a conditioning/extinction framework doesn't suit their experimental needs.

It is reasonable to develop a new task to answer their experimental questions. By no means is there a requirement to use a conditioning/extinction paradigm to address their questions. As they say, "it is not necessary to adopt a learning paradigm to study omission responses", which I agree with.

But the authors seem to want to have it both ways: they frame their paper around how important prediction errors are to extinction processes, but then go out of their way to say how they can't test their hypotheses with a learning paradigm.

Part of their argument that they needed to develop their own task "outside of a learning context" goes as follows:<br /> (1) "...conditioning paradigms generally only include one level of aversive outcome: the electrical stimulation is either delivered or omitted. As a result, the magnitude-related axiom cannot be tested."<br /> (2) "....in conditioning tasks people generally learn fast, rendering relatively few trials on which the prediction is violated. As a result, there is generally little intra-individual variability in the PE responses"<br /> (3) "...because of the relatively low signal to noise ratio in fMRI measures, fear extinction studies often pool across trials to compare omission-related activity between early and late extinction, which further reduces the necessary variability to properly evaluate the probability axiom"

These points seem to hinge on how tasks are "generally" constructed. However, there are many adaptations to learning tasks:<br /> (1) There is no rule that conditioning can't include different levels of aversive outcomes following different cues. In fact, their own design uses multiple cues that signal different intensities and probabilities. Saying that conditioning "generally only include one level of aversive outcome" is not an explanation for why "these paradigms are not tailored" for their research purposes. There are also several conditioning studies that have used different cues to signal different outcome probabilities. This is not uncommon, and in fact is what they use in their study, only with an instruction rather than through learning through experience, per se.<br /> (2) Conditioning/extinction doesn't have to occur fast. Just because people "generally learn fast" doesn't mean this has to be the case. Experiments can be designed to make learning more challenging or take longer (e.g., partial reinforcement). And there can be intra-individual differences in conditioning and extinction, especially if some cues have a lower probability of predicting the US than others. Again, because most conditioning tasks are usually constructed in a fairly simplistic manner doesn't negate the utility of learning paradigms to address PE-axioms.<br /> (3) Many studies have tracked trial-by-trial BOLD signal in learning studies (e.g., using parametric modulation). Again, just because other studies "often pool across trials" is not an explanation for these paradigms being ill-suited to study prediction errors. Indeed, most computational models used in fMRI are predicated on analyzing data at the trial level.

Again, the authors are free to develop their own task design that they think is best suited to address their experimental questions. For instance, if they truly believe that omission-related responses should be studied independent of updating. The question I'm still left puzzling is why the paper is so strongly framed around extinction (the word appears several times in the main body of the paper), which is a learning process, and yet the authors go out of their way to say that they can only test their hypotheses outside of a learning paradigm.

The authors did address other areas of concern, to varying extents. Some of these issues were somewhat glossed over in the rebuttal letter by noting them as limitations. For example, the issue with comparing 100% stimulation to 0% stimulation, when the shock contaminates the fMRI signal. This was noted as a limitation that should be addressed in future studies, bypassing the critical point.

Reviewer #3 (Public Review):

Kang, Huang, and colleagues have provided new data to address concerns regarding confirmation of LRRK1 and LRRK2 deletion in their mouse model and the functional impact of the modest loss of TH+ neurons observed in the substantia nigra of their double KO mice. In the revised manuscript, the new data around the characterization of the germline-deleted LRRK1 and LRRK2 mice add confidence that LRRK1 and LRRK2 can be deleted using the genetic approach. They have also added new text to the discussion to try and address some of the comments and questions raised regarding how LRRK1/2 loss may impact cell survival and the implications of this work for PD-linked variants in LRRK2 and therapeutic approaches targeting LRRK2. The new data provides additional support for the author's claims.

Reviewer #3 (Public Review):

Summary:

Unlike most eukaryotes Blastocystis has a branched glycolysis pathway, which is split between the cytoplasm and the mitochondrial matrix. An outstanding question was how the glycolytic intermediates generated in the 'preparatory' phase' are transported into the mitochondrial matrix for the 'pay off' phase. Here, the authors use bioinformatic analysis to identify two candidate solute carrier genes, bGIC-1 and bGIC-2, and use biochemical and biophysical methods to characterise their substrate specificity and transport properties. The authors demonstrate that bGIC-2 can transport dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and phosphoenolpyruvate, establishing this protein as the 'missing link' connecting the two split branches of glycolysis in this branch of single celled eukaryotes. The authors also present their data on bGIC-1, which suggests a role in anion transport and bOGC, which is a close functional homologue of the human oxoglutarate carrier (hOGC, SLC25A11) and human dicarboxylate carrier (hDIC, SLC25A10).

Strengths:

The results are presented in a clear and logical arrangement, which nicely leads the reader through the process of gene identification and subsequent ligand screening and functional reconstitution. The results are compelling and well supported - the thermal stabilisation data is supported by the exchange studies. Caveats, where apparent, are discussed and rational explanations given.

Weaknesses:

The study does not contain any significant weaknesses in my view. I would like to see the authors include the initial rate plots used in the main figures (possibly as insets), so we can observe the data points used for these calculations. It would also have been interesting to include the AlphaFold models for bGIC-1 and bGIC-2 and a discussion/rationalisation for the substrate specificity discussed in the study.

Reviewer #3 (Public Review):

Summary:

This is an elegant study investigating possible mechanisms underlying the hysteresis effect in the perception of perceptually ambiguous Shepard tones. The authors make a fairly convincing case that the adaptation of pitch direction sensitive cells in auditory cortex is likely responsible for this phenomenon.

Strengths:

The manuscript is overall well written. My only slight criticism is that, in places, particularly for non-expert readers, it might be helpful to work a little bit more methods detail into the results section, so readers don't have to work quite so hard jumping from results to methods and back.

The methods seem sound and the conclusions warranted and carefully stated. Overall I would rate the quality of this study as very high, and I do not have any major issues to raise.

Weaknesses:

I think this study is about as good as it can be with the current state of the art. Generally speaking, one has to bear in mind that this is an observational, rather than an interventional study, and therefore only able to identify plausible candidate mechanisms rather than making definitive identifications. However, the study nevertheless represents a significant advance over the current state of knowledge, and about as good as it can be with the techniques that are currently widely available.

Reviewer #3 (Public Review):

Summary:

How short-term isolation acts on the brain to promote social behavior remains incompletely understood. The authors found that social interactions after a period of acute isolation increased investigation promoted mounting, and increased the production of ultrasonic vocalizations (USVs). This was true for females during same-sex interactions as well as for males interacting with females. Concomitant with these increased behavioral readouts, cFos expression in the preoptic area of the hypothalamus (POA) was found to increase selectively in single-housed females. Chemogenetic silencing of these POA neurons attenuated all three behavioral measures in socially isolated females. Surprisingly, ablation of the same POA neurons decreased mounting duration without impacting social investigation or USV production. While optogenetic activation was sufficient to evoke USV production, it did not affect either mounting or social investigation. In males, chemogenetic silencing of POA neurons decreased mounting but not other behaviors. Together, these data point towards a role of POA neurons in mediating social behaviors after acute isolation but the exact nature of that control appears to depend on the choice of perturbation method, sex, and social context in complex ways that are hard to parse. This study is an essential first step; additional experiments will be needed to explain the apparent discrepancy between the various circuit perturbation results and to gain a more comprehensive understanding of the role of POA in social isolation.

Strengths:

The goal of understanding the neural circuit mechanisms underlying acute social isolation is clearly important and topical. Using a state-of-the-art technique to tag specific neurons that were active during certain behavioral epochs, the authors managed to identify the POA as a critical circuit locus for the effects of social isolation. The experimental design is perfectly reasonable and the quality of the data is good. The control experiments (Figures 2B-D) showing that chemogenetic inactivation of other hypothalamic regions (AH and VMH) do not affect social behavior is indeed quite satisfying and points towards a specific role of POA within the hypothalamus. Using a combination of behavioral assays, activity-dependent neural tagging, and circuit manipulation techniques, the authors present convincing evidence for the role of the preoptic area of the hypothalamus in mediating certain behaviors following social isolation. These data are likely to be a valuable resource for understanding how hypothalamic circuits adjust to the challenges of social isolation.

Weaknesses:

While the authors should be commended for performing and reporting multiple circuit perturbation experiments (e.g., chemogenetics, ablation), the conflicting effects on behavior are hard to interpret without additional experiments. For example, chemogenetic silencing of the POA neurons (using DREADDs) attenuated all three behavioral measures but the ablation of the same POA neurons (using CASPACE) decreased mounting duration without impacting social investigation or USV production. Similarly, optogenetic activation of POA neurons was sufficient to generate USV production as reported in earlier studies but mounting or social investigation remained unaffected. Do these discrepancies arise due to the efficiency differences between DREADD-mediated silencing vs. Casp3 ablation? Or does the chemogenetic result reflect off-manifold effects on downstream circuitry whereas a more permanent ablation strategy allows other brain regions to compensate due to redundancy? It is important to resolve whether these arise due to technical reasons or whether these reflect the underlying (perhaps messy) logic of neural circuitry. Therefore, while it is clear that POA neurons likely contribute to multiple behavioral readouts of social isolation, understanding their exact roles in any greater detail will require further experiments.

Reviewer #3 (Public Review):

Summary:

This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

- The manuscript is well written.

- The methods are clearly described and appropriate.

- Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes.

- The results are interesting and novel.

Weaknesses:

- Analyses were conducted for task-based data rather than resting-state data. It was unclear whether groups differed in task performance. If congenitally deaf individuals found the task more difficult this could lead to changes in FC.

- No differences in overall activation between groups were reported. Activation differences between groups could lead to differences in FC. For example, lower activation may be associated with more noise in the data, which could translate to reduced FC.

- Figure 2B shows higher FC for congenitally deaf individuals than normal-hearing individuals in the insula, supplementary motor area, and cingulate. These regions are all associated with task effort. If congenitally deaf individuals found the task harder (lower performance), then activation in these regions could be higher, in turn, leading to FC. A study using resting-state data could possibly have provided a clearer picture.

- The correlation between the FC map and the FC variability map is 0.3. While significant using permutation testing, the correlation is low, and it is not clear how great the overlap is.

Reviewer #3 (Public Review):

Summary:

This manuscript by Peng et al. presents intriguing data indicating that high-frequency terahertz stimulation (HFTS) of the anterior cingulate cortex (ACC) can alleviate neuropathic pain behaviors in mice. Specifically, the investigators report that terahertz (THz) frequency stimulation widens the selectivity filter of potassium channels thereby increasing potassium conductance and leading to a reduction in the excitability of cortical neurons. In voltage clamp recordings from layer 5 ACC pyramidal neurons in acute brain slice, Peng et al. show that HFTS enhances K current while showing minimal effects on Na current. Current clamp recording analyses show that the spared nerve injury model of neuropathic pain decreases the current threshold for action potential (AP) generation and increases evoked AP frequency in layer 5 ACC pyramidal neurons, which is consistent with previous studies. Data are presented showing that ex-vivo treatment with HFTS in slice reduces these SNI-induced changes to excitability in layer 5 ACC pyramidal neurons. The authors also confirm that HFTS reduces the excitability of layer 5 ACC pyramidal neurons via in vivo multi-channel recordings from SNI mice. Lastly, the authors show that HFTS is effective at reducing mechanical allodynia in SNI using both the von Frey and Catwalk analyses. Overall, there is considerable enthusiasm for the findings presented in this manuscript given the need for non-pharmacological treatments for pain in the clinical setting.

Strengths:

The authors use a multifaceted approach that includes modeling, ex-vivo and in-vivo electrophysiological recordings, and behavioral analyses. Interpretation of the findings is consistent with the data presented. This preclinical work in mice provides new insight into the potential use of directed high-frequency stimulation to the cortex as a primary or adjunctive treatment for chronic pain.

Weaknesses:

There are a few concerns noted that if addressed, would significantly increase enthusiasm for the study.

(1) The left Na current trace for SNI + HFTS in Figure 2B looks to have a significant series resistance error. Time constants (tau) for the rate of activation and inactivation for Na currents would be informative.

(2) It is unclear why an unpaired t-test was performed for paired data in Figure 2. Also, statistical methods and values for non-significant data should be presented.

(3) It would seem logical to perform HFTS on ACC-Pyr neurons in acute slices from sham mice (i.e. Figure 3 scenario). These experiments would be informative given the data presented in Figure 4.

(4) As the data are presented in Figure 4g, it does not seem as if SNI significantly increased the mean firing rate for ACC-Pyr neurons, which is observed in the slice. The data were analyzed using a paired t-test within each group (sham and SNI), but there is no indication that statistical comparisons across groups were performed. If the argument is that HFTS can restore normal activity of ACC-Pyr neurons following SNI, this is a bit concerning if no significant increase in ACC-Pyr activity is observed in in-vivo recordings from SNI mice.

(5) The authors indicate that the effects of HFTS are due to changes in Kv1.2. However, they do not directly test this. A blocking peptide or dendrotoxin could be used in voltage clamp recordings to eliminate Kv1.2 current and then test if this eliminates the effects of HFTS. If K current is completely blocked in VC recordings then the authors can claim that currents they are recording are Kv1.1 or 1.2.

(6) The ACC is implicated in modulating the aversive aspect of pain. It would be interesting to know whether HFTS could induce conditioned place preference in SNI mice via negative reinforcement (i.e. alleviation of spontaneous pain due to the injury). This would strengthen the clinical relevance of using HFTS in treating pain.

Reviewer #3 (Public Review):

This paper builds on the authors' original development of a near infrared (NIR) FRET sensor by reporting in vivo real-time measurements for gamma-secretase activity in the mouse cortex. The in vivo application of the sensor using state of the art techniques is supported by a clear description and straightforward data, and the project represents significant progress because so few biosensors work in vivo. Notably, the NIR biosensor is detectable to ~ 100 µm depth in the cortex. A minor limitation is that this sensor has a relatively modest ΔF as reported in Houser et al, which is an additional challenge for its use in vivo. Thus, the data is fully dependent on post-capture processing and computational analyses. This can unintentionally introduce biases but is not an insurmountable issue with the proper controls that the authors have performed here.

The observation of gamma-secretase signaling that spreads across cells is potentially quite interesting, but it can be better supported. An alternative interpretation is that there exist pre-formed and clustered hubs of high gamma-secretase activity, and that DAPT has stochastic or differential accessibility to cells within the cluster. This could be resolved by an experiment of induction, for example, if gamma-secretase activity is induced or activated at a specific locale and there was observed coordinated spreading to neighboring neurons with their sensor.

Furthermore, to rule out the possibility that uneven viral transduction was not simply responsible for the observed clustering, it would be helpful to see an analysis of 670nm fluorescence alone.

Reviewer #3 (Public Review):

Summary:

The authors use in vitro grown cells and mouse xenografts to show that a combination of drugs, Sulfopin and Vorinostat, can impact the growth of cells derived from Diffuse midline gliomas, in particular the ones carrying the H3 K27M-mutations (clinically classified as DMG, H3 K27M-mutant). The authors use gene expression studies, and chromatin profiling to attempt to better understand how these drugs exert an effect on genome regulation. Their main findings are that the drugs reduce cell growth in vitro and in mouse xenografts of patient tumours, that DMG, H3 K27M-mutant tumours are particularly sensitive, identify potential markers of gene expression underlying this sensitivity, and broadly characterize the correlations between chromatin modification changes and gene expression upon treatment, identifying putative pathways that may be affected and underlie the sensitive (and thus how the drugs may affect the tumour cell biology).

Strengths:

It is a neat, mostly to-the-point work without exploring too many options and possibilities. The authors do a good job not overinterpreting data and speculating too much about the mechanisms, which is a very good thing since the causes and consequences of perturbing such broad epigenetic landscapes of chromatin may be very hard to disentangle. Instead, the authors go straight after testing the performance of the drugs, identifying potential markers and characterizing consequences.

Weaknesses:

If anything, the experiments done on Figure 3 could benefit from an additional replicate.

Reviewer #3 (Public Review):

Summary:

Gularte-Mérida and colleagues took advantage of the existence of so-called consomic strains in the mouse, which result from the substitution of one of their chromosomes by that of another strain, to ask through appropriate crosses whether information carried by this substitution chromosome impacts progeny that do not inherit it. With one exception, the authors did not detect any significant effect for any of the four non-transmitted chromosomes tested. Given these results, the authors conclude that such effects, if they exist, must be extremely rare in the mouse.

Strengths:

This is a very convincing and impressive study, with effects assessed in almost 2500 mice. The negative results obtained should put to rest once and for all the notion that intergenerational, let alone transgenerational, non-DNA sequence-based inheritance via the male germline could be substantial in the mouse.

Weaknesses:

The terminology used (epigenetics, nurture-independent TGE, etc. ) is somewhat confusing and unnecessary.

Reviewer #3 (Public Review):

In this important work, the authors show compelling evidence that the Rapid Alkalinisation Factor1 (RALF1) peptide acts as an interlink between pectin methyl esterification status and FERONIA receptor-like kinase in mediating extracellular sensing. Moreover, the RALF1-mediated pectin perception is surprisingly independent of LRX-mediated extracellular sensing in roots. The authors also show that the peptide directly binds demethylated pectin and the positively charged amino acids are required for pectin binding as well as for its physiological activity.

Some present findings are surprising; previously, the FERONIA extracellular domain was shown to bind pectin directly, and the mode of operation in the pollen tube involves the LRX8-RALF4 complex, which seems not the case for RALF1 in the present study. Although some aspects remain controversial, this work is a very valuable addition to the ongoing debate about this elusive complex regulation and signaling.

The authors drafted the manuscript well, so I do not have a lot of criticism or suggestions. The experiments are well-designed, executed, and presented, and they solidly support the authors' claims.

Reviewer #3 (Public Review):

Summary and Strengths:

In this interesting manuscript, the authors identify a large number of alternative transcription start sites (TSS) and focus their functional analysis on an alternative TSS that is expected to produce a micro-protein (miP) encoding the C-terminus of ATHB2 (ATHB2miP). ATHB2miP is expected to comprise the leucine zipper part of ATHB2 and hence interact with the full-length protein through this dimerization motif. Such interactions are shown using yeast two-hybrid and FRET-FLIM assays. ATHB2 is a well-known shade-induced gene that has been implicated in shade-regulated growth responses. The authors then test the potential role for ATHB2miP genetically by comparing several athb2 loss-of-function (LOF) alleles: one does not express either full-length ATHB2 or the short ATHB2miP (t-ATHB2), two CRISPR alleles give rise to frameshift mutations in the full-length transcript but still express a potentially functional short ATHB2miP (athb2deltaLZ and athb2delta). The authors also use plants that over and ectopically express ATHB2miP (35S:miP). Overall, the results are consistent with the hypothesis that ATHB2miP inhibits the function of ATHB2, which constitutes a novel negative feedback loop. Potentially ATHB2miP may also inhibit the activity of other related HD ZIP proteins (based on 35S:miP). The effects of these genetic alterations on shade-regulated hypocotyl growth are relatively modest. Effects on root growth are also investigated and in one intriguing case, the negative feedback model does not appear to explain the data (Figure 4D, effect on lateral roots, because for this phenotype 35S:miP is very different from the lof alleles). The authors also identify a potentially interesting link between shade-regulated hypocotyl growth and iron uptake. A number of text changes and corrections to the figures would be important for clarity. They primarily concern three issues: names of the alleles, names of the studied shade conditions, and statements about significant differences between genotypes. Also, it would be interesting to know whether the effects of ATHB2 on iron uptake are due to local effects of ATHB2. Is ATHB2 expressed in roots?

Weaknesses:

(1) The naming of the different shade conditions is difficult to follow and not consistent with the way most authors in the field call such conditions. Deep shade is ok (low PAR and low R/FR, WL, PAR 13microE, R/FR 0.13). This condition is clearly defined for experiments in Figure 4. However, data in Figure 1 also use Deep shade (line 174) but PAR is not defined there. I suggest that all light conditions are clearly defined in the figure legends and in the M&M (not the case in this ms). Regarding Canopy shade (WL, PAR 45microE, R/FR 0.15) and proximity shade (WL, PAR 45microE, R/FR 0.06), see lines 355-357, this nomenclature is unclear. First proximity shade has a higher R/FR ratio than canopy shade. Second for canopy shade (compared to the WL control) PAR should decrease which is not what is done here. What is called proximity shade and canopy shade are 2 WL conditions with different R/FR ratios, which are compared to WL controls with the same PAR. It would make more sense to call them proximity shade and indicate the different R/FR ratios. Finally, extensive literature from many plant species and numerous labs has shown that hypocotyl elongation increases with R/FR decreasing. In the data shown in Figure 4, it is the opposite. Hypocotyls in Canopy shade (WL, PAR 45microE, R/FR 0.15) are longer than those in proximity shade (WL, PAR 45microE, R/FR 0.06), while with these R/FR ratios the opposite is expected. Could this be a mistake in the text? Please check.

(2) In several instances (in particular regarding data from Figures 4 and 5), the authors write that 2 genotypes are significantly different while the statistical analysis of the data does not support such statements. For example lines 392-395, the authors write that in WL the t-DNA mutant, both CRISPR mutants and 35S:miP lines all had significantly lower number of lateral roots than the WT. This is true for the t-DNA mutant (group bc, while the WT is in group a), however, all other genotypes are in group ab, hence not significantly different from the WT. Please carefully check all such statements about significant differences.

(3) The naming of the CRISPR mutants is problematic. In particular athb2delta, such a name suggests that the gene is deleted (also suggested by Figure 4A), which is not the case in this CRISPR allele leading to a frameshift early in the coding sequence. This is particularly problematic because in this allele ATHB2miP is still expressed, while based on such a name one would expect that in this mutant both the full length and the miP are lost. Both CRISPR alleles lead to a frameshift and this should be clarified in Figure 4A and in the text.

(4) Overall hypocotyl growth phenotypes of athb2 lof mutants and 35S:miP are similar and consistent with a model according to which ATHB2miP inhibits the full-length protein. However, this is not the case for the root phenotype described in 4D. It would be interesting to discuss this.

(5) The authors propose a role for ATHB2 in the root, in particular linked to iron uptake. Is this due to a local effect of ATHB2 in the roots? Is ATHB2 expressed in roots? It would be very informative if the authors would show such data, e.g. using the reporter lines used in Figure 1. Are both the FL and the miP expressed in roots?

(6) From the description regarding 5'PEAT.seq data presented in Figure 1 (see lines 174-177) it is not clear in which light conditions the seedlings were grown. It appears that samples were collected in 3 conditions. WL and after 45 and 90 minutes of low R/FR treatment. However, then the data is discussed collectively. Does the 12398 TSS correspond to what was found in all three conditions together? Are the authors showing shade-regulation of TSS? This is clearly the case for ATHB2miP. This needs to be clarified.

(7) The way gene expression of low F/FR effects is done might conflate circadian effects and low R/FR effects because the samples from different light conditions are not collected at the same ZT. This is how I understood the text. If I'm wrong please clarify the text. If I am right, this potential problem should be mentioned in the text.

(8) Could the authors envisage a way to genetically test the role of ATHB2miP by using an allele that makes the full length but not the miP? Currently, the authors use lof alleles that either make none of the transcripts (t-DNA) or potentially only the miP (CRISPR alleles). Overall, these alleles do not appear to differ in their phenotypes, suggesting that most of the effect of ATHB2miP is through ATHB2 FL. Having an allele only producing the FL would be nice (but technically I'm not sure how one could do that).

Reviewer #3 (Public Review):

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

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

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

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

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

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

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

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

Reviewer #3 (Public Review):

The authors observed phenotypes of ciliopathy model mice and they seem to coincide with those in human patients. They used mutants in which cilial function genes are deleted in cranial neural crest cells, and found the mutants exhibit abnormal cell differentiation in both neural crest- and mesoderm-lineage cells. The finding clearly shows the importance of tissue/cell interaction. The authors mainly observed the mouse in which Ofd1 gene that is coded on the X chromosome is deleted, therefore, Ofd1fl/WT;Wnt1Cre(HET) mice show that about one-fourth of neural crest cells can exhibit Ofd1 function whereas Ofd1fl;Wnt1Cre (HM) shows null Ofd1 function and show severer phenotypes than HET.

For ectopic brown adipose tissue in the tongue is derived from mesoderm and the authors tried to show that the hypoglossal cord failed to obtain myogenic lineage after entering branchial arches in HET and HM due to lack of communication with neural crest cells. For ectopic bone formation, they found that it is due to the lack of Hedgehog signaling in neural crest cells, which was consistent with the reports in the Smofl/fl;Wnt1-Cre (Xu et al., 2019) and Ift88fl/fl;Wnt1Cre (Kitamura et al. 2020). The ectopic bone is connected to the original mandibular bone. The authors attribute the ectopic bone formation to the migration of mandibular bone neural crest cells into the tongue-forming area.

For the poor tongue frenum formation, the authors found the importance of cell migration from the lateral sides of the branchial arch to the midline and its formation relies on non-canonical Wnt signaling. The authors observed similar phenotypes in the human patients as those in the mutants. The adipose tissue in the tongue area is normally found in the salivary gland region and intermuscular space, and it is intriguing to find the brown adipose tissue anterior to the cervical area in which the most anterior brown adipose tissue develops. qRT-PCR indicates that some of the marker genes are expressed in the laser micro-dissected sections of the ectopic brown adipose tissue. However, histology does not show the typical brown adipose tissue feature. In addition, brown adipose tissue is normally recognized in the sixth pharyngeal region as the cervical brown tissue from around E14.5 (Schulz and Tseng 2013), not E12 as the authors observe. Although the mutants develop under abnormal conditions, is it possible to say they are brown adipose tissue? The point has to be further investigated with more marker expression by immunohistochemical detection and other methods. Since the mutants seem to show impaired midline formation (which is consistent with the condition of human ciliopathy), is it possible to hypothesize that the adipose-like tissue is derived from the mesoderm of posterior branchial arch levels if the tissue is brown adipose tissue?

Cranial neural crest cells start migrating around E8.0 and reach their destination by E9.5. The authors show the lack of neural crest cells in the midline, the fluorescence is absent from the midline in HM, however, they studied it in the E11 mandible (Fig. 4E), almost more than two days after neural crest migration completes. Since the mandibular arch seems to form at the beginning in the mutants, is there a failure in allocating the neural crest and mesoderm at the beginning of the mandibular arch formation?<br /> The authors tried to disturb the interaction between the hypoglossal cord and neural crest cells by making incisions in the dorsal area of the branchial arches. That area contains both neural crest and mesoderm but not the hypoglossal cord-derived mesoderm. The hypoglossal cord passed through the posterior edge of the caudal (6th) pharyngeal arch, along the lateral side of the pericardium towards the anterior, ventral to branchial arches, and then inside the 2nd and 1st branchial arches (Adachi et al., 2018). It expresses Pax3 before entering the branchial arches, then Myf5 in the branchial arches. It seems that the migration of the hypoglossal cord does not require interaction with neural crest cells but it has to be confirmed as well as neural crest migration into the branchial arches from the beginning. Although the hypoglossal cord migrates mostly in mesoderm-derived mesenchyme, we cannot exclude the possibility that hypoglossal cord migration is affected.

The lack of Myf5 expression in Ofd1fl;Wnt1Cre (HM) was explained as a failure in the differentiation of the hypoglossal cord into myoblasts on entrance into the branchial arches. Most of the cervical brown adipose tissue is derived from either Myf5- or Pax3- expressing lineage (Sanchez-Gurmaches and Guertin, 2014). Although the authors suggest that brown adipose cells are fate-changed mesoderm in the branchial arches, how do they explain the association with Myf5- or Pax3- expression?

In addition, the cervical brown tissue is supposed to be derived from the branchial arch mesoderm (Mo et al., 2017). Is the formation of the cervical brown tissue affected in the Ofd1fl/WT;Wnt1Cre(HET) or Ofd1fl;Wnt1Cre (HM) if dysfunction of neural crest cells results in the cell fate change of mesoderm?

For the tongue frenum development, it is hard to understand to hypothesize that its formation is unlikely to associate with midline formation. Although Lgr5 and Tbx22 are not expressed in the midline, the defect in midline formation could cause unnecessary interaction between the right and left tissues.

Tissue morphogenesis takes place in three dimensions, which were not considered in the data, especially in the labeling experiments. When the authors labelled the cells, which cells in which area were labelled? In the textbook, tongue formation is a result of the fusion of the midline processes derived from the branchial arches, therefore, it is important to identify which cells in which area are labelled.

The weakest point is that the authors demonstrate many interesting phenotypes but fail to show the mechanism of altered cell differentiation and direct evidence of the tissue origin of ectopic brown tissue. Without the data, suggestion from the authors' argument is weak, which is reflected in the conclusion of the abstract.

Reviewer #3 (Public Review):

This work submitted by Bu et al. investigated mechanisms of how salt stress-induced arginine catabolism, which is catalyzed by arginase and urease, inhibits seed germination and seedling growth in Arabidopsis using a combination of genetic, biochemical, and live-cell imaging approaches. Their results showed that the two steps for the turnover of arginine into ammonia and the transport of urea from the cotyledon to the root are required for the salt-induced inhibition of seed germination (SISG). Further analysis showed that the cellular accumulation of the end product ammonia is not associated with SISG, but it is the cytoplasmic alkaline stress that primarily causes SISG. Interestingly, they found that the mechanism underlying SISG is conserved in other plant species. In general, this work will be valuable for plant biologists to deeply dissect the complex mechanism that controls salt stress-induced inhibition of plant growth and development in the future.

The conclusions derived from this work are well supported by the data, but some aspects of data analysis need to be clarified and extended.

(1) Inhibition of arginine hydrolysis by enzyme inhibitors (NOHA for arginase and PPD for urease) significantly improved seed germination and seedling growth (Figure 2). It seems that the suppressive effect of NOHA against the salt-induced inhibition of seedling growth is dose-dependent (Figure 2b). Whether NOHA effect on SISG is also dose-dependent and application of a certain level of NOHA can fully rescue the phenotype of SISG remains to be answered. The answers may help to explain the genetic data shown in Figure 3c, where either single (argah1 and argah2) or double (argah1/argah2) mutants partially rescued the phenotype of SISG. However, arginase activity, particularly in argah1 and argah2, is not closely correlated to the phenotype shown in Figure 3c and 3d.

(2) The data shown in Figure 4b and 4e were not fully consistent. The percentage of seed germination rate was about 70% when treated with the highest concentration (7.5 μM) of PPD, but was less than 40% for the aturease mutant.

(3) Cellular pH values detected at the seed germination stage were not convincing. In the text, they did not describe the results showing that the cytoplasmic pH values in hypocotyl and cotyledon cells were alkaline and not affected by NaCl treatment, and PPD treatment only restored the alkaline cytoplasmic pH to that of the control (Figure 7b). This raises two questions: is it true that cytoplasmic pH values are different between root and cotyledon/hypocotyl cells under normal growth conditions? and does PPD treatment alter the cytoplasmic pH only in roots?

Reviewer #3 (Public Review):

Correlation of the HLA-B effects with previously demonstrated allelic differences in dependence on the peptide loading complex (PLC) component chaperone/editor tapasin and demonstration that US10 does not bind the PLC reflect on possible mechanisms of US10 function. Thus, this paper adds new information that may be integrated into evolving models of the steps of MHC-I dependent antigen presentation and how viruses counter immune recognition for their own benefit. Clearer focus on the proposed models for the function of US10 and its mechanism--i.e. what experiments address the mechanism and what additional finding might clarify the mechanism would be helpful.

Reviewer #3 (Public Review):

Summary:

In this study, Han and co-authors showed that implantation of Pik3ca deficient KPC cells (aKO) induced clonal expansion of CD8 T cells in the tumor microenvironment. Using aKO cells, they conducted an in vivo genome-wide gene-deletion screen, which showed that deletion of propionyl-CoA carboxylase subunit B gene (Pccb) in αKO cells (p-aKO) leads to immune evasion and tumor progression. Eventually, mice injected with p-aKO but not aKO succumbed to their tumors. Similar to the parental aKO cell line, p-aKO tumors were still infiltrated with clonally expanded CD8+ and CD4+ T cells, as shown by the IHC. Further analyses showed that T cells infiltrating p-aKO tumors expressed high levels of exhaustion markers (PD-1, CTLA-4, TIM3, and TIGIT). Furthermore, PD-1 signaling blockade using PD-1 mAb or genetic depletion of PD-1 reactivated the infiltrated T cells, controlling tumor progression and improving the overall mice survival. Thus, the authors concluded in the abstract that "Pccb can modulate the activity of cytotoxic T cells infiltrating some pancreatic cancers." Although the data clearly showed that the loss of Pccb facilitated the immune evasion of pancreatic cancer cells, there is no clear evidence provided that Pccb deletion can actually modulate the activity of CD8 T cells. One may argue that the deletion of Pccb reduces the immunogenicity of the p-aKO cancer cells, making them less susceptible to killing by normally functional CD8+ T cells.

Strengths:

In vivo, Crisper-Cas-9 screen using tumor cell lines.

Identify a gene that could reduce the immunogenicity of cancer cells.

Weaknesses:

The IHC technique that was used to stain and characterize the exhaustion status of the tumor-infiltrating T cells.

Reviewer #3 (Public Review):

Summary:

Campbell and colleagues use a combination of high-resolution fMRI, cognitive tasks, and different intensities of light illumination to test the hypothesis that the intensity of illumination differentially impacts hypothalamic substructures that, in turn, promote alterations in arousal that affect cognitive and affective performance. The authors find evidence in support of a posterior-to-anterior gradient of increased blood flow in the hypothalamus during task performance that they later relate to performance on two different tasks. The results provide an enticing link between light levels, hypothalamic activity, and cognitive/affective function, however, clarification of some methodological choices will help to improve confidence in the findings.

Strengths:

* The authors' focus on the hypothalamus and its relationship to light intensity is an important and understudied question in neuroscience.

Weaknesses:

* I found it challenging to relate the authors' hypotheses, which I found to be quite compelling, to the apparatus used to test the hypotheses - namely, the use of orange light vs. different light intensities; and the specific choice of the executive and emotional tasks, which differed in key features (e.g., block-related vs. event-related designs) that were orthogonal to the psychological constructs being challenged in each task.

* Given the small size of the hypothalamus and the irregular size of the hypothalamic parcels, I wondered whether a more data-driven examination of the hypothalamic time series would have provided a more parsimonious test of their hypothesis.

Reviewer #3 (Public Review):

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

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

Specific comments:

(1) In the introduction the authors set up the controversy on whether or not motor neurons play an instructive role in determining "pre-motor fate". This statement is somewhat generic and a bit misleading as it is generally accepted that many aspects of interneuron identity are motor neuron-independent. The authors might want to expand on these studies and better define what they mean by "fate", as it is not clear whether the studies they are citing in support of this hypothesis actually make that claim.

(2) Although it appears unchanged from their images, the authors do not explicitly quantitate the number of total projection neurons in phox2a knockouts.

(3) For figures 2C and 3C, please report the proportion of neurons in each animal, either showing individual data points here or in a separate supplementary figure; and please perform and report the results of an appropriate statistical test.

(4) In the topographical mapping of calcium responses (figures 2D, E and 3D), the authors say they see no differences but this is hard to appreciate based on the 3D plotting of the data. Quantitating the strength of the responses across the 3-axes shown individually and including statistical analyses would help make this point, especially since the plots look somewhat qualitatively different.

(5) The transcriptional analysis is very interesting, however, it is not clear why it was performed at 72 hpf, while functional experiments were performed at 5 days. Is it possible that early aspects of projection neuron identity are preserved, while motor neuron-dependent changes occur later? The authors should better justify and discuss their choice of timepoint. The inclusion of heterozygotes as controls is problematic, given that the authors show there are notable differences between phox2a+/+ and phox2a+/- animals; pooling these two genotypes could potentially flatten differences between controls and phox2a-/-.

(6) Projection neurons appear to be topographically organized and this organization is maintained in the absence of motor neurons. Are there specific genes that delineate ventral and dorsal projection neurons? If so, the authors should look at those as candidate genes as they might be selectively involved in connectivity. Showing that generic synaptic markers (Figure 4E) are maintained in the entire population is not convincing evidence that these neurons would choose the correct synaptic partners.

Reviewer #3 (Public Review):

Brugeuera et al present an impressive series of biochemical experiments that address the question of how Tspan12 acts to promote signaling by Norrin, a highly divergent TGF-beta family member that serves as a ligand for Fzd4 and Lrp5/6 to promote canonical Wnt signaling during CNS (and especially retinal) vascular development. The present study is distinguished from those of the past 15 years by its quantitative precision and its high-quality analyses of concentration dependencies, its use of well-characterized nano-disc-incorporated membrane proteins and various soluble binding partners, and its use of structure prediction (by AlphaFold) to guide experiments. The authors start by measuring the binding affinity of Norrin to Tspan12 in nanodiscs (~10 nM), and they then model this interaction with AlphaFold and test the predicted interface with various charge and size swap mutations. The test suggests that the prediction is approximately correct, but in one region (site 1) the experimental data do not support the model. [As noted by the authors, a failure of swap mutations to support a docking model is open to various interpretations. As AlphFold docking predictions come increasingly into common use, the compendium of mutational tests and their interpretations will become an important object of study.] Next, the authors show that Tspan12 and Fzd4 can simultaneously bind Norrin, with modest negative cooperativity, and that together they enhance Norrin capture by cells expressing both Tspan12 and Fzd4 compared to Fzd4 alone, an effect that is most pronounced at low Norrin concentration. Similarly, at low Norrin concentration (~1 nM), signaling is substantially enhanced by Tspan12. By contrast, the authors show that LRP6 competes with Tspan12 for Norrin binding, implying a hand-off of Norrin from a Tspan12+Fzd4+Norrin complex to a LRP5/6+Fzd4+Norrin complex. Thanks to the authors' careful dose-response analyses, they observed that Norrin-induced signaling and Tspan12 enhancement of signaling both have bell-shaped dose-response curves, with strong inhibition at higher levels of Norrin or Tspan12. The implication is that the signaling system has been built for optimal detection of low concentrations of Norrin (most likely the situation in vivo), and that excess Tspan12 can titrate Norrin at the expense of LRP5/6 binding (i.e., reduction in the formation of the LRP5/6+Fzd4+Norrin signaling complex). In the view of this reviewer, the present work represents a foundational advance in understanding Norrin signaling and the role of Tspan12. It will also serve as an important point of comparison for thinking about signaling complexes in other ligand-receptor systems.

Reviewer #3 (Public Review):

Summary:

The manuscript shows SIRT2 can regulate acetylation of ACSS2 at residue 271, acetylation of 271 protects ACSS2 from proteasomal degradation in a SIRT2-dependent manner. Lastly, authors show that ACSS2 acetylation at K271 promotes lipid accumulation.

Strengths:

The author provides solid data showing ACSS2 acetylation can be regulated by targeting SIRT2 and that SIRT2 regulates ACSS2 ubiquitination. They identify K271 as a site of acetylation and show this is a site when mutated alters SIRT2-mediated ubiquitination.

Weaknesses:

However, data for this manuscript seems preliminary as nearly all data is performed in one cell line, some of the conclusions are not well supported by data and the overall role of ACSS2 K271 acetylation is not well characterized.

Reviewer #3 (Public Review):

Distant metastasis is the major cause of death in patients with breast cancer. In this manuscript, Liu et al. show that RGS10 deficiency elicits distant metastasis via epithelial-mesenchymal transition in breast cancer. As a prognostic indicator of breast cancer, RGS10 regulates the progress of breast cancer and affects tumor phenotypes such as epithelial-mesenchymal transformation, invasion, and migration. The conclusions of this paper are mostly well supported by data, but some analyses need to be clarified.

(1) Because diverse biomarkers have been identified for EMT, it is recommended to declare the advantages of using RGS10 as an EMT marker.

(2) The authors utilized databases to study the upstream regulatory mechanisms of RSG10. It is recommended to clarify why the authors focused on miRNAs rather than other epigenetic modifications.

(3) The role of miR-539-5p in breast cancer has been described in previous studies. Hence, it is recommended to provide detailed elaboration on how miR-539-5p regulates the expression of RSG10.

(4) To enhance the clarity and interpretability of the Western blot results, it would be advisable to mark the specific kilodalton (kDa) values of the proteins.

Reviewer #3 (Public Review):

Summary:

Studying evolutionary trajectories provides important insight in genetic architecture of adaptation and provide potential contribution to evaluating the predictability (or unpredictability) in biological processes involving adaptation. While many papers in the field address adaptation to environmental challenges, the number of studies on how genomic contexts, such as large-scale variation, can impact evolutionary outcomes adaptation is relatively low. This research experimentally evolved a genome-reduced strain for ~1000 generations with 9 replicates and dissected their evolutionary changes. Using the fitness assay of OD measurement, the authors claimed there is a general trend of increasing growth rate and decreasing carrying capacity, despite a positive correlation among all replicates. The authors also performed genomic and transcriptomic research at the end of experimental evolution, claiming the dissimilarity in the evolution at the molecular level.

Strengths:

The experimental evolution approach with a high number of replicates provides a good way to reveal the generality/diversity of the evolutionary routes.

The assay of fitness, genome, and transcriptome all together allows a more thorough understanding of the evolutionary scenarios and genetic mechanisms.

Comments on revised version:

5 in the last round of comments: When the authors mentioned no overlapping in single mutation level, I thought the authors would directly use this statement to support their next sentence about no bias of these mutations. As the author's responded, I was suspecting no overlapping for 65 mutation across the entire genome is likely to be not statistically significant. In the revised version, the authors emphasized and specified their simulation and argument in the following sentences, so I do not have questions on this point anymore.

14 in the last round of comments: As what authors responded, "short-term responses" meant transcriptional or physiological changes within a few hours after environmental or genetic fluctuation. "long-term responses" involve new compensatory mutations and selection. The point was that, the authors found that "the transcriptome reorganization for fitness increase triggered by evolution differed from that for fitness decrease caused by genome reduction." That is short vs long-term responses to genetic perturbation. Some other experimental evolution did short vs long-term responses to environmental perturbation and usually also found that the short-term responses are reverted in the long-term responses (e.g., https://academic.oup.com/mbe/article/33/1/25/2579742). I hope this explanation makes more sense. And I think the authors can make their own decisions on whether they would like to add this discussion or not.

Reviewer #3 (Public Review):

Summary:

In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:

The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42 . In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

Reviewer #3 (Public Review):

Summary:

REV7 facilitates the recruitment of Shieldin complex and thereby inhibits end resection and controls DSB repair choice in metazoan cells. Puzzlingly, Shieldin is absent in many organisms and it is unknown if and how Rev7 regulates DSB repair in these cells. The authors surmised that yeast Rev7 physically interacts with Mre11/Rad50/Xrs2 (MRX), the short-range resection nuclease complex, and tested this premise using yeast two-hybrid (Y2H) and microscale thermophoresis (MST). The results convincingly showed that the individual subunits of MRX interact robustly with Rev7. AlphaFold Multimer modelling followed by Y2H confirmed that the carboxy-terminal 42 amino acid is essential for interaction with MR and G4 DNA binding by REV7. The mutant rev7 lacking the binding interface (Rev7-C1) to MR shows moderate inhibition to the nuclease and the ATPase activity of Mre11/Rad50 in biochemical assays. Deletion of REV7 also causes a mild reduction in NHEJ using both plasmid and chromosome-based assays and increases mitotic recombination between chromosomal ura3-01 and the plasmid ura3 allele interrupted by G4. The authors concluded that Rev7 facilitates NHEJ and antagonizes HR even in budding yeast, but it achieves this by blocking Mre11 nuclease and Rad50 ATPase.

Weaknesses:

There are many strengths to the studies and the broad types of well-established assays were used to deduce the conclusion. Nevertheless, I have several concerns about the validity of experimental settings due to the lack of several key controls essential to interpret the experimental results. The manuscript also needs a few additional functional assays to reach the accurate conclusions as proposed.

(1) AlphaFold model predicts that Mre11-Rev7 and Rad50-Rev7 binding interfaces overlap and Rev7 might bind only to Mre11 or Rad50 at a time. Interestingly, however, Rev7 appears dimerized (Figure 1). Since the MR complex also forms with 2M and 2R in the complex, it should still be possible if REV7 can interact with +-*both M and R in the MR complex. The author should perform MST using MR complex instead of individual MR components. The authors should also analyze if Rev7-C1 is indeed deficient in interaction with MR individually and with complex using MST assay.

(2) The nuclease and the ATPase assays require additional controls. Does Rev7 inhibit the other nuclease or ATPase non-specifically? Are these outcomes due to the non-specific or promiscuous activity of Rev7? In Figure 6, the effect of REV7 on the ATP binding of Rad50 could be hard to assess because the maximum Rad50 level (1 uM) was used in the experiments. The author should use the suboptimal level of Rad50 to check if REV7 still does not influence ATP binding by Rad50.

(3) The moderate deficiency in NHEJ using plasmid-based assay in REV7 deleted cells can be attributed to aberrant cell cycle or mating type in rev7 deleted cells. The authors should demonstrate that rev7 deleted cells retain largely normal cell cycle patterns and the mating type phenotypes. The author should also analyze the breakpoints in plasmid-based NHEJ assays in all mutants, especially from rev7 and rev7-C1 cells.

(4) It is puzzling why the authors did not analyze end resection defects in rev7 deleted cells after a DSB. The author should employ the widely used resection assay after a HO break in rev3, rev7, and mre11 rev7 cells as described previously.

(5) Is it possible that Rev7 also contributes to NHEJ as the part of TLS polymerase complex? Although NHEJ largely depends on Pol4, the authors should not rule out that the observed NHEJ defect in rev7 cells is due at least partially to its TLS defect. In fact, both rev3 or rev1 cells are partially defective in NHEJ (Figure 7). Rev7-C1 is less deficient in NHEJ than REV7 deletion. These results predict that rev7-C1 rev3 should be as defective as the rev7 deletion. Additionally, the authors should examine if Rev7-C1 might be deficient in TLS. In this regard, does rev7-C1 reduce TLS and TLS-dependent mutagenesis? Is it dominant? The authors should also check if Rev3 or Rev1 are stable in Rev7 deleted or rev7-C1 cells by immunoblot assays.

(6) Due to the G4 DNA and G4 binding activity of REV7, it is not clear which class of events the authors are measuring in plasmid-chromosome recombination assay in Figure 9. Do they measure G4 instability or the integrity of recombination or both in rev7 deleted cells? Instead, the effect of rev7 deletion or rev7-C1 on recombination should be measured directly by more standard mitotic recombination assays like mating type switch or his3 repeat recombination.

Reviewer #3 (Public Review):

Summary:

The paper attempts to model the functional significance of variants of PLCG2 in a set of patients with variable clinical manifestations.

Strengths:

A study attempting to use the Drosophila system to test the function of variants reported from human patients.

Weaknesses:

Additional experiments are needed to shore up the claims in the paper. These are listed below.

Major Comments:

(1) Does the pLI/ missense constraint Z score prediction algorithm take into consideration whether the gene exhibits monoallelic or biallelic expression?

(2) Figure 1B: Include human PLCG2 in the alignment that displays the species-wide conserved variant residues.

(3) Figure 4A:<br /> Given that<br /> (i) sl is predicted to be the fly ortholog for both mammalian PLCγ isozymes: PLCG1 and PLCG2 [Line 62]<br /> (ii) they are shown to have non-redundant roles in mammals [Line 71] and<br /> (iii) reconstituting PLCG1 is highly toxic in flies, leading to increased lethality.<br /> This raises questions about whether sl mutant phenotypes are specifically caused by the absence of PLG1 or PLCG2 functions in flies. Can hPLCG2 reconstitution in sl mutants be used as a negative control to rule out the possibility of the same?

(4) Do slT2A/Y; UAS-PLCG1Reference flies survive when grown at 22{degree sign}C? Since transgenic fly expressing PLCG1 cDNA when driven under ubiquitous gal4s, Tubulin and Da, can result in viable progeny at 22{degree sign}C, the survival of slT2A/Y; UAS-PLCG1Reference should be possible.<br /> and similarly<br /> Does slT2A flies exhibit the phenotypes of (i) reduced eclosion rate (ii) reduced wing size and ectopic wing veins and (iii) extra R7 photoreceptor in the fly eye at 22{degree sign}C?<br /> If so, will it be possible to get a complete rescue of the slT2A mutant phenotypes with the hPLCG1 cDNA at 22{degree sign}C? This dataset is essential to establish Drosophila as an ideal model to study the PLCG1 de novo variants.

(5) Localisation and western blot assays to check if the introduction of the de novo mutations can have an impact on the sub-cellular targeting of the protein or protein stability respectively.

(6) Analysing the nature of the reported gain of function (experimental proof for the same is missing in the manuscript) variants:<br /> Instead of directly showing the effect of introducing the de novo variant transgenes in the Drosophila model especially when the full-length PLCG1 is not able to completely rescue the slT2A phenotype;<br /> (i) Show that the gain-of-function variants can have an impact on the protein function or signalling via one of the three signalling outputs in the mammalian cell culture system: (i) inositol-1,4,5-trisphosphate production, (ii) intracellular Ca2+ release or (iii) increased phosphorylation of extracellular signal-related kinase, p65, and p38.<br /> OR<br /> (ii) Run a molecular simulation to demonstrate how the protein's auto-inhibited state can be disrupted and basal lipase activity increased by introducing D1019G and D1165G, which destabilise the association between the C2 and cSH2 domains. The H380R variant may also exhibit characteristics similar to the previously documented H335A mutation which leaves the protein catalytically inactive as the residue is important to coordinate the incoming water molecule required for PIP2 hydrolysis.

(7) Clarify the reason for carrying out the wing-specific and eye-specific experiments using nub-gal4 and eyless-gal4 at 29˚C despite the high gal4 toxicity at this temperature.

(8) For the sake of completeness the authors should also report other variants identified in the genomes of these patients that could also contribute to the clinical features.

Reviewer #3 (Public Review):

In this manuscript, Casas-Tintó et al. explore the role of glial cells in the response to a neurodegenerative injury in the adult brain. They used Drosophila melanogaster as a model organism and found that glial cells are able to generate new neurons through the mechanism of transdifferentiation in response to injury.

This paper provides a new mechanism in regeneration and gives an understanding of the role of glial cells in the process.

Now it got it wrong

Reviewer #3 (Public Review):

Summary:

Based on their observation that tumor has an iron-deficient microenvironment, and the assumption that nutritional immunity is important in bacteria-mediated tumor modulation, the authors postulate that manipulation of iron homeostasis can affect tumor growth. This paper uses straightforward in vitro and in vivo techniques to examine a specific and important question of nutritional immunity in bacteria-mediated tumor therapy. They are successful in showing that manipulation of iron regulation during nutritional immunity does affect the virulence of the bacteria, and in turn the tumor. These findings open future avenues of investigation, including the use of different bacteria, different delivery systems for therapeutics, and different tumor types. The authors were also successful in addressing the reviewer's concerns adequately.

Reviewer #3 (Public Review):

Summary:

In the 'bCFS' paradigm, a monocular target gradually increases in contrast until it breaks interocular suppression by a rich monocular suppressor in the other eye. The present authors extend the bCFS paradigm by allowing the target to reduce back down in contrast until it becomes suppressed again. The main variable of interest is the contrast difference between breaking suppression and (re) entering suppression. The authors find this difference to be constant across a range of target types, even ones that differ substantially in the contrast at which they break interocular suppression (the variable conventionally measured in bCFS). They also measure how the difference changes as a function of other manipulations. Interpretation is in terms of the processing of unconscious visual content, as well as in terms of the mechanism of interocular suppression.

Strengths:

Interpretation of bCFS findings is mired in controversy, and this is an ingenuous effort to move beyond the paradigm's exclusive focus on breaking suppression. The notion of using the contrast difference between breaking and entering suppression as an index of suppression depth is interesting. The finding that this difference is similar for a range of target types that do differ in the contrast at which they break suppression, suggests a common mechanism of suppression across those target types.

Reviewer #3 (Public Review):

Summary:

The authors image dopamine axons in medial prefrontal cortex (mPFC) using microprism-mediated two-photon calcium imaging. They image these axons as mice learn that two auditory cues predict two distinct outcomes, tailshock, or water delivery. They find that some axons show a preference for encoding of the shock and some show a preference for encoding of water. The authors report a greater number of dopamine axons in mPFC that respond to shock. Across time, the shock-preferring axons begin to respond preferentially to the cue predicting shock, while there is a less pronounced increase in the water-responsive axons that acquire a response to the water-predictive cue (these axons also increase non-significantly to the shock-predictive cue). These data lead the authors to argue that dopamine axons in mPFC preferentially encode aversive stimuli.

Strengths:

The experiments are beautifully executed and the authors have mastered an impressively complex technique. Specifically, they are able to image and track individual dopamine axons in mPFC across days of learning. And this technique is used the way it should be: the authors isolate distinct dopamine axons in mPFC and characterize their encoding preferences and how this evolves across learning of cue-shock and cue-water contingencies. Thus, these experiments are revealing novel information about how aversive and rewarding stimuli is encoded at the level of individual axons, in a way that has not been done before. This is timely and important.

Weaknesses:

The overarching conclusion of the paper is that dopamine axons preferentially encode aversive stimuli. However, this is confounded by differences in the strength of the aversive and appetitive outcomes. As the authors point out, the axonal response to stimuli is sensitive to outcome magnitude (Supp Fig 3). That is, if you increase the magnitude of water or shock that is delivered, you increase the change in fluorescence that is seen in the axons. Unsurprisingly, the change in fluorescence that is seen to shock is considerably higher than water reward. Further, over 40% of the axons respond to water early in training [yet just a few lines below the authors write: "Previous studies have demonstrated that the overall dopamine release at the mPFC or the summed activity of mPFC dopamine axons exhibits a strong response to aversive stimuli (e.g., tail shock), but little to rewards", which seems inconsistent with their own data]. Given these aspects of the data, it could be the case that the dopamine axons in mPFC encodes different types of information and delegates preferential processing to the most salient outcome across time. The use of two similar sounding tones (9Khz and 12KHz) for the reward and aversive predicting cues are likely to enhance this as it requires a fine-grained distinction between the two cues in order to learn effectively. That is not to say that the mice cannot distinguish between these cues, rather that they may require additional processes to resolve the similarity, which are known to be dependent on the mPFC.

There is considerable literature on mPFC function across species that would support such a view. Specifically, theories of mPFC function (in particular prelimbic cortex, which is where the axon images are mostly taken) generally center around resolution of conflict in what to respond, learn about, and attend to. That is, mPFC is important for devoting the most resources (learning, behavior) to the most relevant outcomes in the environment. This data then, provides a mechanism for this to occur in mPFC. That is, dopamine axons signal to the mPFC the most salient aspects of the environment, which should be preferentially learnt about and responded towards. This is also consistent with the absence of a negative prediction error during omission: the dopamine axons show increases in responses during receipt of unexpected outcomes but do not encode negative errors. This supports a role for this projection in helping to allocate resources to the most salient outcomes and their predictors, and not learning per se. Below are a just few references from the rich literature on mPFC function (some consider rodent mPFC analogous to DLPFC, some mPFC), which advocate for a role in this region in allocating attention and cognitive resources to most relevant stimuli, and do not indicate preferential processing of aversive stimuli.

References:<br /> 1. Miller, E. K., & Cohen, J. D. (2001). An integrative theory of prefrontal cortex function. Annual review of neuroscience, 24(1), 167-202.<br /> 2. Bissonette, G. B., Powell, E. M., & Roesch, M. R. (2013). Neural structures underlying set-shifting: roles of medial prefrontal cortex and anterior cingulate cortex. Behavioural brain research, 250, 91-101.<br /> 3. Desimone, R., & Duncan, J. (1995). Neural mechanisms of selective visual attention. Annual review of neuroscience, 18(1), 193-222.<br /> 4. Sharpe, M. J., Stalnaker, T., Schuck, N. W., Killcross, S., Schoenbaum, G., & Niv, Y. (2019). An integrated model of action selection: distinct modes of cortical control of striatal decision making. Annual review of psychology, 70, 53-76.<br /> 5. Ridderinkhof, K. R., Ullsperger, M., Crone, E. A., & Nieuwenhuis, S. (2004). The role of the medial frontal cortex in cognitive control. science, 306(5695), 443-447.<br /> 6. Nee, D. E., Kastner, S., & Brown, J. W. (2011). Functional heterogeneity of conflict, error, task-switching, and unexpectedness effects within medial prefrontal cortex. Neuroimage, 54(1), 528-540.<br /> 7. Isoda, M., & Hikosaka, O. (2007). Switching from automatic to controlled action by monkey medial frontal cortex. Nature neuroscience, 10(2), 240-248.

Reviewer #3 (Public Review):

Summary:

The authors note that negative ruminations can lead to pathological brain states and mood/anxiety dysregulation. They test this idea by using mouse engram-tagging technology to label dentate gyrus ensembles activated during a negative experience (fear conditioning). They show that chronic chemogenetic activation of these ensembles leads to behavioral (increased anxiety, increased fear generalization, reduced fear extinction) and neural (increases in neuroinflammation, microglia, and astrocytes).

Strengths:

The question the authors ask here is an intriguing one, and the engram activation approach is a powerful way to address the question. Examination of a wide range of neural and behavioral dependent measures is also a strength.

Weaknesses:

The major weakness is that the authors have found a range of changes that are correlates of chronic negative engram reactivation. However, they do not manipulate these outcomes to test whether microglia, astrocytes, or neuroinflammation are causally linked to the dysregulated behaviors.

Reviewer #3 (Public Review):

Summary:

Here Li et al. examine pup-directed behavior in virgin Mandarin voles. Some males and females tend towards infanticide, others tend towards pup care. c-Fos staining showed more oxytocin cells activated in the paraventricular nucleus (PVN) of the hypothalamus in animals expressing pup care behaviors than in infanticidal animals. Optogenetic stimulation of PVN oxytocin neurons (with an oxytocin-specific virus to express the opsin transgene) increased pup-care, or in infanticidal voles increased latency towards approach and attack.

Suppressing the activity of PVN oxytocin neurons promoted infanticide. The use of a recent oxytocin GRAB sensor (OT1.0) showed changes in medial prefrontal cortex (mPFC) signals as measured with photometry in both sexes. Activating mPFC oxytocin projections increased latency to approach and attack in infanticidal females and males (similar to the effects of peripheral oxytocin injections), whereas in pup-caring animals only males showed a decrease in approach. Inhibiting these projections increased infanticidal behaviors in both females and males and had no effect on pup caretaking.

Strengths:

Adopting these methods for Mandarin voles is an impressive accomplishment, especially the valuable data provided by the oxytocin GRAB sensor. This is a major achievement and helps promote systems neuroscience in voles.

Weaknesses:

The study would be strengthened by an initial figure summarizing the behavioral phenotypes of voles expressing pup care vs infanticide: the percentages and behavioral scores of individual male and female nulliparous animals for the behaviors examined here. Do the authors have data about the housing or life history/experiences of these animals? How bimodal and robust are these behavioral tendencies in the population?

Optogenetics with the oxytocin promoter virus is a nice advance here. More details about their preparation and methods should be in the main text, and not simply relegated to the methods section. For optogenetic stimulation in Figure 2, how were the stimulation parameters chosen? There is a worry that oxytocin neurons can co-release other factors- are the authors sure that oxytocin is being released by optogenetic stimulation as opposed to other transmitters or peptides, and acting through the oxytocin receptor (as opposed to a vasopressin receptor)?

Given that they are studying changes in latency to approach/attack, having some controls for motion when oxytocin neurons are activated or suppressed might be nice. Oxytocin is reported to be an anxiolytic and a sedative at high levels.

The OT1.0 sensor is also amazing, these data are quite remarkable. However, photometry is known to be susceptive to motion artifacts and I didn't see much in the methods about controls or correction for this. It's also surprising to see such dramatic, sudden, and large-scale suppression of oxytocin signaling in the mPFC in the infanticidal animals - does this mean there is a substantial tonic level of oxytocin release in the cortex under baseline conditions?

Figure 5 is difficult to parse as-is, and relates to an important consideration for this study: how extensive is the oxytocin neuron projection from PVN to mPFC?

In Figures 6 and 7, the authors use the phrase 'projection terminals'; however, to my knowledge, there have not been terminals (i.e., presynaptic formations opposed to a target postsynaptic site) observed in oxytocin neuron projections into target central regions.

Projection-based inhibition as in Figure 7 remains a controversial issue, as it is unclear if the opsin activation can be fast enough to reduce the fast axonal/terminal action potential. Do the authors have confirmation that this works, perhaps with the oxytocin GRAB OT sensor?

As females and males had similar GRAB OT1.0 responses in mPFC, why would the behavioral effects of increasing activity be different between the sexes?

Local Only website possible?

Reviewer #3 (Public Review):

The authors delved into an important aspect of abortifacient diseases of livestock in Tanzania. The thoughts of the authors on the topic and its significance are implied, and the methodological approach needs further clarity. The number of wards in the study area, statistical selection of wards, type of questionnaire ie open or close-ended. Statistical analyses of outcomes were not clearly elucidated in the manuscript. Fifteen wards were mentioned in the text but 13 used what were the exclusion criteria. Observations were from pastoral, agropastoral, and smallholder agroecological farmers. No sample numbers or questionnaires were attributed to the above farming systems to correlate findings with management systems. The impacts of the research investigation output are not clearly visible as to warrant intervention methods. What were the identified pathogens from laboratory investigation, particularly with the use of culture and PCR not even mentioning the zoonotic pathogens encountered if any? The public health importance of any of the abortifacient agents was not highlighted.

In conclusion, based on the intent of the authors and the content of this research, and the weight of the research topic, there are obvious weaknesses in the critical data analysis to demonstrate cause, effect, and impact.

Reviewer #3 (Public Review):

A classic method to detect recessive disease variants is homozygosity mapping, where affected individuals in a pedigree are scanned for the presence of runs of homozygosity (ROH) intersecting in a given region. The method could in theory be extended to biobanks with large samples of unrelated individuals; however, no efficient method was available (to the best of my knowledge) for detecting overlapping clusters of ROH in such large samples. In this paper, the authors developed such a method based on the PBWT data structure. They applied the method to the UK biobank, finding a number of associations, some of them not discovered in single SNP associations.

Major strengths:<br /> • The method is innovative and algorithmically elegant and interesting. It achieves its purpose of efficiently and accurately detecting ROH clusters overlapping in a given region. It is therefore a major methodological advance.<br /> • The method could be very useful for many other researchers interested in detecting recessive variants associated with any phenotype.<br /> • The statistical analysis of the UK biobank data is solid and the results that were highlighted are interesting and supported by the data.

Major weaknesses:<br /> • The positions and IDs of the ROH clusters in the UK biobank are not available for other researchers. This means that other researchers will not be able to follow up on the results of the present paper.<br /> • The vast majority of the discoveries were in regions already known to be associated with their respective phenotypes based on standard GWAS.<br /> • The running time seems rather long (at least for the UK biobank), and therefore it will be difficult for other researchers to extensively experiment with the method in very large datasets. That being said, the method has a linear running time, so it is already faster than a naïve algorithm.

Reviewer #3 (Public Review):

Summary:

In this article, the authors provide an inventory of the 5' spliced leader sequences, cap structures, and eIF4E isoforms present in the model dinoflagellate species A. carterae. They provide evidence that the 5' cap structure is m7G, as it is in most characterized eukaryotes that do not employ trans-splicing for mRNA maturation, and that there are additional methylated nucleotides throughout the spliced leader RNAs. They then show that of the 8 different eIF4E species in A. carterae, only a subset of eIF4E1 and eIF4E2 proteins are detected and that the levels change according to time of day. Interestingly, while the eIF4E1 proteins bind a canonical cap nucleotide and are able to complement eIF4E-deficiency in yeast, an eIF4E2 paralog does not bind the traditional cap.

Strengths:

A strength of the article is that the authors have clearly presented the findings and by straying away from traditional model organisms, they have highlighted unique and interesting features of an understudied system for translational control. They provide complementary evidence for most findings using multiple techniques. E.g. the evidence that eIF4E1A binds m7GTP is supported by both pulldowns using m7GTP sepharose as well as SPR experiments to directly monitor binding of recombinant protein with affinity measurements. The methods are extremely detailed noting cell numbers, volumes, concentrations, etc. used in the experiments to be easily replicated.

Weaknesses:

While not necessary to support the author's conclusions, the significance of the work would be further enhanced by additional experiments to gain insights into mechanisms for translational control and to link specific SLs to organismal functions or mechanisms of mRNA recruitment.

-Monitoring diel expression of SLs and direct sequencing of mature mRNA would yield insights into whether there is regulated expression of RNAs with different SLs or the SLs themselves. This would also allow the authors to perform gene ontology to link SL expression at different points in the diel cycle to related functions, e.g. photosynthesis.

-In addition, the work would be strengthened by polysome sequencing or ribosome profiling as a function of the diel cycle, with analyses of when various spliced leader sequences are recruited to ribosomes in parallel with western blotting of polysome fractions to determine when various eIF4E isoforms are present on polysomes. This is a substantial expansion though from what the authors focused on in this manuscript, and not having these experiments does not undermine the findings presented. Alternatively, they could attempt to make bioinformatic comparisons with existing ribosome profiling datasets from a related dinoflagellate, Lingulodinium polyedrum, discussed briefly, if there were sufficient overlap between SL RNAs in these organisms.

Reviewer #3 (Public Review):

Summary:

Lichtinger et al. have used an extensive set of molecular dynamics (MD) simulations to study the conformational dynamics and transport cycle of an important member of the proton-coupled oligopeptide transporters (POTs), namely SLC15A2 or PepT2. This protein is one of the most well-studied mammalian POT transporters that provides a good model with enough insight and structural information to be studied computationally using advanced enhanced sampling methods employed in this work. The authors have used microsecond-level MD simulations, constant-PH MD, and alchemical binding free energy calculations along with cell-based transport assay measurements; however, the most important part of this work is the use of enhanced sampling techniques to study the conformational dynamics of PepT2 under different conditions.

The study attempts to identify links between conformational dynamics and chemical events such as proton binding, ligand-protein interactions, and intramolecular interactions. The ultimate goal is of course to understand the proton-coupled peptide and drug transport by PepT2 and homologous transporters in the solute carrier family.

Some of the key results include<br /> (1) Protonation of H87 and D342 initiate the occluded (Occ) to the outward-facing (OF) state transition.

(2) In the OF state, through engaging R57, substrate entry increases the pKa value of E56 and thermodynamically facilitates the movement of protons further down.

(3) E622 is not only essential for peptide recognition but also its protonation facilitates substrate release and contributes to the intracellular gate opening. In addition, cell-based transport assays show that mutation of residues such as H87 and D342 significantly decreases transport activity as expected from simulations.

Strengths:

(1) This is an extensive MD-based study of PepT2, which is beyond the typical MD studies both in terms of the sheer volume of simulations as well as the advanced methodology used. The authors have not limited themselves to one approach and have appropriately combined equilibrium MD with alchemical free energy calculations, constant-pH MD, and geometry-based free energy calculations. Each of these 4 methods provides a unique insight regarding the transport mechanism of PepT2.

(2) The authors have not limited themselves to computational work and have performed experiments as well. The cell-based transport assays clearly establish the importance of the residues that have been identified as significant contributors to the transport mechanism using simulations.

(3) The conclusions made based on the simulations are mostly convincing and provide useful information regarding the proton pathway and the role of important residues in proton binding, protein-ligand interaction, and conformational changes.

Weaknesses:

(1) Some of the statements made in the manuscript are not convincing and do not abide by the standards that are mostly followed in the manuscript. For instance, on page 4, it is stated that "the K64-D317 interaction is formed in only ≈ 70% of MD frames and therefore is unlikely to contribute much to extracellular gate stability." I do not agree that 70% is negligible. Particularly, Figure S3 does not include the time series so it is not clear whether the 30% of the time where the salt bridge is broken is in the beginning or the end of simulations. For instance, it is likely that the salt bridge is not initially present and then it forms very strongly. Of course, this is just one possible scenario but the point is that Figure S3 does not rule out the possibility of a significant role for the K64-D317 salt bridge.

(2) Similarly, on page 4, it is stated that "whether by protonation or mutation - the extracellular gate only opens spontaneously when both the H87 interaction network and D342-R206 are perturbed (Figure S5)." I do not agree with this assessment. The authors need to be aware of the limitations of this approach. Consider "WT H87-prot" and "D342A H87-prot": when D342 residue is mutated, in one out of 3 simulations, we see the opening of the gate within 1 us. When D342 residue is not mutated we do not see the opening in any of the 3 simulations within 1 us. It is quite likely that if rather than 3 we have 10 simulations or rather than 1 us we have 10 us simulations, the 0/3 to 1/3 changes significantly. I do not find this argument and conclusion compelling at all.

(3) While the MEMENTO methodology is novel and interesting, the method is presented as flawless in the manuscript, which is not true at all. It is stated on Page 5 with regards to the path generated by MEMENTO that "These paths are then by definition non-hysteretic." I think this is too big of a claim to say the paths generated by MEMENTO are non-hysteretic by definition. This claim is not even mentioned in the original MEMENTO paper. What is mentioned is that linear interpolation generates a hysteresis-free path by definition. There are two important problems here: (a) MEMENTO uses the linear interpolation as an initial step but modifies the intermediates significantly later so they are no longer linearly interpolated structures and thus the path is no longer hysteresis-free; (b) a more serious problem is the attribution of by-definition hysteresis-free features to the linearly interpolated states. This is based on conflating the hysteresis-free and unique concepts. The hysteresis in MD-based enhanced sampling is related to the presence of barriers in orthogonal space. For instance, one may use a non-linear interpolation of any type and get a unique pathway, which could be substantially different from the one coming from the linear interpolation. None of these paths will be hysteresis-free necessarily once subjected to MD-based enhanced sampling techniques.

Reviewer #3 (Public Review):

Summary:

The authors were trying to elucidate the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of "abnormal" enlarged vesicles when USP8 function was lost. At this specific point, it's not clear what the objective of the authors was. What would have been their hypothesis addressing whether the reduced lysosomal structures in USP8 (-) animals were linked to MVB formation? Then they observed that the abnormally enlarged vesicles, marked by the PI3P biosensor YFP-2xFYVE, are bigger but in the same number in USP8 (-) compared to wild-type animals, suggesting homotypic fusion. They confirmed this result by knocking down USP8 in a human cell line, and they observed enlarged vesicles marked by YFP-2xFYVE as well. At this point, there is quite an important issue. The use of YFP-2xFYVE to detect early endosomes requires the transfection of the cells, which has already been demonstrated to produce differences in the distribution, number, and size of PI3P-positive vesicles (doi.org/10.1080/15548627.2017.1341465). The enlarged vesicles marked by YFP-2xFYVE would not necessarily be due to the loss of UPS8. In any case, it appears relatively clear that USP8 localizes to early endosomes, and the authors claim that this localization is mediated by Rabex-5 (or Rabx-5). They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation.

Weaknesses:

The weaknesses of this study are, on one side, that the results are almost exclusively dependent on the overexpression of fusion proteins. While useful in the field, this strategy does not represent the optimal way to dissect a cell biology issue. On the other side, the way the authors construct the rationale for each approximation is somehow difficult to follow. Finally, the use of two models, C. elegans and a mammalian cell line, which would strengthen the observations, contributes to the difficulty in reading the manuscript.

The findings are useful but do not clearly support the idea that USP8 mediates Rab5-Rab7 exchange and endosome maturation, In contrast, they appear to be incomplete and open new questions regarding the complexity of this process and the precise role of USP8 within it.

Reviewer #3 (Public Review):

Summary:

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

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

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

Strengths:

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

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

Weaknesses:

The manuscript is highly reliant on the use of drugs, or combinations of drugs, for long periods of time (6hr, 18hr..). Wherever possible the authors should test conclusions drawn from experiments involving drugs also using other canonical cell biology approaches (eg siRNA, Crispr). Although suggestive as a first approach, it is not reliable to draw conclusions from experiments where only drug combinations are being advanced (eg LatB + FCF).

F-actin is well known to play a wide variety of roles in cell death and other canonical cell death pathways (PMID: 26292640). The authors show using pharmacological inhibition that F-actin is key for GUVac formation. However, especially when testing for physiological relevance, how can these other roles for F-actin be ruled out?

To test the role of septins in GUVac formation only recruitment studies and no direct functional work is performed. A drug forchlofeneuron (FCF) is used, but this is well known to have off-target effects (PMID: 27473917).

Cells that possess GUVac are resistant to aniokis, but how are these cells resistant? This report is focused on mechanisms underlying GUVac formation and does not directly test for mechanisms underlying aniokis resistance.

Reviewer #3 (Public Review):

Summary:

The mechanism underlying the well-documented CO2-regulated activity of connexin 26 (Cx26) remains poorly understood. This is largely due to the labile nature of CO2-mediated carbamylation, making it challenging to visualize the effects of this reversible posttranslational modification. This paper by Brotherton et al. aims to address this gap by providing structural insights through cryo-EM structures of a carbamylation-mimetic mutant of the gap junction protein.

Strength:

The combination of the mutation, elevated PCO2, and the use of LMNG detergent resulted in high-resolution maps that revealed, for the first time, the structure of the cytoplasmic loop between transmembrane helix (TM) 2 and 3.

Weaknesses:

While the structure of the TM2-TM3 loop may suggest a mechanism for stabilizing the closed conformation, the EM density is not strong enough to support direct interaction with carbamylated or mutated K125.

Overall, the cryo-EM structures presented in this study support their proposing mechanism in which carbamylation at K125 promotes Cx26 gap junction closure. Through careful control of the pH and PCO2 for each cryo-EM sample, the current study substantiated that the more closed conformation observed in high PCO2 is independent of pH but likely triggered by carbamylation. This was unclear from their prior cryo-EM map of wildtype Cx26 at high PCO2.

While the new structures successfully visualize the TM2-TM3 loop, which likely plays significant roles in CO2-regulated Cx26 activity, further studies are necessary to understand the underlying mechanism. For instance, the current study lacks explanation regarding what propels the movement of the N-terminal helix, how carbamylated K125 interacts with the TM2-TM3 loop, the importance of the lipids visualized in the map, or the reason why gap junctions are constitutively open while hemichannels are closed under normal PCO2 levels

Reviewer #3 (Public Review):

Granule cells' axons bifurcate to form parallel fibers (PFs) and ascending axons (AAs). While the significance of PFs on cerebellar plasticity is widely acknowledged, the importance of AAs remains unclear. In the current paper, Conti and Auger conducted electrophysiological experiments in rat cerebellar slices and identified a new form of synaptic plasticity in the AA-Purkinje cell (PC) synapses. Upon simultaneous stimulation of AAs and PFs, AA-PC EPSCs increased, while PFs-EPSCs decreased. This suggests that synaptic responses to AAs and PFs in PCs are jointly regulated, working as an additional mechanism to integrate motor/sensory input. This finding may offer new perspectives in studying and modeling cerebellum-dependent behavior. Overall, the experiments are performed well. However, there are two weaknesses. First, the baseline of electrophysiological recordings is influenced significantly by run-down, making it difficult to interpret the data quantitatively. The amplitude of AA-EPSCs is relatively small and the run-down may mask the change. The authors should carefully reexamine the data with appropriate controls and statistics. Second, while the authors show AA-LTP depends on mGluR, NMDA receptors, and GABA-A receptors, which cell types express these receptors and how they contribute to plasticity is not clarified. The recommended experiments may help to improve the quality of the manuscript.

Reviewer #3 (Public Review):

Summary

The L114P gain of function mutation in the K2P channel TALK-1 encoded by KCNJ16 has been associated with maturity-onset diabetes of the young (MODY). In this study, Nakhe et al. generated mice carrying L114P TALK-1 and evaluated the impact of the mutation on pancreatic islet functions and glucose homeostasis. The authors report that the mutation increases neonatal lethality, owing to hyperglycemia caused by a lack of glucose-stimulated Ca2+ influx and insulin secretion. Adult mutant mice showed glucose intolerance and fasting hyperglycemia, which is attributed to blunted glucose-stimulated insulin secretion as well as increased glucagon secretion. Interestingly, male mice were more affected than female mice. Islets from adult mutant mice were found to have reduced Ca2+ entry upon glucose stimulation but also enhanced IP3-induced ER Ca2+ release, consistent with previous studies from the group showing a role of TALK-1 in ER Ca2+ homeostasis. Finally, comparison of bulk RNA sequencing results from WT and mutant islets revealed altered expression of genes involved in β-cell identify, function and signaling, which also contributes to the observed islet dysfunction.

Strengths

This is a well-executed and rigorous study that will be of great interest to the diabetes and islet biology communities. The findings provide convincing evidence supporting a causal role of the L114P gain of function TALK-1 mutation in glucose-stimulated insulin secretion defects and diabetes. The neonatal diabetes phenotype and the gender difference uncovered by the study have important clinical implications. The complexity of TALK-1 expression and hormone secretion in different endocrine cell types and how it impacts glucose homeostasis is elegantly illustrated in the L114P TALK-1 mouse model. The authors carefully and thoroughly addressed limitations of their study and discussed future directions. The importance of TALK-1 in β-cell and islet function demonstrated by this study will prompt future efforts targeting this important channel for diabetes treatment.

Reviewer #3 (Public Review):

Summary:

This manuscript reports the discovery of new compounds that selectively inhibit SMARCA4/SMARCA2 ATPase activity that work through a different mode as previously developed SMARCA4/SMARCA2 inhibitors. They also demonstrate the anti-tumor effects of the compounds on uveal melanoma cell proliferation and tumor growth. The findings indicate that the drugs exert their effects by altering chromatin accessibility at binding sites for lineage-specific transcription factors within gene enhancer regions. In uveal melanoma, altered expression of the transcription factor, SOX10, and SOX10 target gene underlies the anti-proliferative effects of the compounds. This study is significant because the discovery of new SMARCA4/SMARCA2 inhibitory compounds that can abrogate uveal melanoma tumorigenicity has therapeutic value. In addition, the findings provide evidence for the therapeutic use of these compounds in other transcription factor-dependent cancers.

Strengths:

The strengths of this manuscript include biochemical evidence that the new compounds are selective for SMARCA4/SMARCA2 over other ATPases and that the mode of action is distinct from a previously developed compound, BRM014, which binds the RecA lobe of SMARCA2. There is also strong evidence that FHT1015 suppresses uveal melanoma proliferation by inducing apoptosis. The in vivo suppression of tumor growth without toxicity validates the potential therapeutic utility of one of the new drugs. The conclusion that FHT1015 primarily inhibits SMARCA4 activity and thereby suppresses chromatin accessibility at lineage-specific enhancers is substantiated by ATAC-seq and ChIP-seq studies.

Weaknesses:

The weaknesses include a lack of more precise information on which SMARCA4/SMARCA2 residues the drugs bind. Although the I1173M/I1143M mutations are evidence that the critical residues for binding reside outside the RecA lobe, this site is conserved in CHD4, which is not affected by the compounds. Hence, this site may be necessary but not sufficient for drug binding or specifying selectivity. A more precise evaluation of the region specifying the effect of the new compounds would strengthen the evidence that they work through a novel mode and that they are selective. Another concern is that the mechanisms by which FHT1015 promotes apoptosis rather than simply cell cycle arrest are not clear. Does SOX10 or another lineage-specific transcription factor underlie the apoptotic effects of the compounds?

Reviewer #3 (Public Review):

Summary:

This study presents a new approach of combining two measurements (pHLA binding and pHLA-TCR binding) in order to refine predictions of which patient mutations are likely presented to and recognized by the immune system. Improving such predictions would play an important role in making personalized anti-cancer vaccinations more effective.

Strengths:

The study combines data from pre-existing tools pVACseq and pMTNet and applies them to a CRC patient population, which the authors show may improve the chance of identifying immunogenic, cancer-derived neoepitopes. Making the datasets collected publicly available would expand beyond the current datasets that typically describe caucasian patients.

Weaknesses:

It is unclear whether the pNetMHCpan and pMTNet tools used by the authors are entirely independent, as they appear to have been trained on overlapping datasets, which may explain their similar scores. The pHLA-TCR score seems to be driving the effects, but this not discussed in detail.

Due to sample constraints, the authors were only able to do a limited amount of experimental validation to support their model; this raises questions as to how generalisable the presented results are. It would be desirable to use statistical thresholds to justify cutoffs in ELISPOT data.

Some of the TCR repertoire metrics presented in Figure 2 are incorrectly described as independent variables and do not meaningfully contribute to the paper. The TCR repertoires may have benefitted from deeper sequencing coverage, as many TCRs appear to be supported only by a single read.

Reviewer #3 (Public Review):

The mechanical properties of DNA wrapped in nucleosomes affect the stability of nucleosomes and may play a role in the regulation of DNA accessibility in eukaryotes. In this manuscript, Ngo and coworkers study how the stability of a nucleosome is affected by the introduction of a CC mismatched base pair, which has been reported to increase the flexibility of DNA. Previously, the group has used a sophisticated combination of single-molecule FRET and force spectroscopy with an optical trap to show that the more flexible half of a 601 DNA segment provides for more stable wrapping as compared to the other half. Here, it is confirmed with a single-molecule cyclization essay that the introduction of a CC mismatch increases the flexibility of a DNA fragment. Consistent with the previous interpretation, it also increased the unwrapping force for the half of the 601 segment in which the CC mismatch was introduced, as measured with single-molecule FRET and force spectroscopy. Enhanced stability was found up to 56 bp into the nucleosome. The intricate role of mechanical stability of nucleosomes was further investigated by comparing force-induced unwrapping profiles of yeast and Xenopus histones. Intriguingly, asymmetric unwrapping was more pronounced for yeast histones.

Note from Reviewing Editor:

The authors addressed the points in the reviews by making appropriate text additions and clarifications.

Reviewer #3 (Public Review):

Summary:

Asabuki and Clopath study stochastic sequence learning in recurrent networks of Poisson spiking neurons that obey Dale's law. Inspired by previous modeling studies, they introduce two distinct learning rules, to adapt excitatory-to-excitatory and inhibitory-to-excitatory synaptic connections. Through a series of computer experiments, the authors demonstrate that their networks can learn to generate stochastic sequential patterns, where states correspond to non-overlapping sets of neurons (cell assemblies) and the state-transition conditional probabilities are first-order Markov, i.e., the transition to a given next state only depends on the current state. Finally, the authors use their model to reproduce certain experimental songbird data involving highly-predictable and highly-uncertain transitions between song syllables.

Strengths:

This is an easy-to-follow, well-written paper, whose results are likely easy to reproduce. The experiments are clear and well-explained. The study of songbird experimental data is a good feature of this paper; finches are classical model animals for understanding sequence learning in the brain. I also liked the study of rapid task-switching, it's a good-to-know type of result that is not very common in sequence learning papers.

Weaknesses:

While the general subject of this paper is very interesting, I missed a clear main result. The paper focuses on a simple family of sequence learning problems that are well-understood, namely first-order Markov sequences and fully visible (no-hidden-neuron) networks, studied extensively in prior work, including with spiking neurons. Thus, because the main results can be roughly summarized as examples of success, it is not entirely clear what the main point of the authors is.

Going into more detail, the first major weakness I see in this paper is the heuristic choice of learning rules. The paper studies Poisson spiking neurons (I return to this point below), for which learning rules can be derived from a statistical objective, typically maximum likelihood. For fully-visible networks, these rules take a simple form, similar in many ways to the E-to-E rule introduced by the authors. This more principled route provides quite a lot of additional understanding on what is to be expected from the learning process. For instance, should maximum likelihood learning succeed, it is not surprising that the statistics of the training sequence distribution are reproduced. Moreover, given that the networks are fully visible, I think that the maximum likelihood objective is a convex function of the weights, which then gives hope that the learning rule does succeed. And so on. This sort of learning rule has been studied in a series of papers by David Barber and colleagues [refs. 1, 2 below], who applied them to essentially the same problem of reproducing sequence statistics in recurrent fully-visible nets. It seems to me that one key difference is that the authors consider separate E and I populations, and find the need to introduce a balancing I-to-E learning rule.

Because the rules here are heuristic, a number of questions come to mind. Why these rules and not others - especially, as the authors do not discuss in detail how they could be implemented through biophysical mechanisms? When does learning succeed or fail? What is the main point being conveyed, and what is the contribution on top of the work of e.g. Barber, Brea, et al. (2013), or Pfister et al. (2004)?

The use of a Poisson spiking neuron model is the second major weakness of the study. A chief challenge in much of the cited work is to generate stochastic transitions from recurrent networks of deterministic neurons. The task the authors set out to do is much easier with stochastic neurons; it is reasonable that the network succeeds in reproducing Markovian sequences, given an appropriate learning rule. I believe that the main point comes from mapping abstract Markov states to assemblies of neurons. If I am right, I missed more analyses on this point, for instance on the impact that varying cell assembly size would have on the findings reported by the authors.

Finally, it was not entirely clear to me what the main fundamental point in the HVC data section was. Can the findings be roughly explained as follows: if we map syllables to cell assemblies, for high-uncertainty syllable-to-syllable transitions, it becomes harder to predict future neural activity? In other words, is the main point that the HVC encodes syllables by cell assemblies?

(1) Learning in Spiking Neural Assemblies, David Barber, 2002. URL: https://proceedings.neurips.cc/paper/2002/file/619205da514e83f869515c782a328d3c-Paper.pdf

(2) Correlated sequence learning in a network of spiking neurons usingmaximum likelihood, David Barber, Felix Agakov, 2002. URL: http://web4.cs.ucl.ac.uk/staff/D.Barber/publications/barber-agakov-TR0149.pdf

Reviewer #3 (Public Review):

Summary:

In this paper, the authors sought to evaluate whether the novel TB drug candidate, spectinamide 1599 (S), given via inhalation to mouse TB models, and combined with the drugs B (bedaquiline) and Pa (pretomanid), would demonstrate similar efficacy to that of BPaL regimen (where L is linezolid). Because L is associated with adverse events when given to patients long-term, and one of those is associated with myelosuppression (bone marrow toxicity) the authors also sought to assess blood parameters, effects on bone marrow, immune parameters/cell effects following treatment of mice with BPaS and BPaL. They conclude that BPaL and BPaS have equivalent efficacy in both TB models used and that BPaL resulted in weight loss and anemia (whereas BPaL did not) under the conditions tested, as well as effects on bone marrow.

Strengths:

The authors used two mouse models of TB that are representative of different aspects of TB in patients (which they describe well), intending to present a fuller picture of the activity of the tested drug combinations. They conducted a large body of work in these infected mice to evaluate efficacy and also to survey a wide range of parameters that could inform the effect of the treatments on bone marrow and on the immune system. The inclusion of BPa controls (in most studies) and also untreated groups led to a large amount of useful data that has been collected for the mouse models per se (untreated) as well as for BPa - in addition to the BPaS and BPaL combinations which are of particular interest to the authors. Many of these findings related to BPa, BPaL, untreated groups, etc corroborate earlier findings and the authors point this out effectively and clearly in their manuscript. To go further, in general, it is a well-written and cited article with an informative introduction.

Weaknesses:

The authors performed a large amount of work with the drugs given at the doses and dosing intervals started, but at present, there is no exposure data available in the paper. It would be of great value to understand the exposures achieved in plasma at least (and in the lung if more relevant for S) in order to better understand how these relate to clinical exposures that are observed at marketed doses for B, Pa, and L as well as to understand the exposure achieved at the doses being evaluated for S. If available as historical data this could be included/cited. Considering the great attempts made to evaluate parameters that are relevant to clinical adverse events, it would add value to understand what exposures of drug effects such as anemia, weight loss, and bone marrow effects, are being observed.

It would also be of value to add an assessment of whether the weight loss, anemia, or bone marrow effects observed for BPaL are considered adverse, and the extent to which we can translate these effects from mouse to patient (i.e. what are the limitations of these assessments made in a mouse study?). For example, is the small weight loss seen as significant, or is it reversible? Is the magnitude of the changes in blood parameters similar to the parameters seen in patients given L?

In addition, it is always challenging to interpret findings for combinations of drugs, so the addition of language to explain this would add value: for example, how confident can we be that the weight loss seen for only the BPaL group is due to L as opposed to a PK interaction leading to an elevated exposure and weight loss due to B or Pa?

Turning to the evaluations of activity in mouse TB models, unfortunately, the evaluations of activity in the BALB/c mouse model as well as the spleens of the Kramnik model resulted in CFU below/at the limit of detection and so, to this reviewer's understanding of the data, comparisons between BPaL and BPaS cannot be made and so the conclusion of equivalent efficacy in BALB/c is not supported with the data shown. There is no BPa control in the BALB/c study, therefore it is not possible to discern whether L or S contributed to the activity of BPaL or BPaS; it is possible that BPa would have shown the same efficacy as the 3 drug combinations. It would be valuable to conduct a study including a BPa control and with a shorter treatment time to allow comparison of BPa, BPaS, and BPaL. In the Kramnik lungs, as the authors rightly note, the studies do not support any contribution of S or L to BPa - i.e. the activity observed for BPa, BPaL, and BPaS did not significantly differ. Although the conclusions note equivalency of BPaL and BPaS, which is correct, it would be helpful to also include BPa in this statement; it would be useful to conduct a study dosing for a longer period of time or assessing a relapse endpoint, where it is possible that a contribution of L and/or S may be seen - thus making a stronger argument for S contributing an equivalent efficacy to L. The same is true for the assessment of lesions - unfortunately, there was no BPa control meaning that even where equivalency is seen for BPaL and BPaS, the reader is unable to deduce whether L or S made a contribution to this activity.

Reviewer #3 (Public Review):

Summary:

The article by Huang et.al. presents an in-depth study on the role of DNA methylation in regulating virulence and metabolism in Pseudomonas syringae, a model phytopathogenic bacterium. This comprehensive research utilized single-molecule real-time (SMRT) sequencing to profile the DNA methylation landscape across three model pathovars of P. syringae, identifying significant epigenetic mechanisms through the Type-I restriction-modification system (HsdMSR), which includes a conserved sequence motif associated with N6-methyladenine (6mA). The study provides novel insights into the epigenetic mechanisms of P. syringae, expanding the understanding of bacterial pathogenicity and adaptation. The use of SMRT sequencing for methylome profiling, coupled with transcriptomic analysis and in vivo validation, establishes a robust evidence base for the findings

Strengths:

The results are presented clearly, with well-organized figures and tables that effectively illustrate the study's findings.

Weaknesses:

It would be helpful to add more details, especially in the methods, which make it easy to evaluate and enhance the manuscript's reproducibility.

Reviewer #3 (Public Review):

Summary:

Pal et al. provide valuable evidence supporting distinct vascular bed-specific VEGF-A mediated vascular permeability function of Neuropilin-1 (NRP1) in adult mice. Using a suite of genetic mice models and state-of-the-art vascular permeability assays the authors demonstrate that ear skin vasculature of EC-specific NRP1 adult knockout mice is hypersensitive to VEGF-A mediated high-molecular weight dye leakage from venules, as opposed to back skin and tracheal vasculature where EC-specific NRP1 loss had a more classical negative effect on permeability. Interestingly, both whole organism KO of NRP1 and a blocking antibody treatment, attenuated VEGF-A mediated permeability in ear skin and had the usual attenuation of permeability phenotype in back skin and tracheal vasculature. Using a pericyte promoter specific reporter mice line, the authors characterize NRP1 expression in the vascular beds of the ear dermis and back skin and conclude that NRP1 expression is higher in perivascular cells in the ear dermis as opposed to back skin vasculature, thus indicating a juxtracrine NRP1-VEGFR2 signaling model in adult mice. Further, they use a Vegfr2 phosphosite mutant homozygous mice model in the background of NRP1 iECKO to find the hypersensitivity to VEGF-A stimulation in ear skin is abrogated and therefore, prove the juxtracrine NRP1 control of VEGFR2 mediated downstream signaling leading to vascular permeability. Further, they successfully show distinctive vascular bed-specific results as above using a well-characterized VE-Cadherin Y685 antibody staining which corresponds to vascular leakage downstream of VEGF-A/VEGFR2 signaling in ear dermis and back skin vascular beds.

Strengths:

The question of the in vivo role of NRP1 in VEGF-A-induced hyper-permeability is an unresolved one and the elegant use of genetic mice models to demonstrate the phenotypes is valuable to the field. The organotypic differences observed in vascular permeability upon VEGF-A treatment in ear skin versus back skin and tracheal vasculature are solid. The subsequent investigation to validate heightened VEGFR2 signaling in ear dermis downstream of VEGF-A stimulation using Vegfr2 Y949F mice, VEC Y685 antibody, and pPLCγ antibody is also very convincing.

Weaknesses:

The mechanism proposed by the authors by which EC-specific loss of NRP1 caused hypersensitivity to VEGF-A in ear dermis is through elevated juxtracrine signaling of NRP1 expressed in pericytes in trans binding and retaining VEGFR2 on the cell surface of ECs to sustain downstream signaling for longer time, in corroboration to earlier findings in Koch et al., 2014, where NRP1 was studied in the context of tumor angiogenesis. To support their claim, the authors stain the ear dermis and back skin vasculature of Pdgfrb-GFP reporter mice, with NRP1 and CD31 antibodies and find out that ear skin vasculature has higher perivascular cells as opposed to back skin vasculature. While this is a good experiment to prove the above point, there are no functional experiments to support this model.

Overall, although the paper presents very useful findings in the field of NRP1-VEGFR2 biology, and most of the conclusions are well supported by the data, there are a few points if addressed can significantly substantiate the model of juxtracrine signaling proposed by the authors. They are:

(1) It will be important to know if the perivascular to vascular NRP1 expression (such as in Figure 3B) increases further in ear skin vasculatures of NRP1 iECKO mice compared to otherwise WT mice.

(2) Does knocking out NRP1 in pericytes attenuate the VEGF-A mediated hyperpermeability observed in ear skin of NRP1 iECKO mice (similar to experiments in 1C, 2C)?

(3) What is the status of VEGFR2 expression in ECs of ear skin and back skin of NRP1 iECKO and NRP1 iKO mice? This experiment is a proof-of-concept and is not essential to prove the point of juxtracrine NRP1 signaling since downstream readouts - pPLCγ and VEC Y685 staining have already been shown to correlate in the ear dermis.

Reviewer #3 (Public Review):

Summary:

This paper by Portela Catani et al examines the antigenic relationships (measured using monotypic ferret and mouse sera) across a panel of N2 genes from the past 14 years, along with the underlying sequence differences and phylogenetic relationships. This is a highly significant topic given the recent increased appreciation of the importance of NA as a vaccine target, and the relative lack of information about NA antigenic evolution compared with what is known about HA. Thus, these data will be of interest to those studying the antigenic evolution of influenza viruses. The methods used are generally quite sound, though there are a few addressable concerns that limit the confidence with which conclusions can be drawn from the data/analyses.

Strengths:

-The significance of the work, and the (general) soundness of the methods.<br /> -Explicit comparison of results obtained with mouse and ferret sera

Weaknesses:

- Machine learning analyses neither experimentally validated nor shown to be better than simple, phylogenetic-based inference.

Reviewer #3 (Public Review):

Summary:

Single unit neural activity tuned to environmental or behavioral variables gradually changes over time. This phenomenon, called representational drift, occurs even when all external variables remain constant, and challenges the idea that stable neural activity supports the performance of well-learned behaviors. While a number of studies have described representational drift across multiple brain regions, our understanding of the underlying mechanism driving drift is limited. Ratzon et al. propose that implicit regularization - which occurs when machine learning networks continue to reconfigure after reaching an optimal solution - could provide insights into why and how drift occurs in neurons. To test this theory, Ratzon et al. trained a recurrent neural network (RNN) trained to perform the oft-utilized linear track behavioral paradigm and compare the changes in hidden layer units to those observed in hippocampal place cells recorded in awake, behaving animals.

Ratzon et al. clearly demonstrate that hidden layer units in their model undergo consistent changes even after the task is well-learned, mirroring representational drift observed in real hippocampal neurons. They show that the drift occurs across three separate measures: the active proportion of units (referred to as sparsification), spatial information of units, and correlation of spatial activity. They continue to address the conditions and parameters under which drift occurs in their model to assess the generalizability of their findings to non-spatial tasks. Last, they investigate the mechanism through which sparsification occurs, showing that flatness of the manifold near the solution can influence how the network reconfigures. The authors suggest that their findings indicate a three stage learning process: 1) fast initial learning followed by 2) directed motion along a manifold which transitions to 3) undirected motion along a manifold.

Overall, the authors' results support the main conclusion that implicit regularization in machine learning networks mirrors representational drift observed in hippocampal place cells. Their findings promise to open new fields of inquiry into the connection between machine learning and representational drift in other, non-spatial learning paradigms, and to generate testable predictions for neural data.

Strengths:

(1) Ratzon et al. make an insightful connection between well-known phenomena in two separate fields: implicit regularization in machine learning and representational drift in the brain. They demonstrate that changes in a recurrent neural network mirror those observed in the brain, which opens a number of interesting questions for future investigation.

(2) The authors do an admirable job of writing to a large audience and make efforts to provide examples to make machine learning ideas accessible to a neuroscience audience and vice versa. This is no small feat and aids in broadening the impact of their work.

(3) This paper promises to generate testable hypotheses to examine in real neural data, e.g., that drift rate should plateau over long timescales (now testable with the ability to track single-unit neural activity across long time scales with calcium imaging and flexible silicon probes). Additionally, it provides another set of tools for the neuroscience community at large to use when analyzing the increasingly high-dimensional data sets collected today.

Weaknesses:

The revised manuscript addresses all the weaknesses outlined in my initial review. However, there is one remaining (minor) weakness regarding how "sparseness" is used and defined.

Sparseness can mean different things to different fields. For example, for engram studies, sparseness could be measured at the population level by the proportion of active cells, whereas for a physiology study, sparseness might be measured at the neuron level by the change in peak firing rate of each cell as an animal enters that cell's place field. In this manuscript, the idea of "sparseness" is introduced indirectly in the last paragraph of the introduction as "...changes in activity statistics (sparseness)...", but it is unclear from the preceding text if the referenced "activity statistics" used to define sparseness are the "fraction of active units," or their "tuning specificity," or both. While sparseness is clearly defined in the Methods section for the RNN, there is no mention of how it is defined for neural data, and spatial information is not mentioned at all. For clarity, I suggest explicitly defining sparseness for both the RNN and real neural data early in the main text, e.g. "Here, we measure sparseness in neural data by A and B, and by the analogous metric(s) of X and Y in our RNN..." This is a small but important nuance that will enhance the ease of reading for a broad neuroscience audience.

Reviewer #3 (Public Review):

Summary:

The manuscript by Nagel, et al. describes studies of mouse vomeronasal sensory neuron (VSN) tuning to mouse urine samples across different sexes and strains, including wild mice, alongside mass spectrometry analysis of the same samples. The authors performed live Ca2+ imaging (CAL520 dye) of VSNs in acute vomeronasal organ (VNO) slices to determine how VSNs are tuned to pairs of stimuli that differ in their origin (e.g. male C57BL/6 versus male BALB/c urine, male C57BL/6 versus female C57BL/6, etc.). For each pair of tested odorants, the results measure the proportion of VSNs that respond to both stimuli ("generalists") or just one of the two ("specialists"), as well as metrics of tuning preference and response reliability. The authors find in most cases that generalists make up a larger proportion of responsive VSNs than specialists, but several pairwise comparisons showed a high degree of strain selectivity. Notably, the authors evaluated VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding strain-dependent differences in the representation of mouse urine. Alongside these measurements of VSN tuning, the authors report results of mass spectrometry analyses of volatiles and proteins in the same urine samples. These analyses indicated a number of molecules in each category that vary across sex and strain, and therefore represent candidate vomeronasal ligands. However, this study did not directly test whether any of these candidate molecules drives VSN activity. Overall, this work provides solid information related to mouse vomeronasal chemosensation.

Strengths:

A strength of the current study is its focus on characterizing the neural responses of the VNO to urine derived from wild mice. The majority of existing vomeronasal system research has relied on the use of inbred strains for both neural response recordings and investigations of candidate vomeronasal system ligands. Inbreeding in laboratory environments may alter the chemical composition of bodily secretions, thereby potentially changing the information they contain. Moreover, the more homogeneous nature of inbred strains could be critical when studying the AOS mediated social aspects. If there exist noticeable differences in the chemical composition of secretions from wild animals compared to inbred strains, this would suggest that future research must consider natural sources of candidate ligands outside of inbred strains. This work identifies some intriguing differences, worthy of further exploration, between the urine composition of wild mice versus inbred mice, as well as disparities in how the VNO responds to urine from these different sources. However, the molecular composition and VNO responsiveness to wild mouse urine was found to be highly overlapping with inbred mouse urine, supporting the continued investigation of candidate ligands found in inbred mouse urine.

Another positive aspect of this work is its use of the same set of stimuli as a previous study by the same authors (Bansal et al., 2021) in the downstream accessory olfactory bulb. The consistency in stimulus selection facilitates a comparison of information processing of sex and strain information from the sensory periphery to the brain. Although comparisons between the two connected regions are not a focus of this work, and methodological differences (e.g., Ca2+ imaging versus electrophysiology) may introduce caveats into comparisons, the support of "apples to apples" comparisons across connected circuits is critical to progress in the field.

Finally, this study directly measured VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding subtle but important differences in the representation of mouse urine in these two recipient strains. Given that there is a long history of behavioral research into strain-specific differences in social behavior, this research paves the way for future studies into how different mouse strains detect and process social chemosignals.

Weaknesses:

One of the primary objectives in this study is to ascertain the extent to which the response profiles of VSNs are specific to sex and strain. The design of these Ca2+ imaging experiments uses a simple stimulus design, using two interleaved bouts of stimulation with pairs of urine (e.g., male versus female C57BL/6, male C57BL/6 versus male BALB/c) at a single dilution factor (1:100). This introduces two significant limitations: (1) the "generalist" versus "specialist" descriptors pertain only to the specific pairwise comparisons made and (2) there is no information about the sensitivity/concentration-dependence of the responses.

The functional measurements of VSN tuning to various pairs of urine stimuli are presented alongside mass spectrometry-based comparisons. However, the mass spectrometry-based analysis was performed separately from VSN tuning experiments/analysis. The juxtaposition of these measurements may give some readers the impression that VSN tuning measurements were integrated with molecular profiling (i.e., that the molecular diversity was causally related physiological responses). This is a hypothesis raised by the parallel studies, but not a supported conclusion of the current work.

The impact of mass spectrometry findings is acknowledged to be limited to nonvolatile organic compounds and proteins/peptides, and that it is possible that few of these candidate molecules are active in the VNO. Moreover, it remains possible that the VSN responses are driven mostly by small nonvolatiles (e.g., polar steroids), a class of strong VSN ligands that were excluded from molecular analysis.

Reviewer #3 (Public Review):

A brain region called the retrotrapezoid nucleus (RTN) regulates breathing in response to changes in CO2/H+, a process termed central chemoreception. A transcription factor called PHOX2B is important for RTN development and mutations in the PHOX2B gene result in a severe type of sleep apnea called Congenital Central Hypoventilation Syndrome. PHOX2B is also expressed throughout life, but its postmitotic functions remain unknown. This study shows that knockdown of PHOX2B in the RTN region in adult rats decreased expression of Task2 and Gpr4 in Nmb-expressing RTN chemoreceptors and this corresponded with a diminished ventilatory response to CO2 but did not impact baseline breathing or the hypoxic ventilatory response. These results provide novel insight regarding postmitotic functions of PHOX2B in RTN neurons.

I have two main concerns and several points of clarification.

Main issues:<br /> (1) The experimental approach was not targeted to Nmb+ neurons and since other cells in the area also express Phox2b, conclusions should be tempered to focus on Phox2b expressing parafacial neurons NOT specifically RTN neurons

(2) It's not clear whether PHOX2B is important for transcription of pH sensing machinery, cell health or both. If knockdown of PHOX2B knockdown results in loss of RTN neurons this is also expected to decrease Task2 and Gpr4 levels, albeit by a transcription-independent mechanism.

Other points:

(3) All individual data points should be visible in floating bar graphs in Figs 1 and 4. For example, I don't see any dots for naïve animals in any of the panels in Fig. 1.

(4) the C1 and facial partly overlap with the RTN at this level of the medulla and these cells should appear as Phox2b+/Nmb- cells so it is not clear to me why these cells are not evident in the control tissue in figs 2B and 3B. Also, some of the bregma levels shown in Fig. 5A overlap with Figs 2-3 so again it's not clear to me how this non-cell type specific viral approach was targeted to Nmb cells but not near by TH+ cells. Please clarify.

(5) How do you get a loss of Nmb+ neurons (Figs 2-3) with no change in Nmb fluorescence (Fig. 5B)? In the absence of representative images these results are not compelling and should be substantiated by more readily quantifiable approaches like qPCR.

Reviewer #3 (Public Review):

Summary:

The manuscript addresses a question inspired by the Baroceptor Hypothesis and its links to visual awareness and interoception. Specifically, the reported study aimed to determine if the effects of cardiac contraction (systole) on binocular rivalry (BR) are facilitatory or suppressive. The main experiment - relying on a technically challenging procedure of presenting stimuli synchronised with the heartbeats of participants - has been conducted with great care, and numerous manipulation checks the authors report convincingly show that the methods they used work as intended. Moreover, the control experiment allows for excluding alternative explanations related to participants being aware of their heartbeats. Therefore, the study convincingly shows the effect of cardiac activity on BR - and this is an important finding. The results, however, do not allow for unambiguously determining if this effect is facilitatory or suppressive (see details below), which renders the study not as informative as it could be.

While the authors strongly focus on interoception and awareness, this study will be of interest to researchers studying BR as such. Moreover, the code and the data the authors share can facilitate the adoption of their methods in other labs.

Strengths:

(1) The study required a complex technical setup and the manuscript both describes it well and demonstrates that it was free from potential technical issues (e.g. in section 3.3. Manipulation check).

(2) The sophisticated statistical methods the authors used, at least for a non-statistician like me, appear to be well-suited for their purpose. For example, they take into account the characteristics of BR (gamma distributions of dominance durations). Moreover, the authors demonstrate that at least in one case their approach is more conservative than a more basic one (Binomial test) would be.

(3) Finally, the control experiment, and the analysis it enabled, allow for excluding a multitude of alternative explanations of the main results.

(4) The authors share all their data and materials, even the code for the experiment.

(5) The manuscript is well-written. In particular, it introduces the problem and methods in a way that should be easy to understand for readers coming from different research fields.

Weaknesses:

(1) The interpretation of the main result in the context of the Baroceptor hypothesis is not clear. The manuscript states: The Baroreceptor Hypothesis would predict that the stimulus entrained to systole would spend more time suppressed and, conversely, less time dominant, as cortical activity would be suppressed each time that stimulus pulses. The manuscript does not specify why this should be the case, and the term 'entrained' is not too helpful here (does it refer to neural entrainment? or to 'being in phase with'?). The answer to this question is provided by the manuscript only implicitly, and, to explain my concern, I try to spell it out here in a slightly simplified form.

During systole (cardiac contraction), the visual system is less sensitive to external information, so it 'ignores' periods when the systole-synchronised stimulus is at the peak of its pulse. Conversely, the system is more sensitive during diastole, so the stimulus that is at the peak of its pulse then should dominate for longer, because its peaks are synchronised with the periods of the highest sensitivity of the visual system when the information used to resolve the rivalry is sampled from the environment. This idea, while indeed being a clever test of the hypothesis in question, rests on one critical assumption: that the peak of the stimulus pulse (as defined in the manuscript) is the time when the stimulus is the strongest for the visual system. The notion of 'stimulus strength' is widely used in the BR literature (see Brascamp et al., 2015 for a review). It refers to the stimulus property that, simply speaking, determines its tendency to dominate in the BR. The strength of a stimulus is underpinned by its low-level visual properties, such as contrast and spatial frequency content. Coming back to the manuscript, the pulsing of the stimuli affected at least spatial frequency (and likely other low-level properties), and it is unknown if it was in phase with the pulsing of the stimulus strength, or not. If my understanding of the premise of the study is correct, the conclusions drawn by the authors stand only if it was.

In other words, most likely the strength of one of the stimuli was pulsating in sync with the systole, but is it not clear which stimulus it was. It is possible that, for the visual system, the stimulus meant to pulse in sync with the systole was pulsing strength-wise in phase with the diastole (and the one intended to pulse with in sync with the diastole strength-wise pulsed with the systole). If this is the case, the predictions of the Baroceptor Hypothesis hold, which would change the conclusion of the manuscript.

(2) Using anaglyph goggles necessitates presenting stimuli of a different colour to each eye. The way in which different colours are presented can impact stimulus strength (e.g. consider that different anaglyph foils can attenuate the light they let through to different degrees). To deal with such effects, at least some studies on BR employed procedures of adjusting the colours for each participant individually (see Papathomas et al., 2004; Patel et al., 2015 and works cited there). While I think that counterbalancing applied in the study excludes the possibility that colour-related effects influenced the results, the effects of interest still could be stronger for one of the coloured foils.

(3) Several aspects of the methods (e.g. the stimuli), are not described at the level of detail some readers might be accustomed to. The most important issue here is the task the participants performed. The manuscript says that they pressed a button whenever they experienced a switch in perception, but it is only implied that there were different buttons for each stimulus.

Brascamp, J. W., Klink, P. C., & Levelt, W. J. M. (2015). The 'laws' of binocular rivalry: 50 years of Levelt's propositions. Vision Research, 109, 20-37. https://doi.org/10.1016/j.visres.2015.02.019<br /> Papathomas, T. V., Kovács, I., & Conway, T. (2004). Interocular grouping in binocular rivalry: Basic attributes and combinations. In D. Alais & R. Blake (Eds.), Binocular Rivalry (pp. 155-168). MIT Press<br /> Patel, V., Stuit, S., & Blake, R. (2015). Individual differences in the temporal dynamics of binocular rivalry and stimulus rivalry. Psychonomic Bulletin and Review, 22(2), 476-482. https://doi.org/10.3758/s13423-014-0695-1

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors propose that astrocytic water channel AQP4 represents the dominant pathway for tonic water efflux without which astrocytes undergo cell swelling. The authors measure changes in astrocytic sulforhodamine fluorescence as the proxy for cell volume dynamics. Using this approach, they perform a technically elegant series of ex vivo and in vivo experiments exploring changes in astrocytic volume in response to AQP4 inhibitor TGN-020 and/or neuronal stimulation. The key finding is that TGN-020 produces an apparent swelling of astrocytes and modifies astrocytic cell volume regulation after spreading depolarizations. Additionally, systemic application of TGN-020 produced changes in diffusion-weighted MRI signal, which the authors interpret as cellular swelling. This study is perceived as potentially significant. However, several technical caveats should be strongly considered and perhaps addressed through additional experiments.

Strengths:

(1) This is a technically elegant study, in which the authors employed a number of complementary ex vivo and in vivo techniques to explore functional outcomes of aquaporin inhibition. The presented data are potentially highly significant (but see below for caveats and questions related to data interpretation).

(2) The authors go beyond measuring cell volume homeostasis and probe for the functional significance of AQP4 inhibition by monitoring Ca2+ signaling in neurons and astrocytes (GCaMP6 assay).

(3) Spreading depolarizations represent a physiologically relevant model of cellular swelling. The authors use ChR2 optogenetics to trigger spreading depolarizations. This is a highly appropriate and much-appreciated approach.

Weaknesses:

(1) The main weakness of this study is that all major conclusions are based on the use of one pharmacological compound. In the opinion of this reviewer, the effects of TGN-020 are not consistent with the current knowledge on water permeability in astrocytes and the relative contribution of AQP4 to this process.

Specifically: Genetic deletion of AQP4 in astrocytes reduces plasmalemmal water permeability by ~two-three-fold (when measured a 37oC, Solenov et al., AJP-Cell, 2004). This is a significant difference, but it is thought to have limited/no impact on water distribution. Astrocytic volume and the degree of anisosmotic swelling/shrinkage are unchanged because the water permeability of the AQP4-null astrocytes remains high. This has been discussed at length in many publications (e.g., MacAulay et al., Neuroscience, 2004; MacAulay, Nat Rev Neurosci, 2021) and is acknowledged by Solenov and Verkman (2004).

Keeping this limitation in mind, it is important to validate astrocytic cell volume changes using an independent method of cell volume reconstruction (diameter of sulforhodamine-labeled cell bodies? 3D reconstruction of EGFP-tagged cells? Else?)

(2) TGN-020 produces many effects on the brain, with some but not all of the observed phenomena sensitive to the genetic deletion of AQP4. In the context of this work, it is important to note that TGN-020 does not completely inhibit AQP4 (70% maximal inhibition in the original oocyte study by Huber et al., Bioorg Med Chem, 2009). Thus, besides not knowing TGN-020 levels inside the brain, even "maximal" AQP4 inhibition would not be expected to dramatically affect water permeability in astrocytes.

This caveat may be addressed through experiments using local delivery of structurally unrelated AQP4 blockers, or, preferably, AQP4 KO mice.

(3) This reviewer thinks that the ADC signal changes in Figure 5 may be unrelated to cellular swelling. Instead, they may be a result of the previously reported TGN-020-induced hyphemia (e.g., H. Igarashi et al., NeuroReport, 2013) and/or changes in water fluxes across pia matter which is highly enriched in AQP4. To amplify this concern, AQP4 KO brains have increased water mobility due to enlarged interstitial spaces, rather than swollen astrocytes (RS Gomolka, eLife, 2023). Overall, the caveats of interpreting DW-MRI signal deserve strong consideration.

Reviewer #3 (Public Review):

Summary:

Neurogenesis in the mammalian olfactory epithelium persists throughout the life of the animal. The process replaces damaged or dying olfactory sensory neurons. It has been tacitly that replacement of the OR subtypes is stochastic, although anecdotal evidence has suggested that this may not be the case. In this study, Santoro and colleagues systematically test this hypothesis by answering three questions: is there enrichment of specific OR subtypes associated with neurogenesis? Is the enrichment dependent on sensory stimulus? Is the enrichment the result of differential generation of the OR type or from differential cell death regulated by neural activity? The authors provide some solid evidence indicating that musk odor stimulus selectively promotes the OR types expressing the musk receptors. The evidence argues against a random selection of ORs in the regenerating neurons.

Strengths:

The strength of the study is a thorough and systematic investigation of the expression of multiple musk receptors with unilateral naris occlusion or under different stimulus conditions. The controls are properly performed. This study is the first to formulate the selective promotion hypothesis and the first systematic investigation to test it. The bulk of the study uses in situ hybridization and immunofluorescent staining to estimate the number of OR types. These results convincingly demonstrate the increased expression of musk receptors in response to male odor or muscone stimulation.

Weaknesses:

A major weakness of the current study is the single-cell RNASeq result. The authors use this piece of data as a broad survey of receptor expression in response to unilateral nasal occlusion. However, several issues with this data raise serious concerns about the quality of the experiment and the conclusions. First, the proportion of OSNs, including both the immature and mature types, constitutes only a small fraction of the total cells. In previous studies of the OSNs using the scRNASeq approach, OSNs constitute the largest cell population. It is curious why this is the case. Second, the authors did not annotate the cell types, making it difficult to assess the potential cause of this discrepancy. Third, given the small number of OSNs, it is surprising to have multiple musk receptors detected in the open side of the olfactory epithelium whereas almost none in the closed side. Since each OR type only constitutes ~0.1% of OSNs on average, the number of detected musk receptors is too high to be consistent with our current understanding and the rest of the data in the manuscript. Finally, unlike the other experiments, the authors did not describe any method details, nor was there any description of quality controls associated with the experiment. The concerns over the scRNASeq data do not diminish the value of the data presented in the bulk of the study but could be used for further analysis.

A weakness of the experiment assessing musk receptor expression is that the authors do not distinguish immature from mature OSNs. Immature OSNs express multiple receptor types before they commit to the expression of a single type. The experiments do not reveal whether mature OSNs maintain an elevated expression level of musk receptors.

There are also two conceptual issues that are of concern. The first is the concept of selective neurogenesis. The data show an increased expression of musk receptors in response to male odor stimulation. The authors argue that this indicates selective neurogenesis of the musk receptor types. However, it is not clear what the distinction is between elevated receptor expression and a commitment to a specific fate at an early stage of development. As immature OSNs express multiple receptors, a likely scenario is that some newly differentiated immature OSNs have elevated expression of not only the musk receptors but also other receptors. The current experiments do not distinguish the two alternatives. Moreover, as pointed out above, it is not clear whether mature OSNs maintain the increased expression. Although a scRNASeq experiment can clarify it, the authors, unfortunately, did not perform an in-depth analysis to determine at which point of neurogenesis the cells commit to a specific musk receptor type. The quality of the scRNASeq data unfortunately also does not lend confidence for this type of analysis.

A second conceptual issue, the idea of homeostasis in regeneration, which the authors presented in the Introduction, needs clarification. In its current form, it is confusing. It could mean that a maintenance of the distribution of receptor types, or it could mean the proper replacement of a specific OR type upon the loss of this type. The authors seem to refer to the latter and should define it properly.

Reviewer #3 (Public Review):

In this manuscript, Magnuson and colleagues investigate the meiotic functions of ARID1A, a putative DNA binding subunit of the SWI/SNF chromatin remodeler BAF. The authors develop a germ cell specific conditional knockout (cKO) mouse model using Stra8-cre and observe that ARID1A-deficient cells fail to progress beyond pachytene, although due to inefficiency of the Stra8-cre system the mice retain ARID1A-expressing cells that yield sperm and allow fertility. Because ARID1A was found to accumulate at the XY body late in Prophase I, the authors suspected a potential role in meiotic silencing and by RNAseq observe significant misexpression of sex-linked genes that typically are silenced at pachytene. They go on to show that ARID1A is required for exclusion of RNA PolII from the sex body and for limiting promoter accessibility at sex-linked genes, consistent with a meiotic sex chromosome inactivation (MSCI) defect in cKO mice. The authors proceed to investigate the impacts of ARID1A on H3.3 deposition genome-wide. H3.3 is known be regulated by ARID1A and is linked to silencing, and here the authors find that upon loss of ARID1A, overall H3.3 enrichment at the sex body as measured by IF failed to occur, but H3.3 was enriched specifically at transcriptional start sites of sex-linked genes that are normally regulated by ARID1A. The results suggest that ARID1A normally prevents H3.3 accumulation at target promoters on sex chromosomes and based on additional data, restricts H3.3 to intergenic sites. Finally, the authors present data implicating ARID1A and H3.3 occupancy in DSB repair, finding that ARID1A cKO leads to a reduction in focus formation by DMC1, a key repair protein. Overall the paper provides new insights into the process of MSCI from the perspective of chromatin composition and structure, and raises interesting new questions about the interplay between chromatin structure, meiotic silencing and DNA repair.

In general the data are convincing. The conditional KO mouse model has some inherent limitations due to incomplete recombination and the existence of 'escaper' cells that express ARID1A and progress through meiosis normally. This reviewer feels that the authors have addressed this point thoroughly and have demonstrated clear and specific phenotypes using the best available animal model. The data demonstrate that the mutant cells fail to progress past pachytene, although it is unclear whether this specifically reflects pachytene arrest, as accumulation in other stages of Prophase also is suggested by the data in Table 1.

The revised manuscript more appropriately describes the relationship between ARID1A and DNA damage response (DDR) signaling. The authors don't see defects in a few DDR markers in ARID1A CKO cells (including a low resolution assessment of ATR), suggesting that ARID1A may not be required for meiotic DDR signaling. However, as previously noted the data do not rule out the possibility that ARID1A is downstream of DDR signaling, and the authors note the possibility of a role for DDR signaling upstream of ARID1A.

A final comment relates to the impacts of ARID1A loss on DMC1 focus formation and the interesting observation of reduced sex chromosome association by DMC1. The authors additionally assess the related recombinase RAD51 and suggest that it is unaffected by ARID1A loss. However, only a single image of RAD51 staining in the cKO is provided (Fig. S11) and there are no associated quantitative data provided. The data are suggestive and conclusions about the impacts of ARID1A loss on RAD51 must be considered as preliminary until more rigorously assessed.

Reviewer #3 (Public Review):

Summary:

This work describes a new pathway by which malaria parasites, P. falciparum, may regulate their growth and virulence (i.e. their expression of virulence-linked cytoadhesins). This is a topic of considerable interest in the field - does this important parasite sense factor(s) in its host bloodstream and regulate itself accordingly? Several fragments of evidence have come out on this topic in the past decade, showing, for example, reduced parasite growth under calorie restriction (in mice); parasite dormancy in response to amino acid starvation (in culture and in mice), and also reduced virulence in dry-season, low-parasitaemia infections in humans. The molecular mechanisms that may underlie this interesting biology remain only poorly understood.

Here, the authors show that dry-season P. falciparum parasites have reduced expression of Pol3-transcribed tRNAs and ncRNAs that positively regulate virulence gene expression. They link the level of Pol3 activity to PfMaf1, a remnant of the largely-absent nutrient-sensing TOR pathway in this parasite. They propose that in the dry season, human hosts may be calorie-restricted, leading to Maf1 moving to the nucleus and suppressing Pol3, thus downregulated growth and virulence of parasites. The evidence is intriguing and the idea is conceptually elegant.

Strengths:

The use of dry/wet-season field samples from The Gambia is a strength, showing potential real-world relevance. The generation of an inducible knockdown of Maf1 in lab-cultured parasites is also a strength, allowing this pathway to be studied somewhat in isolation.

Weaknesses:

(1) The signals upstream of Maf1 remain rather a black box. 4 are tested - heatshock and low-glucose, which seem to suppress ALL transcription; low-Isoleucine and high magnesium, which suppress Pol3. Therefore the authors use Mg supplementation throughout as a 'starvation type' stimulus. They do not discuss why they didn't use amino acid limitation, which could be more easily rationalised physiologically. It may for experimental simplicity (no need for dropout media) but this should be discussed, and ideally sample experiments with low-IsoLeu should be done too, to see if the responses (e.g. cytoadhesion) are all the same.

(2) The proteomics, conducted to seek partners of Maf1, is probably the weakest part. From Fig S4 it is clear that the proteins highlighted in the text are highly selected (as ones that might be relevant, e.g. phosphatases), but many others are more enriched. It would be good to see a) the top hits from the whole list provided as a short table within the main proteomics figure, along with the GO terms that actually came top in enrichment; b) the whole list provided as a supp. spreadsheet for easy re-analysis, rather than a PDF which cannot be easily re-used.

(3) Fig 3 shows the Maf1-low line has very poor growth after only 5 days but it is stated that no dead parasites are seen even after 8 cycles and the merozoites number is down only ~18 to 15... is this too small to account for such poor growth (~5-fold reduced in a single cycle, day 3-5)? It would additionally be interesting to see a cell-cycle length assessment and invasion assay, to see of Maf1-low parasite have further defects in growth.

Other weaknesses, which are more restricted but were not addressed in revision, are highlighted below:

Fig S1B - The downregulation of RNAPol3 transcripts caused by a commercial Pol3 inhibitor is pretty weak - mostly non-significant. The authors might comment on why they think this is, when interfering with PfMaf1 evidently has a greater effect.

Fig 2D: the legend states ' Expressed transcripts from three replicates between control and addition of MgCl2 that are significantly up-regulated are highlighted in red while significantly down-regulated RNA Pol III genes are highlighted in blue (FDR corrected p-value of <0.05) and a FC {greater than or equal to}{plus minus} 1.95) with examples listed as text'. This isn't very clear. The authors could clarify whether they took ALL (Pol3 or not) upregulated genes to show in red, but only putative Pol3-regulated genes to show in blue? If so, why? Or did they take all significantly downregulated genes, and found they were all annotated as pol3 transcribed? (I cannot see any dots that are not blue. If there are some, a clearer figure is needed?)

Line 227: 'PfMaf1 levels were shown to decrease by approximately 57% in total extracts after one cycle' - the provenance of this very precise percentage isn't clear (it does not appear on the figure). Is it densitometry of a western blot? And if so, is it an average of the 3 replicates that are stated in the legend (but not shown), or from the single example blot shown in Figure 3?

Fig 4A: the western blot, as shown, lacks controls, both for loading and for completeness of cyto/nuclear fractionation. To avoid confusion, these should be shown in the main figure, as is standard in the field, rather than separately in a supp figure. Ideally, 3 repeats should be done, with densitiometry quantification.