Reviewer #1 (Public Review):

The goal of the authors is to use whole-exome sequencing to identify genomic factors contributing to asthenoteratozoospermia and male infertility. Using whole-exome sequencing, they discovered homozygous ZMYND12 variants in four unrelated patients. They examined the localization of key sperm tail components in sperm from the patients. To validate the findings, they knocked down the ortholog in Trypanosoma brucei. They further dissected the complex using co-immunoprecipitation and comparative proteomics with samples from Trypanosoma and Ttc29 KO mice. They concluded that ZMYND12 is a new asthenoteratozoospermia-associated gene, bi-allelic variants of which cause severe flagellum malformations and primary male infertility.

The major strengths are that the authors used the cutting-edge technique, whole-exome sequencing, to identify genes associated with male infertility, and used a new model organism, Trypanosoma brucei to validate the findings, together with other high-throughput tools, including comparative proteomics to dissect the protein complex essential for normal sperm formation/function. The major weakness is that limited samples could be collected from the patients for further characterization by other approaches, including western blotting and TEM.

In general, the authors achieved their goal, and the conclusion is supported by their results. The findings not only provide another genetic marker for the diagnosis of asthenoteratozoospermia but also enrich the knowledge of cilia/flagella.

Reviewer #1 (Public Review):

It has been shown previously that maternal aging in mice is associated with an increase in accumulation of damaged mitochondria and activation of parkin-mediated autophagy (see DOI: 10.1080/15548627.2021.1946739). It has also been shown that C-natriuretic peptide (CNP) regulates oocyte meiotic arrest and that its use during in vitro oocyte maturation can improve parameters associated with decreased oocyte quality. Here the authors tested whether use of CNP treatment in vivo could improve oocyte quality and fertility of aged mice, for which they provided convincing evidence. They also attempted to determine how CNP improves oocyte developmental competence. They showed a correlation between CNP use in vivo and the appearance (and some functional qualities) of cytoplasmic organelles more closely approximating those of oocytes from young mice. However, this correlation could not be interpreted to imply causation. Additional experiments performed using CNP during in vitro maturation were not properly controlled and so are not possible to interpret.

A strength of the manuscript is that the authors use an in vivo treatment to improve oocyte quality rather than just using CNP during oocyte maturation in vitro as has been done previously. This strategy provides more potential for improving oocyte quality - over the course of oocyte growth and maturation - rather than just the final few hours of maturation alone. This strategy also has the potential to be translated into a more generally useful clinical therapeutic method that using CNP during in vitro maturation. However, it is difficult to glean information regarding how CNP might have its effects in vivo. A range of models are used in the manuscript with a mix of in vivo studies with in vitro experiments, which results in some disconnect between systemic CNP and its reported intrafollicular action as well as in the short-term versus longer-term actions of CNP on oocyte quality. Specifically, CNP was shown to be reduced in the plasma of aged mice, but this was not shown in the granulosa cells, which are the reported source of CNP that acts on oocytes. Whether the ovarian source of CNP is reduced in aged females was not demonstrated, and CNP is not known to act on oocytes through an endocrine effect. In vivo treatments with CNP by i.p. injection were performed, but the dose (120 ug/kg) and time (14 days) of treatment were not validated by any prior experiments to give them physiological relevance.

Weaknesses:

1. The Results section is not always clear regarding what CNP treatment was done - in vivo injections or in vitro maturation. For example, what is the difference, if any, between Figures 2C-D and Figures S2A-B?

This remains unclear in the revised manuscript.

2. Immature oocytes from aged females (~1 year) were treated with a two-step culture system with a pre-IVM step with CNP. Controls included oocytes from young (6-8 weeks) females or oocytes from aged females treated by conventional IVM. The description of these methods suggests that control oocytes did not receive an equivalent pre-IVM culture, hence the relevance of comparisons of CNP-treated versus control oocyte is questionable. This concern has not been addressed in the revised manuscript. It was observed that aged oocytes pre-cultured in CNP improved polar body extrusion rates and meiotic spindle morphology compared to oocytes in conventional IVM, as has been well established. The description of statistical methods does not make clear whether the PBE rate in CNP-treated old oocytes remained significantly lower than young controls.

This concern has not been addressed in the revised manuscript.

3. The main effect of the CNP 2-week treatment appears to be increasing the number of follicles that grow into secondary and antral stages, but there is no attempt made to discover the mechanism by which this occurs and therefore to understand why there might be an increase in the number of ovulated eggs, quality of the eggs, and litter size. It is also not clear how an intraperitoneal injection can guarantee its effectiveness because the half-life of CNP is very short, only a few minutes.

This concern has not been addressed in the revised manuscript.

4. Meiotic spindle morphology, as well as a number of putative markers of cytoplasmic maturation are also suggested to be improved after pre-culture with CNP. In each case a subjective interpretation of "normal" morphology of these markers is derived from observations of the young controls and the proportions of oocytes with normal or abnormal appearance is evaluated. However, parameters that define abnormal patterns of these markers appear to be subjective judgements, and whether these morphological patterns can be mechanistically attributed to the differences in developmental potential cannot be concluded.

This concern has not been addressed in the revised manuscript.

5. In addition to the localization patterns of mitochondria, the mitochondrial membrane potential, oocyte ATP content and ROS levels were assessed through more objective quantitative methods. These are well known to be defective in oocytes of aged females and CNP treatment improved these measures. Mitochondrial dysfunction is the most obvious link between oocyte apoptosis, autophagy, cytoplasmic organelle miss-localization and aberrant spindle morphology. Among the most intriguing results is the finding that CNP mediated a cAMP-dependent protein kinase (PKA) dependent reduction in mitochondrial autophagy mediators PINK and Parkin and reduced the recruitment of Parkin to mitochondria in oocytes. However, it may not be possible to directly link this observation to the improvements in IVM oocyte quality, since PINK/Parkin assessments were performed in oocytes from cultured follicles treated with CNP for 6 days.

This weakness has not been addressed in the revised manuscript.

6. The gold standard assay for oocyte quality is embryo transfer and live birth. The authors assessed the impact of maturing oocytes in vitro in the presence of CNP on oocyte quality by less robust assays (e.g., preimplantation embryo development in vitro), so the impact on oocyte quality is less certain.

This weakness has not been addressed in the revised manuscript.

7. The numbers of embryos should have been corrected for the number of eggs fertilized as a starting point so that the percentage that developed to each stage could be expressed as a percentage of successfully fertilized eggs rather than overall percentages. As currently shown in the Figures and described in the Legend, there is no information regarding what the percentage on the y-axis means. For example, does Figure 4B show the number of 2C embryos divided by the number of eggs inseminated? Or is it divided by the number of successfully fertilized eggs, and if so, how was that assessed?

There is no additional information provided in the revised manuscript to address these concerns.

8. When fewer eggs are fertilized, the numbers of embryos per group are lower and so the impact of culturing multiple embryos together is lost. As a result, it is possible that culture conditions rather than oocyte quality drove the differences in the numbers of embryos that achieved each stage of development.

This concern has not been addressed in the revised manuscript. Similar numbers of oocytes were cultured together, but not similar numbers of fertilized oocytes, or embryos.

9. Not all claims in the Discussion are supported by the evidence provided. For example, "In addition, the findings demonstrated that CNP improved cytoplasmic maturation events by maintaining normal CG, ER and Golgi apparatus distribution and function in aged oocytes" but it was never demonstrated that the altered distribution had any functional impact.

This concern has not been addressed in the revised manuscript.

10. Incompleteness and errors in the Methods section reduce confidence in many of the results reported.

This concern has not been addressed in the revised manuscript.

11. The methods used for Statistical Analysis are never explained in either the Methods or the Figure legends. It is unclear whether appropriate analyses were done, and it is frequently unclear what was the sample size and how many times a particular experiment was repeated. These weaknesses detract from confidence in the data.

This concern has not been addressed adequately in the revised manuscript.

Reviewer #1 (Public Review):

This manuscript describes a series of experiments documenting trophic egg production in a species of harvester ant, Pogonomyrmex rugosus. In brief, queens are the primary trophic egg producers, there is seasonality and periodicity to trophic egg production, trophic eggs differ in many basic dimensions and contents relative to reproductive eggs, and diets supplemented with trophic eggs had an effect on the queen/worker ratio produced (increasing worker production).

The manuscript is very well prepared and the methods are sufficient. The outcomes are interesting and help fill gaps in knowledge, both on ants as well as insects, more generally. More context could enrich the study and flow could be improved.

Reviewer #1 (Public Review):

The authors report a study, where they have sequenced whole genomes of four individuals of an extinct species of butterfly from western North America (Glaucopsyche xerces), along with seven genomes of a closely related species (Glaucopsyche lygdamus), mainly from museum specimens, several to many decades old. They then compare these fragmented genomes to a high-quality, chromosome-level assembly of a genome of a European species in the same genus (Glaucopsyche alexis). They find that the extinct species shows clear signs of declining population sizes since the last glacial period and an increase in inbreeding, perhaps exacerbating the low viability of the populations and contributing to the extinction of the species.

The study really highlights how museum specimens can be used to understand the genetic variability of populations and species in the past, up to a century or more ago. This is an incredibly valuable tool, and can potentially help us to quickly identify whether current populations of rare and declining species are in danger due to inbreeding, or whether at least their genetic integrity is in good condition and other factors need to be prioritised in their conservation. In the case of extinct species, sequencing museum specimens is really our only window into the dynamics of genomic variability prior to extinction, and such information can help us understand how genetic variation is related to extinction.

I think the authors have achieved their goal admirably, they have used a careful approach to mapping their genomic reads to a related species with a high-quality genome assembly. They might miss out on some interesting genetic information in the unmapped reads, but by and large, they have captured the essential information on genetic variability within their mapped reads. Their conclusions on the lower genetic variability in the extinct species are sound, and they convincingly show that Glaucopyche xerces is a separate species to Glaucopsyche lygdamus (this has been debated in the past).

Reviewer #1 (Public Review):

First, I agree with the authors of this manuscript that conformational changes in the XFEL structures with 2.8 A resolution are not reliable enough for demonstrating the subtle changes in the electron transfer events in this bacterial photosynthesis system. Actually, the data statistics in the paper by Dods et al. showed that the high-resolution range of some of the XFEL datasets may include pretty high noise (low CC1/2 and high Rsplit) so the comparison of the subtle conformational changes of the structures is problematic.

The manuscript by Gai Nishikawa investigated time-dependent changes in the energetics of the electron transfer pathway based on the structures by Dods et al. by calculating redox potential of the active and inactive branches in the structures and found no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures published by Dods, R.et al. (2021). This study provided validation for the interpretation of the structures of those electron-transferring proteins.

The paper was well prepared.

Comments on latest version:

The revisions the authors have made have improved the manuscript.

Reviewer #1 (Public Review):

Summary: This paper performs fine-mapping of the silkworm mutants bd and its fertile allelic version, bdf, narrowing down the causal intervals to a small interval of a handful of genes. In this region, the gene orthologous to mamo is impaired by a large indel, and its function is later confirmed using expression profiling, RNAi, and CRISPR KO. All these experiments are convincingly showing that mamo is necessary for the suppression of melanic pigmentation in the silkworm larval integument.

The authors also use in silico and in vitro assays to probe the potential effector genes that mamo may regulate.

Strengths: The genotype-to-phenotype workflow, combining forward (mapping) and reverse genetics (RNAi and CRISPR loss-of-function assays) linking mamo to pigmentation are extremely convincing.

Weaknesses:

1) The last section of the results, entitled "Downstream target gene analysis" is primarily based on in silico genome-wide binding motif predictions.<br /> While the authors identify a potential binding site using EMSA, it is unclear how much this general approach over-predicted potential targets. While I think this work is interesting, its potential caveats are not mentioned. In fact the Discussion section seems to trust the high number of target genes as a reliable result. Specifically, the authors correctly say: "even if there are some transcription factor-binding sites in a gene, the gene is not necessarily regulated by these factors in a specific tissue and period", but then propose a biological explanation that not all binding sites are relevant to expression control. This makes a radical short-cut that predicted binding sites are actual in vivo binding sites. This may not be true, as I'd expect that only a subset of binding motifs predicted by Positional Weight Matrices (PWM) are real in vivo binding sites with a ChIP-seq or Cut-and-Run signal. This is particularly problematic for PWM that feature only 5-nt signature motifs, as inferred here for mamo-S and mamo-L, simply because we can expect many predicted sites by chance.

2) The last part of the current discussion ("Notably, the industrial melanism event, in a short period of several decades ... a more advanced self-regulation program") is flawed with important logical shortcuts that assign "agency" to the evolutionary process. For instance, this section conveys the idea that phenotypically relevant mutations may not be random. I believe some of this is due to translation issues in English, as I understand that the authors want to express the idea that some parts of the genome are paths of least resistance for evolutionary change (e.g. the regulatory regions of developmental regulators are likely to articulate morphological change). But the language and tone is made worst by the mention that in another system, a mechanism involving photoreception drives adaptive plasticity, making it sound like the authors want to make a Lamarckian argument here (inheritance of acquired characteristics), or a point about orthogenesis (e.g. the idea that the environment may guide non-random mutations).<br /> Because this last part of the current discussion suffers from confused statements on modes and tempo of regulatory evolution and is rather out of topic, I would suggest removing it.

In any case, it is important to highlight here that while this manuscript is an excellent genotype-to-phenotype study, it has very few comparative insights on the evolutionary process. The finding that mamo is a pattern or pigment regulatory factor is interesting and will deserve many more studies to decipher the full evolutionary study behind this Gene Regulatory Network.

Minor Comment :

The gene models presented in Figure 1 are obsolete, as there are more recent annotations of the Bm-mamo gene that feature more complete intron-exon structures, including for the neighboring genes in the bd/bdf intervals. It remains true that the mamo locus encodes two protein isoforms.<br /> An example of the Bm-mamo locus annotation, can be found at : https://www.ncbi.nlm.nih.gov/gene/101738295<br /> RNAseq expression tracks (including from larval epidermis) can be displayed in the embedded genome browser from the link above using the "Configure Tracks" tool.

Based on these more recent annotations, I would say that most of the work on the two isoforms remains valid, but FigS2, and particularly Fig.S2C, need to be revised.

Reviewer #1 (Public Review):

This manuscript presents an important study that contributes to our understanding of the reliability of ancient environmental DNA (aeDNA) extracted from sediment cores. The authors address the potential biases and challenges associated with using aeDNA to infer past ecosystems, specifically focusing on the case of mammoths and woolly rhinoceroses in the Yamal peninsula, West Siberia.

The introduction provides an overview of the significance of sedimentary deposits as archives of past ecosystem changes and illustrates the remarkable insights gained from previous studies using aeDNA, highlighting its potential for reconstructing paleoecology, phylogeography, and understanding extirpation and extinction events of keystone taxa. The authors then report the detection of DNA and near complete mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals in the sampled sediment cores (which are dated to the last few centuries). The authors then employed additional methods to confirm the presence of ancient DNA from mammoths in these sediment cores. Conventional PCR and Sanger sequencing of a mammoth COI fragment confirmed the amplification of mammoth DNA. Mammal metabarcoding and droplet digital PCR (ddPCR) further supported the detection of mammoth DNA in both cores.

The hybridisation enrichment experiment results showed high read counts assigned to Mammuthus, ranging from 2,852 to 72,919 reads per library in core LK-001. Negative controls did not produce any reads assigned to mammals, indicating the absence of contamination. The study also revealed the presence of woolly rhinoceros sequences in the sediment cores, with 12 out of 23 libraries producing more than 100 reads assigned to woolly rhinoceros. The total number of woolly rhinoceros reads was 2,737, and the cumulative mitogenome coverage reached 44%.

The authors carefully addressed the incongruity between the temporal occurrence of these extinct species and the presence of their DNA in recent sediments. They proposed several mechanisms that could explain the recovery of Pleistocene megafaunal DNA in the sediment cores. The minor amount of ancient DNA post mortem damage observed in the mammoth sequences indicates exceptional preservation, consistent with an origin from (recent) permafrost. The dynamics of permafrost thawing and redeposition in the study area provide a plausible explanation for the presence of ancient DNA in the sediments. The authors discuss potential mechanisms for the redistribution of Late Pleistocene material in the sediments, including thermo-denudation processes, methane emissions from degrading permafrost, and the formation of taliks and methane seepage. These processes can disturb the stratigraphy of lake sediments and potentially mix ancient material within the modern sediments. I believe the conclusions are supported by the data and the manuscript is well-written and clear to follow for the reader.

Reviewer #1 (Public Review):

This manuscript by Neininger-Castro and colleagues presents a novel automatic image analysis method for assessing sarcomeres, the basic units of myofibrils and validates this tool in a couple of experimental approaches that interfere with sarcomere assembly in iPSC-cardiomyocytes (iPSC-CM).

Automatic quantification of sarcomeres is definitely something that is useful to the field. I am surprised that there is no reference in the manuscript to SarcTrack, published by Toepfer and colleagues in 2019 (PMID 30700234), which has exactly the same purpose. The advantage of the image analysis software presented in the current manuscript appears to me to be that it can cover both mature sarcomeres and nascent sarcomeres in premyofibrils effectively.

Reviewer #1 (Public Review):

The authors use electrophysiological and behavioral measurements to examine how animals could reliably determine odor intensity/concentration across repeated experiences. Because stimulus repetition leads to short-term adaptation evidenced by reduced overall firing rates in the antennal lobe and firing rates are otherwise concentration-dependent, there could be an ambiguity in sensory coding between reduced concentration or more recent experience. This would have a negative impact on the animal's ability to generate adaptive behavioral responses that depend on odor intensities. The authors conclude that changes in concentration alter the constituent neurons contributing to the neural population response, whereas adaptation maintains the 'activated ensemble' but with scaled firing rates. This provides a neural coding account of the ability to distinguish odor concentrations even after extended experience. Additional analyses attempt to distinguish hypothesized circuit mechanisms for adaptation but are inconclusive. A larger point that runs through the manuscript is that overall spiking activity has an inconsistent relationship with behavior and that the structure of population activity may be the more appropriate feature to consider.

To my knowledge, the dissociation of effects of odor concentration and adaptation on olfactory system population codes was not previously demonstrated. This is a significant contribution that improves on any simple model based on overall spiking activity. The primary result is most strikingly supported by visualization of a principal components analysis in Figure 4. However, there are some weaknesses in the data and analyses that limit confidence in the overall conclusions.

1) Behavioral work interpreted to demonstrate discrimination of different odor concentrations yields inconsistent results. Only two of the four odorants follow the pattern that is emphasized in the text (Figure 1F). Though it's a priori unlikely that animals are incapable of distinguishing odor concentrations at any stage in adaptation, the evidence presented is not sufficient to reach this conclusion.<br /> 2) While conclusions center on concepts related to the combination of activated neurons or the "active ensemble", this specific level of description is not directly demonstrated in any part of the results. We see individual neural responses and dimensional reduction analyses, but we are unable to assess to what extent the activated ensemble is maintained across experience.<br /> 3) There is little information about the variance or statistical strength of results described at the population level. While the PCA presents a compelling picture, the central point that concentration changes and adaptation alter population responses across separable dimensions is not demonstrated quantitatively. The correlation analysis that might partially address this question is presented to be visually interpreted with no additional testing.<br /> 4) Results are often presented separately for each odor stimulus or for separate datasets including two odor stimuli. An effort should be made to characterize patterns of results across all odor stimuli and their statistical reliability. This concern arises throughout all data presentations.<br /> 5) The relevance of the inconclusive analysis of inferred adaptation mechanisms in Figure 2d-f and the single experiment including a complex mixture in Figure 7 to the motivating questions for this study are unclear.<br /> 6) Throughout the description of the results, typical standards for statistical reporting (sample size, error bars, etc.) are not followed. This prevents readers from assessing effect sizes and undermines the ability to assign a confidence to any particular conclusion.

DOI: 10.1371/journal.pbio.3002223

Resource: RRID:ZFIN_ZDB-ALT-160830-1

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-160830-1

What is this?

(~14:00) The way to gain massive results is to have massive irrational goals complemented by small reasonable steps or milestones.

Big goals motivate. Big goals give focus and clarity, they are a filter (see Dr. Benjamin Hardy's content); they allow for easy application of the power law.

(~13:00) Koe argues for making information relevant (Dr. Sung always says you must make info relevant) through the learning for the solving of a particular problem, either for a client, your business, or your personal life. Your problem becomes the lense through which you learn.

For self-education this is ideal.

Dr. Sung's approach differs in that he advocates for the creation of relevancy through inquiry (the asking of relational questions) which is also incredibly powerful, however this is more suited to gaining more motivation for forced learning, i.e., in the formal education system.

In addition, Koe's lense is, I think, more of a high-level filter, whereas Sung's questioning is applicable on the content level. Therefore, both approaches could be, and should be, combined into the same overall (self-)educational system.

(~10:20) Koe makes a very, very, very valid point about education:

I quote: "There is one thing that the school system did get right which is consistent, daily education in hopes for a better future. But, schools don't prioritize curiosity, so most people hate learning by the time they graduate." (emphasis added by me)

The larger point that Koe is making is that if we own anything in life, it is our mind; for everything else can be taken away from us; as such, we must spend a significant amount of effort to cultivate it, grow it, care for it, and make it unique.

(~6:07) Koe argues that specializing, or focusing on one aspect only, limits your potential in every conceivable way.

I think I agree, yet I do also think there is a place for that... It depends on the person and what they enjoy. However, I might still be mistaken.

Koe argues for the following trait of a modern renaissance man (or woman):

• Self-Educated
• Pursue Interest
• Leverage the Digital World
• Exercise (physical training)
• Conscious about their health
• Social
• Doing meaningful work
• Acknowledgement of the Spiritual

Dan Koe seems to argue against a specialistic education based on the argument that it is nigh-impossible for a teenager to decide what they want (to be) for the rest of their lives. He also gives the argument that it results in a lack of creativity and underlying knowledge (that which connects the dots, instead of compartmentalization) which would result in abnormal performance.

I can bypass the limitation of the first point by giving the counter-point that when one has an insane amount of metacognition, which can be trained, it does not matter if one changes path later; why? Because one can easily learn the new subject matter and skills.

However, the second point is interesting and I think I agree with it. That said, I think there is a continuum, instead of only two points, between super-specialists and super-generalists. I myself enjoy specializing. And I believe a team of specialists (that can also work together) can accomplish much more than one (or even multiple) generalist.

Ten minutes before sleep, do the following: PRAY

It's a combination of visualization, commitment, and meditation

Request the subconscious through this act of prayer.

Also visualize the outcome and process of that which you aspire to do the following day, and even that which you want to achieve the following month(s). Thus, visualize the following: Big Picture, Milestones, and yourself the next day.

In the morning, process your subconscious state by instead of immediately inputting, you start outputting!

This can be done through journaling.

Put the phone on airplane mode (in addition to blocking blue light) before sleep, for quite some time before sleep, in order to avoid (over)stimulation and the creation of dopamine which negatively impacts (falling a)sleep

What is done right before and right after sleep sets the stage for literally everything.

How you do anything is how you do everything.

Reviewer #1 (Public Review):

The authors put forth the hypothesis that hepatocyte and/or non-parenchymal liver MCT1 may be responsible for physiologic effects (lower body weight gain and less hepatic steatosis) in MCT1 global heterozygote mice. They generate multiple tools to test this hypothesis, which they combine with mouse diets that induce fatty liver, steatohepatitis and fibrosis. Novel findings include that deletion of hepatocyte MCT1 does not change liver lipid content, but increases liver fibrosis. Deletion of hepatic stellate cell (HSC) MCT1 does not substantially affect any liver parameter, but concomitant HSC MCT1 deletion does reverse fibrosis seen with hepatocyte MCT1 knockout or knockdown. In both models, plasma lactate levels do not change, suggesting that liver MCT1 does not substantially affect systemic lactate. In general, the data match the conclusions of the manuscript, and the studies are well-conducted and well-described. Further work would be necessary to dissect mechanism of fibrosis with hepatocyte MCT1, and whether this is due to changes in local lactate (as speculated by the authors) or another MCT1 substrate. This would be important to understand this novel potential cross-talk between hepatocytes and HSCs.

A parallel and perhaps more important advance is the generation of new methodology to target HSC in mice, using modified siRNA and by transduction of AAV9-Lrat-Cre. Both methods would reduce the need to cross floxed mice with the Lrat-Cre allele, saving time and resources. These tools were validated to an extent by the authors, but not sufficiently to ensure that there is no cross-reactivity with other liver cell types. For example, AAV9-Lrat-Cre-transduced MCT1 floxed mice show compelling HSC but not hepatocyte Mct1 knockdown, but other liver cell types should be assessed to ensure specificity. This is particularly important as overall liver Mct1 decreased by ~30% in AAV9-Lrat-Cre-transduced mice, which may exceed HSC content of these mice, especially when considering a 60-70% knockdown efficiency. This same issue also affects Chol-MCT1-siRNA, which the authors demonstrate to affect hepatocytes and HSC, but likely affects other cell types not tested. As this is a new and potentially valuable tool, it would be important to assess Mct1 expression across more non-parenchymal cells (i.e. endothelial, cholangiocytes, immune cells) to determine penetration and efficacy.

Reviewer #1 (Public Review):

The cerebral cortex, or surface of the brain, is where humans do most of their conscious thinking. In humans, the grooves (sulci) and bumps (convolutions) have a particular pattern in a region of the frontal lobe called Broca's area, which is important for language. Specialists study features imprinted on the internal surfaces of braincases in early hominins by casting their interiors, which produces so-called endocasts. A major question about hominin brain evolution concerns when, where, and in which fossils a humanlike Broca's area first emerged, the answer to which may have implications for the emergence of language. The researchers used advanced imaging technology to study the endocast of a hominin (KNM-ER 3732) that lived about 1.9 million years ago (Ma) in Kenya to test a recently published hypothesis that Broca's remained primitive (apelike) prior to around 1.5 Ma. The results are consistent with the hypothesis and raise new questions about whether endocasts can be used to identify the genus and/or species of fossils.

Reviewer #1 (Public Review):

This work describes a structural analysis of the tripartite HipBST toxin-antitoxin (TA) system, which is related to the canonical two-component HipBA system composed of the HipA serine-threonine kinase toxin and the HipB antitoxin. The crystal structure of the kinase-inactive HipBST complex of the Enteropathogenic E. coli O127:H6 was solved and revealed that HipBST forms a hetero-hexameric complex composed of a dimer of HipBST heterotrimers that interact via the HipB subunit. The HipS antitoxin shows a structural resemblance to HipA N-terminal region and the HipT toxin represents to the core kinase domain of HipA, indicating that in HipBST the hipA toxin gene was likely split in two genes, namely hipS and hipT.

The structure also reveals a conserved and essential Trp residue within the HipS antitoxin, which likely prevents the conserved "Gly-rich loop" of HipT from adopting an inward conformation needed for ATP binding. This work also shows that the regulating Gly-rich loop of the HipT toxin contains conserved phosphoserine residues essential for HipT toxicity that are key players within the HipT active site interacting network and which likely control antitoxin binding and/or activity.

Strengths:

The manuscript is well written and the experimental work well executed. It shows that major features of the classical two-component HipAB TA system have somehow been rerouted in the case of the tripartite HipBST. This includes the N-terminal domain of the HipA toxin, which now functions as bona fide antitoxin, and the partly relegated HipB antitoxin, which could only function as a transcription regulator. In addition, this work shows a new mode of inhibition of a kinase toxin and highlights the impact of the phosphorylation state of key toxin residues in controlling the activity of the antitoxin.

Weaknesses:

A major weakness of this work is the lack of data concerning the role of HipB, which likely does not act as an antitoxin. Does it act as a transcriptional regulator of the hipBST operon and to what extent both HipS and HipT contribute to such regulation? These are still open questions.

In addition, there is no in-depth structural comparison between the structure of the HipBST solved in the work and the two recent structures of HipBST from Legionella. This is also a major weakness of this work.

Reviewer #1 (Public Review):

In this manuscript, Knecht, Sirias et al describe toxin-immunity pair from Proteus mirabilis. Their observations suggest that the immunity protein could protect against non-cognate effectors from the same family. They analyze these proteins by dissecting them into domains and constructing chimeras which leads them to the conclusion that the immunity can be promiscuous and that the binding of immunity is insufficient for protective activity.

Strengths:

The manuscript is well written and the data are very well presented and could be potentially interesting. The phylogenetic analysis is well done, and provides some general insights.

Weaknesses:

1) Conclusions are mostly supported by harsh deletions and double hybrid assays. The later assays might show binding, but this method is not resolutive enough to report the binding strength. Proteins could still bind, but the binding might be weaker, transient, and out-competed by the target binding.

2) While the authors have modeled the structure of toxin and immunity, the toxin-immunity complex model is missing. Such a model allows alternative, more realistic interpretation of the presented data. Firstly, the immunity protein is predicted to bind contributing to the surface all over the sequence, except the last two alpha helices (very high confidence model, iPTM>0.8). The N terminus described by the authors contributes one of the toxin-binding surfaces, but this is not the sole binding site. Most importantly, other parts of the immunity protein are predicted to interact closer to the active site (D-E-K residues). Thus, based on the AlphaFold model, the predicted mechanism of immunization remains physically blocking the active site. However, removing the N terminal part, which contributes large interaction surface will directly impact the binding strength. Hence, the toxin-immunity co-folding model suggests that proper binding of immunity, contributed by different parts of the protein, is required to stabilize the toxin-immunity complex and to achieve complete neutralization. Alternative mechanisms of neutralization might not be necessary in this case and are difficult to imagine for a DNAse.

3) Dissection of a toxin into two domains is also not justified from a structural point of view, it is probably based on initial sequence analyses. The N terminus (actually previously reported as Pone domain in ref 21) is actually not a separate domain, but an integral part of the protein that is encased from both sides by the C terminal part. These parts might indeed evolve faster since they are located further from the active site and the central core of the protein. I am happy to see that the chimeric toxins are active, but regarding the conservation and neutralization, I am not surprised, that the central core of the protein fold is highly conserved. However, "deletion 2" is quite irrelevant - it deletes the central core of the protein, which is simply too drastic to draw any conclusions from such a construct - it will not fold into anything similar to an original protein, if it will fold properly at all.

4) Regarding the "promiscuity" there is always a limit to how similar proteins are, hence when cross-neutralization is claimed authors should always provide sequence similarities. This similarity could also be further compared in terms of the predicted interaction surface between toxin and immunity.

Overall, it looks more like a regular toxin-immunity couple, where some cross-reactions with homologues will, of course, be possible, depending on how far the sequences have deviated. Nevertheless, taking all of the above into account, these results do not challenge toxin-immunity specificity dogma.

Joint Public Review

Introduction - Well written and placed within the current trends of unprecedented biodiversity loss, with emphasis in freshwater ecosystems. The authors identify three important points as to why biodiversity action plans have failed. Namely, community changes occur over large spatio-temporal scales and monitoring programs capture a fraction of these long-term dynamics (e.g. few decades) which although good at capturing trends in biodiversity change, they often fail at identifying the drivers of these changes. Additionally, most of these rely on manual sorting of samples, overlooking cryptic diversity, or state-of-the-art techniques such as sedimentary DNA (sedaDNA) which allow studying decade-long dynamics, usually focus on specific taxonomic groups unable to represent community-level changes. Secondly, the authors identify that biodiversity is threatened by multiple factors and are rarely studied in tandem. Finally, the authors stress the need for high-throughput approaches to study biodiversity changes since historically, most conservation efforts rely on highly specialized skills for biodiversity monitoring, and even well studied species have relatively short time series data. The authors identify a model freshwater lake (Lake Ring, Denmark) - suitable due to its well documented history over the last 100 years - to present a comprehensive framework using metabarcoding, chemical analysis and climatic records for identifying past and current impacts on this ecosystem arising from multiple abiotic environmental stressors.

Results - They are brief and should expand some more. Particularly, there are no results regarding metabarcoding data (number of reads, filtering etc.). These details are important to know the quality of the data which represents the bulk of the analyses. Even the supplementary material gives little information on the metabarcoding results (e.g. number of ASVs - whether every ASV of each family were pooled etc.). The drivers of biodiversity change section could be restructured and include main text tables showing the families positively or negatively correlated with the different variables (akin to table S2 but simplified).

Discussion<br /> The discussion is well written identifying first, some of the possible caveats of this study, particularly regarding classification of metabarcoding data, its biases and the possible DNA degradation of ancient sediment DNA. The authors discuss how their results fit to general trends showing how agricultural runoff and temperature drive changes in freshwater functional biodiversity primarily due to their synergistic effects on bioavailability, adsorption, etc. The authors highlight the advantage of using a system-level approach rather than focusing on taxa-specific studies due to their indicator status. Similarly, the authors justify the importance of studying community composition as far back as possible since it reveals unexpected patterns of ecosystem resilience. Lake Ring, despite its partially recovered status, has not returned to its semi-pristine levels of biodiversity and community assemblage. Additionally, including enzyme activity allows to assess functional diversity of the studied environment although reference databases of these pathways are still lacking. Finally, the authors discuss the implications of their findings under a conservation and land management framework suggesting that by combining these different approaches, drivers of biodiversity stressors can be derived with high accuracy allowing for better informed mitigation and conservation efforts.

Reviewer #1 (Public Review):

In the best genetically and biochemically understood model of eukaryotic DNA replication, the budding yeast, Saccharomyces cerevisiae, the genomic locations at which DNA replication initiates are determined by a specific sequence motif. These motifs, or ARS elements, are bound by the origin recognition complex (ORC). ORC is required for loading of the initially inactive MCM helicase during origin licensing in G1. In human cells, ORC does not have a specific sequence binding domain and origin specification is not specified by a defined motif. There have thus been great efforts over many years to try to understand the determinants of DNA replication initiation in human cells using a variety of approaches, which have gradually become more refined over time.

In this manuscript Tian et al. combine data from multiple previous studies using a range of techniques for identifying sites of replication initiation to identify conserved features of replication origins and to examine the relationship between origins and sites of ORC binding in the human genome. The authors identify a) conserved features of replication origins e.g. association with GC-rich sequences, open chromatin, promoters and CTCF binding sites. These associations have already been described in multiple earlier studies. They also examine the relationship of their determined origins and ORC binding sites and conclude that there is no relationship between sites of ORC binding and DNA replication initiation. While the conclusions concerning genomic features of origins are not novel, if true, a clear lack of colocalization of ORC and origins would be a striking finding. However, the majority of the datasets used do not report replication origins, but rather broad zones in which replication origins fire. Rather than refining the localisation of origins, the approach of combining diverse methods that monitor different objects related to DNA replication leads to a base dataset that is highly flawed and cannot support the conclusions that are drawn, as explained in more detail below.

Methods to determine sites at which DNA replication is initiated can be divided into two groups based on the genomic resolution at which they operate. Techniques such as bubble-seq, ok-seq can localise zones of replication initiation in the range ~50kb. Such zones may contain many replication origins. Conversely, techniques such as SNS-seq and ini-seq can localise replication origins down to less than 1kb. Indeed, the application of these different approaches has led to a degree of controversy in the field about whether human replication does indeed initiate at discrete sites (origins), or whether it initiates randomly in large zones with no recurrent sites being used. However, more recent work has shown that elements of both models are correct i.e. there are recurrent and efficient sites of replication initiation in the human genome, but these tend to be clustered and correspond to the demonstrated initiation zones (Guilbaud et al., 2022).

These different scales and methodologies are important when considering the approach of Tian et al. The premise that combining all available data from five techniques will increase accuracy and confidence in identifying the most important origins is flawed for two principal reasons. First, as noted above, of the different techniques combined in this manuscript, only SNS-seq can actually identify origins rather than initiation zones. It is the former that matters when comparing sites of ORC binding with replication origin sites if a conclusion is to be drawn that the two do not co-localise.

Second, the authors give equal weight to all datasets. Certainly, in the case of SNS-seq, this is not appropriate. The technique has evolved over the years and some earlier versions have significantly different technical designs that may impact the reliability and/or resolution of the results e.g. in Foulk et al. (Foulk et al., 2015), lambda exonuclease was added to single stranded DNA from a total genomic preparation rather than purified nascent strands), which may lead to significantly different digestion patterns (ie underdigestion). Curiously, the authors do not make the best use of the largest SNS-seq dataset (Akerman et al., 2020) by ignoring these authors separation of core and stochastic origins. By blending all data together any separation of signal and noise is lost. Further, I am surprised that the authors have chosen not to use data and analysis from a recent study that provides subsets of the most highly used and efficient origins in the human genome, at high resolution (Guilbaud et al., 2022).

References:

Akerman I, Kasaai B, Bazarova A, Sang PB, Peiffer I, Artufel M, Derelle R, Smith G, Rodriguez-Martinez M, Romano M, Kinet S, Tino P, Theillet C, Taylor N, Ballester B, Méchali M (2020) A predictable conserved DNA base composition signature defines human core DNA replication origins. Nat Commun, 11: 4826

Foulk MS, Urban JM, Casella C, Gerbi SA (2015) Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res, 25: 725-735

Guilbaud G, Murat P, Wilkes HS, Lerner LK, Sale JE, Krude T (2022) Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation. Nucleic Acids Res, 50: 7436-7450

Reviewer #1 (Public Review):

The authors sought to resolve the coordinated functions of the two muscles that primarily power flight in birds (supracoracoideus and pectoralis), with particular focus on the pectoralis. Technology has limited the ability to resolve some details of pectoralis function, so the authors developed a model that can make accurate predictions about this muscle's function during flight. The authors first measured aerodynamic forces, wing shape changes, and pectoralis muscle activity in flying doves. They used cutting-edge techniques for the aerodynamic and wing shape measurements and they used well-established methods to measure activity and length of the pectoralis muscle. The authors then developed two mathematical models to estimate the instantaneous force vector produced by the pectoralis throughout the wing stroke. Finally, the authors applied their mathematical models to other-sized birds in order to compare muscle physiology across species.

The strength of the methods is that they smoothly incorporate techniques from many complementary fields to generate a comprehensive model of pectoralis muscle function during flight. The high-speed structured-light technique for quantifying surface area during flight is novel and cutting-edge, as is the aerodynamic force platform used. These methods push the boundaries of what has historically been used to quantify their respective aspects of bird flight and their use here is exciting. The methods used for measuring muscle activation and length are standard in the field. Together, these provide both a strong conceptual foundation for the model and highlight its novelty. This model allows for estimations of muscle function that are not feasible to measure in live birds during flight at present. The weakness of this approach is that it relies heavily on a series of assumptions. While the research presented in this paper makes use of powerful methods from multiple fields, those methods each have assumptions inherent to them that simplify the biological system of study. This reduction in the complexity of phenomena allows the specific measurements to be made. In joining the techniques of multiple fields to study the greater complexity of the phenomenon of interest, the assumptions are all incorporated also. Furthermore, assumptions are inherent to mathematical modeling of biological phenomena. That being said, the authors acknowledge and justify their assumptions at each step and their model seems to be quite good at predicting muscle function.

Indeed, the authors achieve their aims. They effectively integrate methods from multiple disciplines to explore the coordination and function of the pectoralis and supracoracoideus muscles during flight. The conclusions that the authors derive from their model address the intended research aim.

The authors demonstrate the value of such interdisciplinary research, especially in studying complex behaviors that are difficult or infeasible to measure in living animals. Additionally, this work provides predictions for muscle function that can be tested empirically. These methods are certainly valuable for understanding flight but also have implications for biologists studying movement and muscle function more generally.

Reviewer #1 (Public Review):

In this paper, Schalcher et al. examined how barn owls' landing force affects their hunting success during two hunting strategies: strike hunting and sit-and-wait hunting. They tracked tens of barn owls that raised their nestlings in nest boxes and utilized high-resolution GPS and acceleration loggers to monitor their movements. In addition, camcorders were placed near their nest boxes and used to record the prey they brought to the nest, thus measuring their foraging success.

This study generated a unique dataset and provided new insights into the foraging behavior of barn owls. The researchers discovered that the landing force during hunting strikes was significantly higher compared to the sit-and-wait strategy. Additionally, they found a positive relationship between landing force and foraging success during hunting strikes, whereas, during the sit-and-wait strategy, there was a negative relationship between the two. This suggests that barn owls avoid detection by generating a lower landing force and producing less noise. Furthermore, the researchers observed that environmental characteristics affect barn owls' landing force during sit-and-wait hunting. They found a greater landing force when landing on buildings, a lower landing force when landing on trees, and the lowest landing force when landing on poles. The landing force also decreased as the time to the next hunting attempt decreased. These findings collectively suggest that barn owls reduce their landing force as an acoustic camouflage to avoid detection by their prey.

The main strength of this work is the researchers' comprehensive approach, examining different aspects of foraging behavior, including high-resolution movement, foraging success, and the influence of the environment on this behavior, supported by impressive data collection. The weakness of this study is that the results only present a partial biological story contained within the data. The focus is on acoustic camouflage without addressing other aspects of barn owls' foraging strategy, leaving the reader with many unanswered questions. These include individual differences, direct measurements of owls' fitness, a detailed analysis of the foraging strategy of males and females, and the collective effort per nest box. However, it is possible that these data will be published in a separate paper.

The results presented support the authors' conclusion that lower landing force during sit-and-wait hunting increases hunting success, likely due to a decreased probability of detection by their prey, resulting in acoustic camouflage. The authors also argue that hunting success is crucial for survival, and thus, acoustic camouflage has a direct link to fitness. While this statement is reasonable, it should be presented as a hypothesis, as no direct evidence has been provided here. However, since information about nestling survival is typically monitored when studying behavior during the breeding period, the authors' knowledge of the effect of acoustic camouflage on owls' fitness can probably be provided. Furthermore, it will be interesting to further examine the foraging strategies used by different individuals during foraging, the joint foraging success of both males and females within each nest box, and the link between landing force and foraging success if the data are available. However, even without this additional analysis on survival, this paper provides an unprecedented dataset and the first measurement of landing force during hunting in the wild. It is likely to inspire many other researchers currently studying animal foraging behavior to explore how animals' movements affect foraging success.

Reviewer #1 (Public Review):

After revision, the manuscript is clearly improved and I thank the authors for their efforts. Yet, two contentious issues remain.

Firstly, I am skeptical whether the circularity issue has been resolved.

The authors equate uncertainty in the outcome of interactions with social complexity and they then diagnose for these three species that higher social complexity correlates with higher communicative complexity. Yet, there is still an inherent link between the occurrence of signals and other behaviours that allow the authors to determine the outcome of an interaction.

I do agree with the authors' conclusion that the three species vary in terms of the predictability of their signaling behaviour and the outcome of interactions. I just think the observed link between the two is not very surprising or informative, but rather inevitable.

Secondly, I am still not convinced that visual communication is more prevalent in situations with higher predation pressure. There are two reasons: relying on visual communication requires that the recipients, typically one's group members, are actually looking at the signaler when they produce the signal. The vocal-auditory channel in contrast, has a much higher potential to reach all recipients, even when visual communication in impaired. In addition, the idea that predators use acoustic signals to single out individuals and preferentially attack them, is poorly corroborated by data, especially for terrestrial predators. In contrast, there is ample evidence that prey species direct their calls at terrestrial predators (mobbing calls against snakes, antelope vigorously snorting against lions and leopards). See also this paper by Griesser (PMID 23941356).

Reviewer #1 (Public Review):

There are a number of outstanding questions concerning how cohesin turnover on DNA is controlled by various accessory factors and how such turnover is controlled by post-translational modification. In this paper, Nasmyth et al. perform a series of AlphaFold structure predictions that aim to address several of these outstanding questions. Their structure predictions suggest that the release factor WAPL forms a ternary complex with PDS5 and SA/SCC3. This ternary complex appears to be able to bind the N-terminal end of SCC1, suggesting how formation of such a complex could stabilize an open state of the cohesin ring. Additional calculations suggest how the Eco/ESCO acetyltransferases and Sororin engage the SMC3 head domain presumably to protect against WAPL-mediated release.

This work thus demonstrates the power of AF prediction methods and how they can lead to a number of interesting and testable hypotheses that can transform our understanding of cohesin regulation. These findings require orthogonal experimental validation, but authors argue convincingly that such validation should not be a pre-requisite to publication.

In their revised version, the authors did not systematically include model confidence scores, and it therefore remains difficult for the reader to evaluate the reliability of the models obtained. The authors correctly point out that such metrics are available on Figshare. It is therefore possible to obtain such information. The caveat is that it remains to the user to identify and extract the relevant information. While they claim that they have labeled N- and C-termini in their figures, no such labeling can be seen in the revised version. Addition of such labels, at least for some of the figures, would help the user to navigate the models.

Also, PAE plots still contain chain labels (A,B,C etc.) and it is not always clear which protein is being referred to. Which segment does the reader need to focus on? The authors claim that PAE plots are now amended but no such changes can be seen. At least for the key models proposed, the authors should have facilitated access and help readers interpret the different plots/models. While it is possible to load the different PDB files from Figshare, the current version still requires that the reader then works out what segments are used and how they correspond to chain labels shown.

It is exciting to see AF-multimer predictions being applied to cohesin. As some of the reported interactions are not universally conserved and some involve relatively small interfaces the possibility arises that these interfaces show poor or borderline confidence scores. As some of these interfaces map to mutants that have previously been obtained by hypothesis-free genetic screens and mutational analyses, they appear nevertheless valid. Thus, an important point to make is that even interfaces that show modest confidence scores may turn out to be valid while others may be not.

Reviewer #1 (Public Review):

Cell death plays a critical role on regulating organogenesis. During tooth morphogenesis, apoptosis of embryonic dental tissue plays critical roles on regulating tooth germ development. The current study focused on ferroptosis, another way of cell death which has rarely been investigated in tooth development, and showed it may also play an important role on regulating the tooth dimension. The topic is novel and interesting, but the experimental design has many flaws which significantly compromised the study.

1. The entire study was based on ex vivo tooth germ explant culture. Mandibular tooth germs of E15.5 (bell stage) were isolated for ex vivo culture. Most tooth germ explant culture experiments were actually using tooth germ of much earlier stages (E11.5-E13.5) for organ culture. After E16.5, both the large size and initially formed enamel/dentin could prevent nutrition from penetrating inside. Also, using tooth germ of earlier stage will help identify impact of ferroptosis upon early tooth development.<br /> 2. Due to limited penetration, the ex vivo culture in the study lasted for no more than 5 days. I would recommend the authors to perform kidney capsule transplantation as an alternative approach, which can support tooth germ development much longer even into root formation.<br /> 3. The major justification of using tooth germ ex vivo culture as the model in the study was to "conduct high-throughput analysis". However, the study could hardly be qualified as a high-throughput analysis. I would recommend the authors perform RNA sequencing for comparing tooth germs before/after erastin treatment. Such experiments won't take too much time or resource.<br /> 4. Although the study mostly used molars as the model, the in vivo iron concentration was only demonstrated on incisors, but not molars (Figure 1).<br /> 5. Phenotype analysis in Figure 2 is too superficial. Only dimensional information was provided. Cusps number, cusps distribution pattern and rooth/furcation formation were not evaluated. Differentiation of amelobast/odontoblast was not evaluated. The proliferation rate in the dental epithelium/mesenchyme was not analyzed.<br /> 6. Low magnification images should be included in Figure 3 to display the entire tooth germs.<br /> 7. In Figure 4, does ferroptotic inhibitor eliminate the iron accumulation in the tooth germ? How about the expression level of several target genes shown in Figure 3?<br /> 8. The manuscript has many typos and grammar mistakes. All "submandibular" should be simply "mandibular". "eastin" should be "erastin" (line 92). "partly" should be "partially" (line 611).

Reviewer #1 (Public Review):

Comments on the original submission:

Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) has shown that PIP5pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulate the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch".

Comments on revised manuscript:

In this revised manuscript, Touray et al. have responded to reviewers' comments with some revisions satisfactorily. However, the authors still haven't addressed some key scientific rigor issues, which are listed below:

1. It is critical to clearly state whether the observations are made for the endogenous WT protein or the tagged protein. It is good that the authors currently clearly indicate the results observed in vivo are for the RAP1-HA protein. However, this is not as clearly stated for in vitro EMSA analyses. In addition, in discussion, the authors simply assumed that the c-terminally tagged RAP1 behaves the same as WT RAP1 and all conclusions were made about WT RAP1.

There are two choices here. The authors can validate that RAP1-HA still retains RAP1's essential function as a sole allele in T. brucei cells (as was recommended previously). Indeed, HA-tagged RAP1 has been studied before, but it is the N-terminally HA-tagged RAP1 that has been shown to retain its essential functions. Adding the HA tag to the C-terminus of RAP1 may well cause certain defects to RAP1. For example, N-terminally HA-tagged TERT does not complement the telomere shortening phenotype in TERT null T. brucei cells, while C-terminally GFP-tagged TERT does, indicating that HA-TERT is not fully functional while TERT-GFP likely has its essential functions (Dreesen, RU thesis). Although RAP1-HA behaves similar to WT RAP1 in many ways, it is still not fully validated that this protein retains essential functions of RAP1. By the way, it has been published that cells lacking one allele of RAP1 behave as WT cells for cell growth and VSG silencing (Yang et al. 2009, Cell; Afrin et al. 2020, mSphere). In addition, although RAP1 may interact with TRF weakly, the interaction is direct, as shown in yeast 2-hybrid analysis in (Yang et al. 2009, Cell).

Alternatively, if the authors do not wish to validate the functionality of RAP1-HA, they need to add one paragraph at the beginning of the discussion to clearly state that RAP1-HA may not behave exactly as WT RAP1. This is important for readers to better interpret the results. In addition, the authors need to tune down the current conclusions dramatically, as all described observations are made on RAP1-HA but not the WT RAP1.

For a similar reason, the current EMSA results truly reflect how C-terminally His6-tagged RAP1 and RAP1 fragments behave. If the authors choose not to remove the His6 tag, it is essential that they use "RAP1-His6" to refer to these recombinant proteins. It is also essential for the authors to clearly state in the discussion that the tagged RAP1 fragments bind DNA, but the current data do not reveal whether WT RAP1 binds DNA. In addition, the authors incorrectly stated that "disruption of the MybL domain sequence did not eliminate RAP1-telomere binding in vivo" (lines 165-166). In ref 29, deletion of Myb domain did not abolish RAP1-telomere association. However, point mutations in MybL domain that abolish RAP1's DNA binding activities clearly disrupted RAP1's association with the telomere chromatin. Therefore, the current observation is not completely consistent with that published in ref 29.

2. There is no evidence, in vitro or in vivo, that binding PI(3,4,5)P3 to RAP1 causes conformational change in RAP1. The BRCT domain of RAP1 is known for its ability to homodimerize (Afrin et al. 2020, mSphere). It is therefore possible that binding of PI(3,4,5)P3 to RAP1 simply disrupts its homodimerization function. The authors clearly have extrapolated their conclusions based on available data. It is therefore important to revise the discussion and make appropriate statements.

Reviewer #1 (Public Review):

This paper describes the role of WRNIP1 AAA+ ATPase, particularly its UBZ domain for ubiquitin-binding, but not ATPase, to prevent the formation of the R-loop when DNA replication is mildly perturbated. By combining cytological analysis for DNA damage, R-loop, and chromosome aberration with the proximity ligation assay for colocalization of various proteins involved in DNA replication and transcription, the authors provide solid evidence to support the claim. The authors also revealed a distinct role of WRNIP1 in the prevention of R-loop-induced DNA damage from FANCD2, which is inconsistent with the known relationship between WRNIP1 and FANCD2 in the repair of crosslinks.

Reviewer #1 (Public Review):

Summary:<br /> This research offers an in-depth exploration and quantification of social vocalization within three families of Mongolian gerbils. In an enlarged, semi-natural environment, the study continuously monitored two parent gerbils and their four pups from P14 to P34. Through dimensionality reduction and clustering, a diverse range of gerbil call types was identified. Interestingly, distinct sets of vocalizations were used by different families in their daily interactions, with unique transition structures exhibited across these families. The primary results of this study are compelling, although some elements could benefit from clarification

Strengths:<br /> Three elements of this study warrant emphasis. Firstly, it bridges the gap between laboratory and natural environments. This approach offers the opportunity to examine natural social behavior within a controlled setting (such as specified family composition, diet, and life stages), maintaining the social relevance of the behavior. Secondly, it seeks to understand short-timescale behaviors, like vocalizations, within the broader context of daily and life-stage timescales. Lastly, the use of unsupervised learning precludes the injection of human bias, such as pre-defined call categories, allowing the discovery of the diversity of vocal outputs.

Weaknesses:<br /> 1. While the notable differences in vocal clusters across families are convincing, the drivers of these differences remain unclear. Are they attributable to "dialect," call usage, or specific vocalizing individuals (e.g., adults vs. pups)? Further investigation, via a literature review or additional observation, into acoustic differences between adult and pup calls is recommended. Moreover, a consistent post-weaning decrease in the bottom-left cluster (Fig. S3) invites interpretation: could this reflect drops in pup vocalization?

2. Developmental progression, particularly during pre-weaning periods when pup vocal output remains unstable, might be another factor influencing cross-family vocal differences. Representing data from this non-stationary process as an overall density map could result in the loss of time-dependent information. For instance, were dominating call types consistently present throughout the recording period, or were they prominent only at specific times? Displaying the evolution of the density map would enhance understanding of this aspect.

3. Family-specific vocalizations were credited to the transition structure, a finding that may seem obvious if the 1-gram (i.e., the proportion of call types) already differs. This result lacks depth unless it can be demonstrated that, firstly, the transition matrix provides a robust description of the data, and secondly, different families arrange the same set of syllables into unique sequences.

Reviewer #1 (Public Review):

In this manuscript, Davidsen and coworkers describe the development of a novel aspartate biosensor jAspSNFR3. This collaborative work supports and complements what was reported in a recent preprint by Hellweg et al., (bioRxiv; doi: 10.1101/2023.05.04.537313). In both studies, the newly engineered aspartate sensor was developed from the same glutamate biosensor previously developed by the authors of this manuscript. This coincidence is not casual but is the result of the need to find tools capable of measuring aspartate levels in vivo. Therefore, it is undoubtedly a relevant and timely work carried out by groups experienced in aspartate metabolism and in the generation of metabolite biosensors.

Reviewer #1 (Public Review):

The authors have developed an open-source high-resolution microscope that is easily accessible to scientists, students, and the general public. The microscope is specifically designed to work with incubators and can image cells in culture over long periods. The authors provide detailed instructions for building the microscope and the necessary software to run it using off-the-shelf components. The system has great potential for studying cell biology and various biological processes.

The authors' work will make scientific instruments more accessible and remove obstacles to the free diffusion of capabilities and know-how in science. This important contribution will enable more people to conduct scientific research.

Reviewer #1 (Public Review):

This is a very interesting paper which addresses how auditory cortex represents sound while an animal is performing an auditory task. The study involves psychometric and neurophysiological measurement from ferrets engaged in a challenging tone in noise discrimination task, and relates these measurements using neurometric analysis. A novel neural decoding technique (decoding-based dimensionality reduction or dDR, introduced in a previous paper by two of the authors) is used to reduce bias so that stimulus parameters can be read out from neuronal responses.

The central finding of the study is that, when an animal is engaged in a task, non-primary auditory cortex represents task-relevant sound features in a categorical way. In primary cortex, task engagement also affects representations, but in a different way - the decoding is improved (suggesting that representations have been enhanced), but is not categorical in nature. The authors argue that these results are compatible with a model where early sensory representations form an overcomplete representation of the world, and downstream neurons flexibly read out behaviourally relevant information from these representations.

I find the concept and execution of the study very interesting and elegant. The paper is also commendably clear and readable. The differences between primary and higher cortex are compelling and I am largely convinced by the authors' claim that they have found evidence that broadly supports a mixed selectivity model of neural disentanglement along the lines of Rigotti et al (2013). I think that the increasing body of evidence for these kinds of representations is a significant development in our understanding of higher sensory representations. I also think that the dDR method is likely to be useful to researchers in a variety of fields who are looking to perform similar types of neural decoding analysis.

Reviewer #1 (Public Review):

Summary:<br /> Tian et al. investigated the effects of emotional signals in biological motion on pupil responses. In this study, subjects were presented with point-light biological motion stimuli with happy, neutral, and sad emotions. Their pupil responses were recorded with an eye tracker. Throughout the study, emotion type (i.e., happy/sad/neutral) and BM stimulus type (intact/inverted/non-BM/local) were systematically manipulated. For intact BM stimuli, happy BM induced a larger pupil diameter than neutral BM, and neutral BM also induced a larger pupil diameter than sad BM. Importantly, the diameter difference between happy and sad BM correlated with the autistic trait of individuals. These effects disappeared for the inverted BM and non-BM stimuli. Interestingly, both happy and sad emotions show superiority in pupil diameter.

Strengths:<br /> 1. The experimental conditions and results are very easy to understand.<br /> 2. The writing and data presentation are clear.<br /> 3. The methods are sound. I have no problems with the experimental design and results.

Weaknesses:<br /> 1. My main concern is the interpretation of the intact and local condition results. The processing advantage of happy emotion is not surprising given a number of existing studies. However, the only difference here seems to be the smaller (or larger) pupil diameter for sad compared to neutral in the intact (or local, respectively) condition. The current form only reports this effect but lacks in-depth discussions and explanations as to why this is the case.

2. I also found no systematic discussion and theoretical contributions regarding the correlation with the autistric trait. If the main point of this paper is to highlight an implicit and objective behavioral marker of the autistric trait, more interpretation and discussion of the links between the results and existing findings in ASD are needed.

Reviewer #1 (Public Review):

The manuscript by Zhu and colleagues aimed to clarify the importance of isoform diversity of PCDHg in establishing cortical synapse specificity. The authors optimized 5' single-cell sequencing to detect cPCDHg isoforms and showed that the pyramidal cells express distinct combinations of PCDHg isoforms. Then, the authors conducted patch-clamp recordings from cortical neurons whose PCDHg diversity was disrupted. In the elegant experiment in Figure 3, the authors demonstrated that the neurons expressing the same sets of cPCDHg isoforms are less likely to form synapses with each other, suggesting that identical cPCDHg isoforms may have a repulsive effect on synapse formation. Importantly, this phenomenon was dependent on the similarity of the isoforms present in neurons but not on the amount of proteins expressed.

One of the major concerns in an earlier version was whether PCDHg isoforms, which are expressed at a much lower level than C-type isoforms, have true physiological significance. The authors conducted additional experiments to address this point by using PCDHg cKO and provided convincing data supporting their conclusion. The results from PCDHg C4 overexpression, showing no impact on synaptic connectivity, further clarified the importance of isoforms. I have no further concerns, however, I would like to point out that the evidence for the necessity of the PCDHg isoform is still lacking because most experiments were done by overexpression. It would be helpful for the readers if the authors could add this point to the discussion.

Reviewer #1 (Public Review):

Spikol et al. investigate the roles of two distinct populations of neurons in the nucleus incertus (NI). The authors established two new transgenic lines that label gsc2- and rln3a-expressing neurons. They show that the gsc2+ and rln3a+ NI neurons show divergent projection patterns and project to different parts of the interpeduncular nucleus (IPN), which receive inputs from habenula (Hb). Furthermore, calcium imaging shows that gsc2 neurons are activated by the optogenetic activation of the dorsal Hb-IPN and respond to aversive electric shock stimuli, while rln3a neurons are highly spontaneously active. The ablation of rln3a neurons, but not gsc2 neurons, alters locomotor activity of zebrafish larvae.

The strength of the paper is their genetic approach that enabled the authors to characterize many different features of the two genetically targeted populations in the NI. These two neuronal populations are anatomically closely apposed and would have been indistinguishable without their genetic tools. Their analyses provide valuable information on the diverse anatomical, physiological and behavioral functions of the different NI subtypes. On the other hand, these pieces of evidence are only loosely linked with each other to reach a mechanistic understanding of how the NI works in a circuit. For example, the anatomical study revealed the connections from the NI to the IPN, while the optogenetic mapping experiments investigate the other way around, i.e. the connection from the IPN to the NI.

Reviewer #1 (Public Review):

This study examines whether the human brain uses a hexagonal grid-like representation to navigate in a non-spatial space constructed by competence and trustworthiness. To test this, the authors asked human participants to learn the levels of competence and trustworthiness for six faces by associating them with specific lengths of bar graphs that indicate their levels in each trait. After learning, participants were asked to extrapolate the location from the partially observed morphing bar graphs. Using fMRI, the authors identified brain areas where activity is modulated by the angles of morphing trajectories in six-fold symmetry. The strength of this paper lies in the question it attempts to address. Specifically, the question of whether and how the human brain uses grid-like representations not only for spatial navigation but also for navigating abstract concepts, such as social space, and guiding everyday decision-making. This question is of emerging importance.

The weak points of this paper are that its findings are not sufficiently supporting their arguments, and there are several reasons for this:

1. Does the grid-like activity reflect 'navigation over the social space' or 'navigation in sensory feature space'? The grid-like representation in this study could simply reflect the transition between stimuli (the length of bar graphs). Participants in this study associated each face with a specific length of two bars, and the 'navigation' was only guided by the morphing of a bar graph image. Moreover, any social cognition was not required to perform the task where they estimate the grid-like activity. To make social decision-making that was conducted separately, we do not know if participants needed to navigate between faces in a social space. Instead, they can recall bar graphs associated with faces and compute the decision values by comparing the length of bars. Notably, in the trust game in this study, competence and trustworthiness are not equally important to make a decision (Equation 1). The expected value is more sensitive to one over the other. This also suggests that the space might not reflect social values but perceptual differences.

2. Does the brain have a common representation of faces in a social space? In this study, participants don't need to have a map-like representation of six faces according to their levels of social traits. Instead, they can remember the values of each trait. The evidence of neural representations of the faces in a 2-dimensional social space is lacking. The authors argued that the relationship between the reaction times and the distances between faces provides evidence of the formation of internal representations. However, this can be found without the internal representation of the relationships between faces. If the authors seek internal representations of the faces in the brain, it would be important to show that this representation is not simply driven by perceptual differences between bar graphs that participants may recall in association with each face.

Considering these caveats, it is hard for me to agree if the authors provide evidence to support their claims.

Reviewer #1 (Public Review):

Summary:

In this study, the authors show that a long-non coding RNA lncDACH1 inhibits sodium currents in cardiomyocytes by binding to and altering the localization of dystrophin. The authors use a number of methodologies to demonstrate that lncDACH1 binds to dystrophin and disrupts its localization to the membrane, which in turn downregulates NaV1.5 currents. Knockdown of lncDACH1 upregulates NaV1.5 currents. Furthermore, in heart failure, lncDACH1 is shown to be upregulated which suggests that this mechanism may have pathophysiolgoical relevance.

Strengths:

1. This study presents a novel mechanism of Na channel regulation which may be pathophysiologically important.

2. The experiments are comprehensive and systematically evaluate the physiological importance of lncDACH1.

Weaknesses:

1. What is indicated by the cytoplasmic level of NaV1.5, a transmembrane protein? The methods do not provide details regarding how this was determined. Do you authors means NaV1.5 retained in various intracellular organelles?

2. What is the negative control in Fig. 2b, Fig. 4b, Fig. 6e, Fig. 7c? The maximum current amplitude in these seem quite different. -40 pA/pF in some, -30 pA/pF in others and this value seems to be different than in CMs from WT mice (<-20 pA/pF). Is there an explanation for what causes this variability between experiments and/or increase with transfection of the negative control? This is important since the effect of lncDACH1 is less than 50% reduction and these could fall in the range depending on the amplitude of the negative control.

3. NaV1.5 staining in Fig. 1E is difficult to visualize and to separate from lncDACH1. Is it possible to pseudocolor differently so that all three channels can be visualized/distinguished more robustly?

4. The authors use shRNA to knockdown lncDACH1 levels. It would be helpful to have a scrambled ShRNA control.

5. Is there any measurement on the baseline levels of LncDACH1 in wild-type mice? It seems quite low and yet is a substantial increase in NaV1.5 currents upon knocking down LncDACH1. By comparison, the level of LncDACH1 seems to be massively upregulated in TAC models. Have the authors measured NaV1.5 currents in these cells? Furthermore, does LncDACH1 knockdown evoke a larger increase in NaV1.5 currents?

6. What do error bars denote in all bar graphs, and also in the current voltage relationships?

Reviewer #1 (Public Review):

Sukumar et al build on a body of work from the Palmer lab that seeks to unravel the transcriptional targets of Alk signaling (a receptor tyrosine kinase). Having uncovered its targets in the mesoderm in an earlier study, they seek to determine its targets in the central nervous system. To do this, they use Targeted DamID (TaDa) in the wild-type and Alk dominant negative background and identify about 1700 genes that might be under the control of Alk signalling. Using their earlier data and applying a set of criteria - upregulated in gain-of-Alk, downregulated in loss-of-Alk, and co-expressed with Alk positive cells in single cell datasets - they arrive upon a single gene, Sparkly, which is predicted to be a neuropeptide precursor.

They generate antibodies and mutants for Sparkly and determine that it is responsive to Alk signalling and is expressed in many neuroendocrine cells, as well as in clock neurons. Though the mutants survive, they have reduced lifespans and are hyperactive. In summary, the authors identify a previously unidentified transcriptional target of Alk signalling, which is likely cleaved into a neuropeptide and is involved in regulating circadian activity.

The data support claims made, are generally well presented and the manuscript clearly written. The link between circadian control of Alk signalling in Clock neurons > Spar expression > ultimately controlling circadian activity, however, was not clear.

Reviewer #1 (Public Review):

This study was designed to examine the bypass of Ras/Erk signaling defects that enable limited regeneration in a mouse model of hepatic regeneration. The authors show that this hepatocyte proliferation is marked by expression of CD133 by groups of cells. The CD133 appears to be located on intracellular vesicles associated with microtubules. These vesicles are loaded with mRNA. The authors conclude that the CD133 vesicles mediate an intercellular signaling pathway that supports cell proliferation. These are new observations that have broad significance to the fields of regeneration and cancer.

The primary observation is that the limited regeneration observed in livers with Ras/Erk signaling defects is associated with CD133 expression by groups of cells. The functional significance of CD133 was tested using Prom1 KO mice - the data presented are convincing.

The major weakness of the study is that some molecular mechanistic details are unclear - this is, in part, due to the extensive new biology that is described. Nevertheless, the data used to support some key points in this study are unclear:

a) What is the evidence that the observed CD133 groups of cells are not due to clonal growth. Is this conclusion based on the time course (the groups appear more rapidly than proliferation) or is this based on the GFP clonal analysis?

b) What is the evidence that the CD133 vesicles mediate intercellular communication. This is an exciting hypothesis, but what is the evidence that this happens? Is this inferred from IEG mRNA diversity? or some other data. Is there direct evidence of transfer - for example, the does the GFP clonal analysis show transfer of GFP that is not mediated by clonal proliferation? Moreover, since the hepatocytes are isogenic, what distinguishes the donor and recipient cells?

Increased clarity concerning what is hypothesis and what is directly supported by data - would improve the presentation of this study.

Reviewer #1 (Public Review):

Summary:<br /> The authors use a combination of ChIP-seq, RNA-seq and ATAC-seq on FACS-purified germ cells to understand the changes in transcription and chromatin landscape of germline stem cells (GSCs) and their progeny during adult oogenesis.

Strengths:<br /> The major strengths of the paper include high quality -omics data, robust analyses of the data, and a well-written manuscript. The data strongly support the conclusions (1) that GSCs have more open chromatin than its differentiating daughter cells, (2) that H3K9me3 heterochromatin forms in 16-cell cyst stage and silences GSC-enriched genes, transposons, and testis-biased and somatic genes; (3) that GSC-enriched genes encoding cell cycle control, protein synthesis and signal transduction reside in clusters in autosomal pericentric regions; and (4) that there is a transcriptionally-driven metabolic reprogramming of nurse (germline) cells to aerobic glycolysis.

These data sets and analyses will have a high impact of the field of germline lifecycle (from GSC to primordial germ cells to GSCs again). The authors will make these data sets available through NCBI GEO and on Github. However, these are not incredibly user friendly and these data sets are extremely useful. I wonder if it is possible to incorporate these valuable data set into existing websites?

Weaknesses:<br /> There are no obvious weaknesses.

Reviewer #1 (Public Review):

The current manuscript builds on a previous publication from the same group(s) (https://doi.org/10.7554/eLife.77779) that identified several new interacting partners of the ESCRT pathway. The authors show via fluorescence polarization anisotropy (FPA) and NMR spectroscopy that the microtubule-interacting and trafficking (MIT) domains of CALPAIN7 bind to the IST1 subunit of the ESCRT-III complex. The authors used a powerful combination of biochemical, structural, and cell biological tools. The experiments are designed well and are performed to a high standard. The vast majority of the conclusions are supported by the data.

The authors report the X-ray crystal structure of the MIT-IST complex show the exact residues involved in the interaction and the mode of binding. They validate their findings and put them in a biological context by introducing mutations into the key residues in the MIT domain of CALPAIN7 and IST domain of ESCRT-III that can disrupt and restore the molecular interactions using in vitro biochemical assays and in vivo immunofluorescence microscopy imaging.

The writing is exceptional. I could not spot a single typographical or grammatical error. The manuscript is easy to read. It is concise and is nicely supported by high-quality figures. It was a pleasure to read.

The authors provide a great amount of detailed information about the experiments performed and have deposited the majority (if not all) of the plasmids used in the study in a public depository. They should be commended for this decision.

Reviewer #1 (Public Review):

Iskusnykh et al. present an elegant and thorough analysis of the role of transcription factor Lmx1a as a master regulator of the cortical hem, which is a secondary organizer in the brain. The authors report that loss of Lmx1a in the hem alters expression levels of Wnts, that Lmx1a is critical for hem progenitors to exit the cell cycle properly, and that Lmx1a loss leads to defects in CR cell differentiation and migration. Furthermore, the authors show that hem-like fate can be induced by overexpressing Lmx1a. This is a fundamental role for a transcription factor that was long used as a hem marker but was never examined for its function in the hem. This study has broader implications for how secondary organizers are created in the embryo and would be of great interest to a wide readership. The conclusions are broadly well supported by the data, though there are a few points of interpretation that need to be addressed.

(1) Figure 3A shows staining intensity in WT and Lmx1a-/- whereas the quantification has Lmx1a+/-. Both genotypes are relevant, -/- and +/-, to test whether the loss of 1 copy of Lmx1a results in a partial diminution of Wnt3a levels. Likewise, it is necessary to examine Wnt3a expression levels in the Wnt3a+/- embryo. Together, these could explain why the Lmx1a+/-; Wnt3a+/- double heterozygote has a DG phenotype, otherwise, it remains an unexplained though interesting observation.

(2) Line 309: "to test Wnt3a as a downstream mediator of Lmx1a function in CH/DG development, we performed an analysis of Lmx1a/Wnt3a double heterozygotes rather than Wnt3a overexpression rescue experiments in Lmx1a -/- mice."

The authors' reasoning is unclear. The double het experiments do not go on to show that one gene acts via the other. It's entirely possible the two act via parallel pathways.<br /> However, since Lmx1a does indeed regulate Wnt3a levels, this is a good argument for suggesting it acts via Wnt3a, even without the overexpression rescue. The authors could reorganize the data and rephrase the definitive "acts via" statement (also in the heading of this section, line 289, and discussion, line 553) to better fit the data.

(3) In the discussion section, the authors should include that trans-hilar and supragranular scaffold is disrupted in Lrp6 and Lef1 single as well as double mutants, which indicates Wnt signaling has a role to play in the morphogenesis of this scaffold. In this context, the author may discuss how Lmx1a could regulate this process via modulating Wnt signaling.

(4) Reduction in Tbr2 levels (Fig4B): E 13.5, not all Tbr2+ cells in the hem show a visible decrease in Tbr2 levels. The CR cells in the marginal zone show faint Tbr2. It would be useful if the staining intensity within the hem was quantified by dividing the section into three bins along the radial axis: Ventricular Zone, "Intermediate" zone, and Marginal zone to get a sense of the intensity profile. Co-labeling with p73 would identify CR cells and distinguish them from hem progenitors.

(5) Are the total number of Prox1+ cells at E14.5 similar between control and Lmx1a-/- ? Might the decrease in Prox1+ cells in the DG of P21 Lmx1a-/- animals occur due to granule cell death or because fewer cells were specified due to lower Wnts from the compromised Lmx1a-/- hem? The authors should examine cell death, labeling with CC3 and Prox1 together to test the cell death angle and discuss if the specification angle applies.

(6) In figure 6, the authors show that Lmx1a OE is sufficient to induce hem-like features, and identify p73+ cells (CR cell lineage). Is the choroid lineage not induced or was it not examined? A line to this effect would be useful. Also, the validation that it is indeed ectopic hem could be stronger with a few additional markers, since this is a striking finding.

Reviewer #1 (Public Review):

Huang, Kevin Y. et al. perform a meta-analysis of single-cell RNA-seq (scRNA-seq) data derived from 11 studies and across six tissues (liver, lung, heart, skin, kidney, endometrium) to address a focused hypothesis: pro-fibrotic SPP1+ macrophages that have been found in liver and lung tissue of idiopathic pulmonary fibrosis patients exist in other human tissues which can result in broader fibrotic disease states. The authors use existing, state-of-the-art single-cell analysis tools to perform the meta-analysis. They convincingly show that the SPP1+ macrophage population can be identified in lung, liver, heart, skin, uterus (endometrium), and kidney clusters derived from each tissues' scRNA-seq data. They further identify three subpopulations of the SPP1+ macrophages: a matrisome-associated macrophages (MAMs) defined as SPP1+MAM+ and two others enriched for inflammatory and ribosomal processes which they group together and define as SPP1+MAM-. Pathway analysis of genes unregulated in SPP1+MAM+ vs SPP1+MAM- cells yields significant enrichment of extracellular matrix remodeling and metabolism-related pathways and genes. This allows them to arrive at SPP1+MAM+ and SPP1+MAM- gene expression signature scores to further highlight the upregulation of these pathways in SPP1+MAM+ macrophages and their role in fibrosis. They explicitly show enrichment for SPP1+MAM+ macrophages in disease compared to healthy control subjects in a variety of tissues and their associated fibrosis-related diseases. Cell differentiation trajectory analysis identified 2 main trajectories: both starting from FCN1+ infiltrating monocytes/macrophages with one moving toward a homeostatic state and another toward SPP1+MAM+. They verified this using an alternative trajectory analysis approach. Importantly, for all tissues and fibrotic diseases, they found SPP1+MAM+ were at the end of the trajectory preceded by the SPP1+MAM- state, suggesting SPP1+MAM+ represents a common polarization state of SPP1+ macrophages. They develop a probability-based score that estimates the propensity of SPP1+MAM- macrophages to differentiate into SPP1+MAM+ and show that this was significantly higher in fibrotic disease subjects compared to healthy controls. They go on to identify the transcription factor networks (regulons) associated with SPP1+MAM+ differentiation and activation. They find a number of enriched regulons/transcription factors and through a linear-modeling trajectory analysis highlight the regulons that are associated specifically with the SPP1+MAM- to SPP1+MAM+ transition. In this way, they prioritize the NFATC1 and HIVEP3 regulations as driving the differentiation of SPP1+MAM- macrophages toward the SPP1+MAM+ polarization state. Finally, given that age is a risk factor for fibrotic disease, they assessed the association of SPP1+MAM+ and SPP1+MAM- gene signatures in healthy control old and young human subjects as well as old and young mice and found SPP1+MAM+ was either exclusively (human) or more significantly (mice) elevated in old versus young compared to SPP1+MAM-.

The strengths of this paper are the authors gathered a number of relevant single-cell RNA-seq data sets from fibrosis-focused studies to address a highly focused hypothesis (stated above). They gained the power to detect the population of SPP1+MAM+ cells by integrating these datasets. The analysis is carried out well using existing state-of-the-art tools. With whatever metric or single cell analysis-based discovery they make about the SPP1+MAM+ subpopulations (e.g., gene signatures, endpoint of trajectory analysis, associated regulons, etc), they compare the relevant scoring metrics in fibrosis and control subjects at every stage of the meta-analysis and find the SPP11+MAM+ is consistently higher across tissues and fibrosis-related diseases.

There are only minor weaknesses in this paper. One is that some of the most highly significant or simply significant results are not shown in main figures but are summarized in supplementary tables (e.g., MYC TARGETS V1 would have appeared as the most significant, highest enriched, and among the largest in terms of set size). Another is analysis criteria that may not yield the most biologically relevant or impactful conclusion (e.g., while the regulon THRA does not display a shift in slopes it shows the strongest, progressive increase going toward the SPP1+MAM+ state).

Reviewer #1 (Public Review):

The manuscript by Salloum and colleagues examines the role of statin-mediated regulation of mitochondrial cholesterol as a determinant of epigenetic programming via JMJD3 in macrophages.

Key strengths of the work include:

1. Mechanistic analysis of how statin treatments can remodel the mitochondrial membrane content via cholesterol depletion which in turn affects JMJD3 levels is a novel concept.

2. Use of RNA-seq and ATAC-seq data provides an avenue for unbiased analysis of the statin effects.

3. Use of methyl-cyclodextrin (MCD) alongside statins increases the robustness of the findings and the use of NFKB inhibitors suggests a mechanistic role for NFKB.

The conclusions are only partially supported by the presented data:

1. There is a lack of any in vivo studies that are required to demonstrate that the concentrations of statins used to induce epigenetic programming of macrophages are physiologically relevant. There have been numerous studies that have examined the anti-inflammatory effects of statins but there is significant debate and controversy regarding the in vivo relevance. Much of the in vivo effects of statins are achieved via changes in systemic cholesterol levels but the direct effects on macrophages are not clear.

2. "Statins" is used globally and it is unclear which statins were used, which doses of statins, and the treatment durations

3. The RNA-seq, ATAC-seq, and selected H3K27 ChIP only show a snapshot of the results without leveraging the power of unbiased analysis. Such an unbiased analysis could show whether the examined genes are indeed the most relevant targets of statins.

4. CCCP depletion can have broad toxic effects and it is difficult to interpret specific roles of ATP synthase from potentially toxic mitochondrial uncoupling.

Reviewer #1 (Public Review):

In this manuscript, Drs. Miura, Mori, and colleagues, first present lineage tracing data using PDGFRa-CreERT2 and Foxa2-Cre drivers to show that PDGFRa+ cells, when lineage-labeled early in development go on to form the lung mesenchyme (but little to none of the epithelium), whereas FOXA2 expressing cells go on to contribute to both the lung epithelium and lung mesenchyme. However, it is already well known that FOXA2 is expressed in the mesendoderm around the time of gastrulation, and that this population generates both endoderm and mesodermal derivatives. As a result, it is not surprising that lineage labeling this population would contribute to both the lung epithelium and lung mesenchyme. The authors use the term bona fide lung (BFL) generative lineage. However, since the mesendoderm contributes to both the endoderm and mesoderm, but is by no means specific to the lung, and as shown in this paper (Figure 2G) the FOXA2 population only generates 30-40% of the mesenchyme, the term BFL is both confusing and misleading.

In the second portion of the manuscript, the authors conditionally delete Fgfr2 using a Foxa2-Cre driver. Although loss of Fgf10 or Fgfr2 is known to result in lung agenesis, deletion of Fgfr2 within the FOXA2+ expressing cells is novel. However, since FOXA2 is broadly expressed within the nascent lung epithelium and Fgfr2 is known to be expressed within the lung epithelium, it isn't entirely clear how much information this adds beyond what already known from other Fgfr2 knockout studies. Perhaps the most interesting aspect of the reported phenotype is that the other organs (e.g. intestine) in these knockout animals appears to be relatively spared. This should be better characterized by the authors, as currently only a few H&E images are shown.

The authors then used conditional blastocyst complementation with nGFP+iPSCs from wild-type mice to rescue the phenotype of the Fgfr2 conditional knockout mice, showing that an embryonic lung is formed. However, blastocyst complementation has previously been performed with other knockout mouse models with severe lung hypoplasia/aplasia, including Dr. Mori's previous Nature Medicine paper. Although most of the previous mouse models target the endoderm/early epithelial cells (e.g. conditional deletion of Ctnnb1, Fgfr2, or global knockout of Nkx2.1; see Li E, et al. Dev Dyn 2021 Jul;250(7):1001-1020; Wen B, Am J Resp Crit Care Med. 2020; in addition to Mori M, Nature Medicine, 2019), Kitahara A, et al (Cell Rep. May 12 2020;31(6):107626) previously reported blastocyst complementation in in Fgf10 null mouse model, so it is not clear what the current study significantly adds contributes to this existing body of literature. The lungs of the mice undergoing blastocyst complementation are also incompletely characterized in the current version of this study. For example, it is unclear how functional these lungs are and whether they are capable of gas exchange after birth.

Reviewer #1 (Public Review):

In this manuscript, the authors examine the role of Sirt2 on cardiac hypertrophy by using 2 in-vivo models- systemic KO of Sirt2 and cardiac-specific KO of Sirt 2. The authors show that Sirt2 is important for the development of heart failure and cardiac hypertrophy. Mechanistically, the authors show that Sirt2 regulates NRF2 and that deletion of Sirt2 is protective through stabilization and increased nuclear translocation of NRF2. The paper is clinically relevant and the data is of high quality.

Reviewer #1 (Public Review):

Sangar and co-workers have shown the role of Syntaxin 6 on prion (PrP) protein aggregation and have claimed a possible molecular mechanism behind its role in pathogenesis of Prion diseases like CJD. Authors have elaborately shown the aggregation kinetics of PrP in absence and presence of Stx6 by their in-house developed NAA assay which allowed them to catch the interaction of PrP with other cellular proteins like Stx6 or Hspa1a. By in vitro aggregation kinetics assays, authors show that Stx6 delays the aggregation kinetics of PrP and leads to amorphous aggregates rather than well-formed fibrils. By a neurite length detection assay with treatment of preformed PrP-fibrils (with or without Stx6 interaction), they show enhanced toxicity of Prp-Stx6 aggregates compared to PrP fibrils. However, there are a number of concerns related to physiological relevance that need to be addressed by the authors.

Reviewer #1 (Public Review):

In this manuscript, the authors compare the behavior of the intracellular ion channel TPC1 in two species of plants. TPC1 channels are cation permeable pseudo tetramers which in plants are expressed in the intracellular vacuole and whose function is not well understood.

It was previously known that calcium ions are modulators of the function of these channels and the authors identify a cluster of negative charges facing the lumen of the vacuole that can regulate the action of calcium and modulate the voltage dependence of the channels. Interestingly, this cluster is not entirely present in the faba bean (Vicia faba) vfTPC1 channel.

Through electrophysiological recordings of transfected channels into TPC1-lacking vacuoles, the authors show that the vfTPC1 channels activate at more negative voltages, even in the presence of high concentrations of calcium, leading to cation entrance into the vacuole and increased excitability, as assessed by action potential firing.

Though the findings are interesting and the methods are of high quality, the authors fail to convey the importance of the problem they are tackling and do not frame their findings in the broad physiology of the plant species they study.

These findings should be of interest to plant physiologists and channel biophysicists interested in TPC1 channels.

(~10:00) "The context determines the meaning of the content."

Thus reframing is very powerful as you recontextualize the past, and therefore see it in a whole new light; the meaning of the past changed.

By asking what you have learned from the past, you become anti-fragile and flexible, as you turn the past into something useful; an asset.

"The past happens for us, not to us."

"How you frame the past influences your expectations for the future."

"You can't disconnect your view of the future from your experience in the present."

"You can't have meaning in the present without hope & purpose in the future."

One of the powerful things about journaling is that you can control the past; reframe it. What is the meaning of the past gets determined by both the present and the future.

Hardy recommends to often (even daily) reflect on the past and notice how different you are now compared to then. What you have achieved, what is possible now that was not possible then, etc.

What did I learn today?

• for: W2W, inequality, 1%, oligarch, elites, kleptocrats, civil society - exploitation, TUSN, Transnational Uncivil Society Network
• title: How the kleptocrats and oligarchs hunt civil society groups to the ends of the Earth
• author: Peter Rinko
• comment
  • This article explores the research work of Alexander Cooley, John Heathershaw and Ricard Soares de Oliverira who coauthored the paper

Transnational Uncivil Society Networks: kleptocracy’s global fightback against liberal activism - reference - https://ora.ox.ac.uk/objects/uuid:5e5a3052-c693-4991-a7cc-bc2b47134467/download_file?file_format=application%2Fpdf&safe_filename=Cooley_et_al_2023_transnational_uncivil_society.pdf&type_of_work=Journal+article

Reviewer #1 (Public Review):

Guan et al. explored the mechanisms responsible for the development, maintenance, and functional properties of a specific subset of unconventional T cells expressing a Va3.2 T cell receptor that recognizes a peptide, QFL, presented by the class Ib protein Qa-1. Prior studies from this group showed that cells from mice deficient in the ER protease ERAAP elicit responses in wild-type animals enriched for Qa-1-restricted CD8 T cells. They further showed that a significant proportion of these responses were directed against the QFL peptide derived from a conserved protein with incompletely understood functions. Many of these so-called QFL T cells expressed Va3.2-Ja21, were present in the spleen of wild-type mice, and exhibited a memory-like phenotype. Due to their relatively low frequency and weak staining with Qa-1 tetramers, analyzing QFL T cells has been challenging. Therefore, the authors generated dextramers, which permitted them to more rigorously identify these cells. They confirmed some of their previous findings and further showed that Va3.2+ and Va3.2- QFL T cells were present in the intestinal epithelium, where they also express CD8alpha homodimers, a characteristic of most small intestinal intraepithelial lymphocytes (siIELs), and most similar to the so-called natural siIELs that acquire their innate functions in the thymus. The authors show that TAP but not Qa-1 or ERAAP expression are required for the development of these cells, and both Qa-1 and ERAAP are required for the natural siIEL phenotype. Some of these findings were confirmed using a new TCR transgenic mouse expressing the QFL TCR. They further show that retention but not homing of QFL T cells to the intestinal epithelium involves commensal microorganisms, and using in silico approaches, they identify a commensal that contains a peptide similar to QFL that can activate QFL T cells. Finally, they show that this organism, P. pentosaceus, can promote gut retention of QFL T cells when it is introduced into germ-free mice. From these findings, the authors conclude that the microbiota influences the maintenance of Qa-1-restricted T cells.

Comments:<br /> 1. Overall, the authors employ a number of new reagents and elegant approaches to explore the development, maintenance, and functional properties of QFL T cells.<br /> 2. Generally, conclusions made are well supported by the data presented.<br /> 3. One limitation of the work is that the immunological functions of QFL T cells remain unclear.<br /> 4. The work covers a lot of ground (intestinal IELs, unconventional T cells, innate/virtual memory T cells, Qa-1/HLA-E, etc) that may not be familiar to many readers.<br /> 5. A few questions remain:<br /> a) Regarding the results for TAP knockout animals, since Qa-1 does not appear to be required for QFL T cell development, the absence of these cells in TAP KO mice cannot be easily explained.<br /> b) The Va3.2 T cells display similarities with previously identified innate/virtual memory T cells, some of which require IL-4 production by CD1d-restricted NKT cells for their intrathymic development, which is not fully discussed.<br /> c) Qa-1/peptide complexes may also be recognized by CD94/NKG2 (both inhibitory and activating) receptors on NK cells and subsets of CD8 T cells, which may complicate data interpretation, but is not noted in the text.<br /> d) Are these conclusions relevant to the human homolog of Qa-1, HLA-E?

Reviewer #1 (Public Review):

By identifying a loss of function mutant of IQCH in an infertile patient, Ruan et al. show that IQCH is essential for spermiogenesis by generating a knockout mouse model of IQCH. Similar to infertile patients with a mutant of IQCH, IQCH knockout mice are characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. Mechanistically, IQCH regulates the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis.

Although this manuscript contains a potentially interesting piece of work that delineates a mechanism of IQCH that associates with spermatogenesis, this reviewer feels that a number of issues require clarification and re-evaluation for a better understanding of the role of IQCH in spermatogenesis. With the shortage of logic and supporting data, causal relationships are still not clear among IQCH, CaM, and HNRPAB. The most serious point in this manuscript could be that the authors try to generalize their interpretations with a model that is too simplified from limited pieces of their data. The way the data and the logic are presented needs to be largely revised, and several interpretations should be supported by direct evidence.

Reviewer #1 (Public Review):

Recent studies in plants and human cell lines argued for a central role of 1,5-InsP8 as the central nutrient messenger in eukaryotic cells, but previous studies concluded that this function is performed by 1-InsP7 in baker's yeast. Chabert et al now performed an elegant set of capillary electrophoresis coupled to mass spectrometry time course experiments to define the cellular concentrations of different inositol pyrophosphosphates (PP-InsPs) in wild-type yeast cells under normal and phosphate (Pi) starvation growth conditions. These experiments, in my opinion, form the center of the present study and clearly highlight that the levels of all major PP-InsPs drop under Pi starvation, with the 1,5-InsP8 isomer showing the most rapid changes.

The analysis of known mutants in the PP-InsP biosynthetic pathways furthermore demonstrate that loss-of-function of the PPIP5K enzymes Kcs1 and Vip1 result in a loss of 1,5-InsP8 and a hyperaccumulation of 5-InsP7, respectively. In line with this, loss-of-function of known PP-InsP phosphatases Ddp1 and Swi14 result in hyperaccumulation of either 1- or 5-InsP7, as anticipated from their in vitro substrate specificities. These experiments are of high technical quality and add to our understanding of the kinetics of PP-InsP metabolism/catabolism in yeast.

Next, the authors use changes in subcellular localisation of the central transcription factor Pho4 to assay at which time point after onset of Pi starvation the PHO pathway becomes activated. The early onset of the response, the behavior of the kcs1D mutant and of the ksc1D/vip1D all strongly argue for 1,5-InsP8 as the central nutrient messenger. I find this part of the manuscript well argued, nicely correlating PP-InsP levels, dynamics and the different mutant phenotypes.

The third part of the manuscript is a structure-function study of the CDK inhibitor Pho81, basically using a reverse genetics approach. This analysis demonstrates at the genetic level that the Pho81 SPX domain is required for activation of the PHO pathway. Next, the authors design point mutations that should block either interaction of Pho81-SPX with 1,5-InsP8 or interaction of Pho81 with the Pho80/Pho85 complex. In my opinion, these data can only provide limited insight into the molecular mechanism, as no complementary in vitro binding assays / in vivo co-IP experiments with the wild-type and mutant forms of Pho81 are presented. This seems to be due to technical limitations in recombinantly expressing and purifying the respective Pho81 protein for in vitro PP-InsP binding and protein - protein interaction assays.

Taken together, the work by Chabert et al, reinvestigates and clarifies the activation of the yeast PHO pathway by PP-InsP nutrient messengers and their cellular SPX receptors. From this work, a more unified eukaryotic mechanism emerges, in which 1,5-InsP8 represents the central signaling molecule in different species, with conserved SPX receptors sensing this signaling molecule.

Reviewer #1 (Public Review):

The authors of the manuscript cultivated a Planctomycetes strain affiliated with Phycisphaerae. The strain was one of the few Planctomycetes from deep-sea environments and demonstrated several unique characteristics, such as being the only known Phycisphaerae using a budding mode of division, extensive involvement in nitrate assimilation, and being able to release phage particles without cell death. The manuscript is generally well-written. However, a few issues need to be more clearly addressed, especially regarding the identification and characterization of the phage.

Reviewer #1 (Public Review):

The authors investigate how the viscoelasticity of the fingertip skin can affect the firing of mechanoreceptive afferents and they find a clear effect of recent physical skin state (memory), which is different between afferents. The manuscript is extremely well-written and well-presented. It uses a large dataset of low threshold mechanoreceptive afferents in the fingertip, where it is particularly noteworthy that the SA-2s have been thoroughly analyzed and play an important role here. They point out in the introduction the importance of the non-linear dynamics of the event when an external stimulus contacts the skin, to the point at which this information is picked up by receptors. Although clearly correlated, these are different processes, and it has been very well-explained throughout. I have some comments and ideas that the authors could think about that could further improve their already very interesting paper. Overall, the authors have more than achieved their aims, where their results very much support the conclusions and provoke many further questions. This impact of the previous dynamics of the skin affecting the current state can be explored further in so many ways and may help us to better understand skin aging and the effects of anatomical changes of the skin.

At the beginning of the Results, it states that FA-2s were not considered as stimuli and did not contain mechanical events with frequency components high enough to reliably excite them. Was this really the case, did the authors test any of the FA-2s from the larger dataset? If FA-2s were not at all activated, this is also relevant information for the brain to signal that it is not a relevant Pacinian stimulus (as they respond to everything). Further, afferent receptive fields that were more distant to the stimulus were included, which likely fired very little, like the FA-2s, so why not consider them even if their contribution was low?

One question that I wondered throughout was whether you have looked at further past history in stimulation, i.e. not just the preceding stimulus, but 2 or 3 stimuli back? It would be interesting to know if there is any ongoing change that can be related back further. I do not think you would see anything as such here, but it would be interesting to test and/or explore in future work (e.g. especially with sticky, forceful, or sharp indentation touch). However, even here, it could be that certain directions gave more effects.

Did the authors analyze or take into account the difference between receptive field locations? For example, did afferents more on the sides have lower responses and a lesser effect of history?

Was there anything different in the firing patterns between the spontaneous and non-spontaneously active SA-2s? For example, did the non-spontaneous show more dynamic responses?

Were the spontaneously active SA-2 afferents firing all the time or did they have periods of rest - and did this relate to recent stimulation? Were the spontaneously active SA-2s located in a certain part of the finger (e.g. nail) or were they randomly spread throughout the fingertip? Any distribution differences could indicate a more complicated role in skin sensing.

Did the authors look to see if the spontaneous firing in SA-2s between trials could predict the extent to which the type 1 afferents encode the proceeding stimulus? Basically, does the SA-2 state relate to how the type 1 units fire?

In the discussion, it is stated that "the viscoelastic memory of the preceding loading would have modulated the pattern of strain changes in the fingertip differently depending on where their receptor organs are situated in the fingertip". Can the authors expand on this or make any predictions about the size of the memory effect and the distance from the point of stimulation?

In the discussion, it would be good if the authors could briefly comment more on the diversity of the mechanoreceptive afferent firing and why this may be useful to the system.

Also, the authors could briefly discuss why this memory (or recency) effect occurs - is it useful, does it serve a purpose, or it is just a by-product of our skin structure? There are examples of memory in the other senses where comparisons could be drawn. Is it like stimulus adaptation effects in the other senses (e.g. aftereffects of visual motion)?

One point that would be nice to add to the discussion is the implications of the work for skin sensing. What would you predict for the time constant of relaxation of fingertip skin, how long could these skin memory effects last? Two main points to address here may be how the hydration of the skin and anatomical skin changes related to aging affect the results. If the skin is less viscoelastic, what would be the implications for the firing of mechanoreceptors?

How long does it take for the effect to end? Again, this will likely depend on the skin's viscoelasticity. However, could the authors use it in a psychophysical paradigm to predict whether participants would be more or less sensitive to future stimuli? In this way, it would be possible to test whether the direction modifies touch perception.

Reviewer #1 (Public Review):

This paper describes the development and initial validation of an approach-avoidance task and its relationship to anxiety. The task is a two-armed bandit where one choice is 'safer' - has no probability of punishment, delivered as an aversive sound, but also lower probability of reward - and the other choice involves a reward-punishment conflict. The authors fit a computational model of reinforcement learning to this task and found that self-reported state anxiety during the task was related to a greater likelihood of choosing the safe stimulus when the other (conflict) stimulus had a higher likelihood of punishment. Computationally, this was represented by a smaller value for the ratio of reward to punishment sensitivity in people with higher task-induced anxiety. They replicated this finding, but not another finding that this behavior was related to a measure of psychopathology (experiential avoidance), in a second sample. They also tested test-retest reliability in a sub-sample tested twice, one week apart and found that some aspects of task behavior had acceptable levels of reliability. The introduction makes a strong appeal to back-translation and computational validity. The task design is clever and most methods are solid - it is encouraging to see attempts to validate tasks as they are developed. The lack of replicated effects with psychopathology may mean that this task is better suited to assess state anxiety, or to serve as a foundation for additional task development.

Reviewer #1 (Public Review):

This manuscript describes identification of influential organisms on rice growth and an attempt of validation. The analysis of eDNA on rice pot and mimic field provides rice growth promoting organisms. This approach is novel for plant ecology field. However current results did not fully support whether eDNA analysis-based detection of influencing organism.

The strength of this manuscript is to attempt application of eDNA analysis-based plant growth differentiation. The weakness is too preliminary data and experimental set-up to make any conclusion. The trials of authors experiments are ideal. However, the process of data analysis did not meet certain level. For example, eDNA analysis of different time points on rice growth stages resulted two influential organisms for rice growth. Then they cultivate two species and applied rice seedlings. Without understanding of fitness and robustness, how we can know the effect of the two species on rice growth.

The authors did not check the fate of two species after introducing into rice. If this is true, it is difficult to link between the rice gene expression after treatments and the effectiveness of two species. I think the validation experiment in 2019 needs to be re-conducted.

As authorized gave answered, no strong rationale to select the two species was found. However, I insist that the method has enough novelty to present to general audiences.

Reviewer #1 (Public Review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with a lipid composition similar to the native virion. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. Additional contacts and conformational restraints imposed by ectodomain regions of the envelope glycoprotein, however, remain unaddressed-the size of such simulations likely runs into technical limitations including sampling and compute time.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive. However, given the large amount of data presented within the manuscript, the text would benefit from clearer subsections that delineate discrete mechanistic discoveries, particularly for experimentalists interested in antibody discovery and design. One area the paper does not address involves the polyreactivity associated with membrane binding antibodies-MD simulations and/or pulling velocity experiments with model membranes of different compositions, with and without model antigens, would be needed. Finally, given the challenges in initializing these simulations and their limitations, the text regarding their generalized use for discovery, rather than mechanism, could be toned down.

Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #1 (Public Review):

The authors of the manuscript "High-resolution kinetics of herbivore-induced plant volatile transfer reveal tightly clocked responses in neighboring plants" assessed the effects of herbivory-induced maize volatiles on receiver plants over a period of time in order to assess the dynamics of the responses of receiver plants. Different volatile compound classes were measured over a period of time using PTR-ToF-MS and GC-MS, under both natural light:dark conditions, and continuous light. They also measured gene expression of related genes as well as defense-related phytohormones. The effects of a secondary exposure to GLVs on primed receiver plants were also measured.

The paper addresses some interesting points, however, some questions arise regarding some of the methods employed. Firstly, I am wondering why VOCs (as measured by GC-MS) were not quantified. While I understand that quantification is time-consuming and requires more work, it allows for comparisons to be made between lines of the same species, as well as across other literature on the subject. As experiments with VOC dispensers were also used in this experiment, I find it even more baffling that the authors didn't confirm the concentration of the emission from the plants they used to make sure they matched. The references cited justifying the concentration used (saying it was within the range of GLVs emitted by their plants) to prepare the dispenser were for either a different variety of maize (delprim versus B73) or arabidopsis. Simply relying on the area under the curve and presenting results using arbitrary units is not enough for analyses like these.

With regards to the correlation analyses shown in Figure 6, the results presented in many of the correlation plots are not actually informative. By blindly reporting the correlation coefficient important trends are being ignored, as there are clearly either bimodal relationships (e.g. upper left panel, HAC/TMTT, HAC/MNT) or even stranger relationships (e.g. upper left panel, IND/SQT, IND/MNT) that are not being well explained by a correlation plot. It is not appropriate to discuss the correlation factors presented here and to draw such strong conclusions on emission kinetics. The comparison between plants under continuous light and normal light:dark conditions is interesting, but I think there are better ways to examine these relationships, for example, multivariate analysis might reveal some patterns.

In Figure 2, the elevated concentrations of beta-caryophyllene found in the control plants at 8h and 16.75h measurement timepoints are curious. Is this something that is commonly seen in B73? While there can be discrepancies between emissions and compounds actually present within leaf tissue, it is a little bit odd that such high levels of b-caryophyllene were found at these timepoints, however, this is not reflected in the PTR-ToF-MS measurements of sesquiterpenes. It would be beneficial to include an overview of the mechanism of production and storage of sesquiterpenes in maize leaves, which would clarify why high amounts were found only in the GC-MS analysis and not the PTR-ToF-MS analysis, which is a more sensitive analytical tool. It is possible that the amounts of b-caryophyllene present in the leaf are actually extremely low, however as the values are not given as a concentration but rather arbitrary units, it is not possible to tell. I would include a line explaining what is seen with b-caryophyllene. Additionally, it seems like the amounts of TMTT within the leaf are extraordinarily high (judging only by the au values given for scale), far higher than one would expect from maize.

Reviewer #1 (Public Review):

In this paper, the authors show that disruption of calcineurin, which is encoded by tax-6 in C. elegans, results in increased susceptibility to P. aeruginosa, but extends lifespan. In exploring the mechanisms involved, the authors show that disruption of tax-6 decreases the rate of defecation leading to intestinal accumulation of bacteria and distension of the intestinal lumen. The authors further show that the lifespan extension is dependent on hlh-30, which may be involved in breaking down lipids following deficits in defecation, and nhr-8, whose levels are increased by deficits in defecation. The authors propose a model in which disruption of the defecation motor program is responsible for the effect of calcineurin on pathogen susceptibility and lifespan, but do not exclude the possibility that calcineurin affects these phenotypes independently of defecation.

Reviewer #1 (Public Review):

In this study the authors first perform global knockout of the gene coding for the polarity protein Crumbs 3 (CRB3) in the mouse and show that this leads to perinatal lethality and anopthalmia. Next, they create a conditional knockout mouse specifically lacking CRB3 in mammary gland epithelial cells and show that this leads to ductal epithelial hyperplasia, impaired branching morphogenesis and tumorigenesis. To study the mechanism by which CRB3 affects mammary epithelial development and morphogenesis the authors turn to MCF10A cells and find that CRB3 shRNA-mediated knockdown in these cells impairs their ability to form properly polarized acini in 3D cultures. Furthermore, they find that MCF10A cells lacking CRB3 display reduced primary ciliation frequency compared to control cells, which is supported by rescue experiments and is in agreement with previous studies implicating CRB3 in primary cilia biogenesis. Using a combination of biochemical, molecular- and imaging approaches the authors then provide evidence indicating that CRB3 promotes ciliogenesis by mediating Rab11-dependent recruitment of gamma-tubulin ring complex (gamma-TuRC) component GCP6 to the centrosome/ciliary base, and they also show that CRB3 itself is localized to the base of primary cilia. Finally, to assess the functional consequences of CRB3 loss on ciliary signaling function, the authors analyze the effect of CRB3 loss on Hedgehog and Wnt signaling using cell-based assays or a mouse model.

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. Although the authors provide evidence that CRB3 promotes ciliogenesis via (indirect) physical association with Rab11 and gamma-TuRC, the precise mechanism by which CRB3 promotes ciliogenesis remains to be clarified.

Reviewer #1 (Public Review):

Summary:<br /> This manuscript provides some valuable findings concerning the hippocampal circuitry and the potential role of adult-born granule cells in an interesting long-term social memory retrieval. The behavior experiments and strategy employed to understand how adult-born granule cells contribute to long-term social discrimination memory are interesting.

I have a few concerns, however with the strength of the evidence presented for some of the experiments. The data presented and the method described is incomplete in describing the connection between cell types in CA2 and the projections from abGCs. Likewise, I worry about the interpretation of the data in Figures 1 and 2 given the employed methodology. I think that the interpretation should be broadened. This second concern does not impact the interest and significance of the findings.

Strengths:<br /> The behavior experiments are beautifully designed and executed. The experimental strategy is interesting.

Weaknesses:<br /> The interpretation of the results may not be justified given the methods and details provided.

Reviewer #1 (Public Review):

The evolution of dioecy in angiosperms has significant implications for plant reproductive efficiency, adaptation, evolutionary potential, and resilience to environmental changes. Dioecy allows for the specialization and division of labor between male and female plants, where each sex can focus on specific aspects of reproduction and allocate resources accordingly. This division of labor creates an opportunity for sexual selection to act and can drive the evolution of sexual dimorphism.

In the present study, the authors investigate sex-biased gene expression patterns in juvenile and mature dioecious flowers to gain insights into the molecular basis of sexual dimorphism. They find that a large proportion of the plant transcriptome is differentially regulated between males and females with the number of sex-biased genes in floral buds being approximately 15 times higher than in mature flowers. The functional analysis of sex-biased genes reveals that chemical defense pathways against herbivores are up-regulated in the female buds along with genes involved in the acquisition of resources such as carbon for fruit and seed production, whereas male buds are enriched in genes related to signaling, inflorescence development and senescence of male flowers. Furthermore, the authors implement sophisticated maximum likelihood methods to understand the forces driving the evolution of sex-biased genes. They highlight the influence of positive and relaxed purifying selection on the evolution of male-biased genes, which show significantly higher rates of non-synonymous to synonymous substitutions than female or unbiased genes. This is the first report (to my knowledge) highlighting the occurrence of this pattern in plants. Overall, this study provides important insights into the genetic basis of sexual dimorphism and the evolution of reproductive genes in Cucurbitaceae.

There are, however, parts of the manuscript that are not clearly described or could be otherwise improved.

- The number of denovo-assembled unigenes seems large and I would like to know how it compares to the number of genes in other Cucurbitaceae species. The presence of alternatively assembled isoforms or assembly artifacts may be still high in the final assembly and inflate the numbers of identified sex-biased genes.

- It is interesting that the majority of sex-biased genes are present in the floral buds but not in the mature flowers. I think this pattern could be explored in more detail, by investigating the expression of male and female sex-biased genes throughout the flower development in the opposite sex. It is also not clear how the expression of the sex-biased genes found in the buds changes when buds and mature flowers are compared within each sex.

- The statistical analysis of evolutionary rates between male-biased, female-biased, and unbiased genes is performed on samples with very different numbers of observations, therefore, a permutation test seems more appropriate here.

- The impact of pleiotropy on the evolutionary rates of male-biased genes is speculative since only two tissue samples (buds and mature flowers) are used. More tissue types need to be included to draw any meaningful conclusions here.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript of Zhao et al. aimed at investigating the relationships between type 2 diabetes, bone mineral density (BMD) and fracture risk using Mendelian Randomization (MR) approach.<br /> The authors found that genetically predicted T2D was associated with higher BMD and lower risk of fracture, and suggested a mediated effect of RSPO3 level. Moreover, when stratified by the risk factors secondary to T2D, they observed that the effect of T2D on the risk of fracture decreased when the number of risk factors secondary to T2D decreased.

Strengths:<br /> - Important question<br /> - Manuscript is overall clear and well-written<br /> - MR analyses have been conducted properly, which include the usage of various MR methods and sensitivity analyses, and likely meet the criteria of the MR-strobe checklist to report MR results.

Weaknesses:<br /> - Previous MR studies on that topic have not been discussed<br /> - Multivariable MR could have been used to better assessed the mediative effect of BMI or RSPO3 on the relationships between T2D and fracture risk.

Reviewer #1 (Public Review):

In this manuscript, the authors investigate differences between Tibetans and Han Chinese at altitude in terms of placental transcriptomes during full-term pregnancy. Most importantly, they found that the inter-population differentiation is mostly male-specific and the observed direction of transcriptional differentiation seems to be adaptive at high altitude. In general, it is of great importance and provides new insights into the functional basis of Tibetan high-altitude adaptations, which so far have been mostly studied via population genetic measures only. More specifically, I firmly believe that we need more phenotype data (including molecular phenotypes such as gene expression data) to fully understand Tibetan adaptations to high altitude, and this manuscript is a rare example of such a study. I have a few suggestions and/or questions with which I hope to improve the manuscript further, especially in terms of 1) testing if the observed DEG patterns are truly adaptive, and 2) how and whether the findings in this study can be linked to EPAS1 and EGLN1, the signature adaptation genes in Tibetans.

Major Comments:

1. The DEG analysis is the most central result in this manuscript, but the discrepancy between sex-combined and sex-specific DEGs is quite mind-boggling. For those that were differentially expressed in the sex-specific sets but not in the sex-combined one, the authors suggest an opposite direction of DE as an explanation (page 11, Figure S5). But Figure S5A does not show such a trend, showing that down-regulated genes in males are mostly not at all differentially expressed in females. Figure S6B does show such a trend, but it doesn't seem to be a dominant explanation. I would like to recommend the authors test alternative ways of analysis to boost statistical power for DEG detection other than simply splitting data into males and females and performing analysis in each subset. For example, the authors may consider utilizing gene-by-environment interaction analysis schemes here biological sex as an environmental factor.

2. Please clarify how the authors handled multiple testing correction of p-values.

3. The "natural selection acts on the placental DEGs ..." section is potentially misleading readers to assume that the manuscript reports evidence for positive selection on the observed DEG pattern between Tibetans and Han, which is not.<br /> a) Currently the section simply describes an overlap between DEGs and a set of 192 genes likely under positive selection in Tibetans (TSNGs). The overlap is quite small, leading to only 13 genes in total (Figure 6). The authors are currently not providing any statistical measure of whether this overlap is significantly enriched or at the level expected for random sampling.<br /> b) The authors are describing sets of DEGs that seem to affect important phenotypic changes in a consistent and adaptive direction. A relevant form of natural selection for this situation may be polygenic adaptation while the authors only consider strong positive selection at a single variant/gene level.<br /> c) The manuscript is currently providing no eQTL information that can explain the differential expression of key genes. The authors can actually do this based on the genotype and expression data of the individuals in this study. Combining eQTL info, they can set up a test for polygenic adaptation (e.g. Berg and Coop; https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004412). This will provide a powerful and direct test for the adaptiveness of the observed DEG pattern.

4. The manuscript is currently only minimally discussing how findings are linked to EPAS1 and EGLN1 genes, which show the hallmark signature of positive selection in Tibetans. In fact, the authors' group previously reported male-specific association between EPAS1 SNPs and blood hemoglobin level. Many readers will be intrigued to see a discussion about this point.

Reviewer #1 (Public Review):

The authors report a new bioinformatics pipeline ("SPICE") to predict pairwise cooperative binding-sites based on input ChIP-seq data for transcription factor (TF)-of-interest, analyzed against DNA-binding sites (DNA motifs) in a database (HOCOMOCO). The pipeline also predicts the optimal distance between the paired binding sites. The pipeline correctly predicted known/reported transcription factor cooperations, and also predicted new cooperations, not yet reported in literature. The authors choose to follow up on the predicted interaction between Ikaros and Jun. Using ChIP-seq in mouse B cells, they show extensive overlap in binding regions between Ikaros and Jun in LPS+IL21 stimulated cells. In a human B-lineage cell line (MINO) they show that anti-Ikaros Ab can co-immunoprecipitate Jun protein, and that the MINO cell extracts contain protein(s) that can bind to the CNS9 probe (conserved region upstream of IL10 gene), and that binding is lost upon mutation of two basepairs in the AP1 binding motif, and reduced upon mutation of two basepairs in the non-canonical Ikaros binding motif. Part of this protein complex is super-shifted with an anti-Jun antibody, and more DNA is shifted with addition of an anti-Ikaros antibody.

The authors perform EMSA showing that recombinant Jun can bind to the tested DNA-region (IL10 CNS9) and that addition of recombinant Ikaros (or anti-Ikaros antibody in Fig 3E) can enhance binding (increase amount of DNA shifted). The authors lastly show that the IL10 CNS9 DNA region can enhance transcription in B- and T-cells with a luciferase reporter assay, and that 2 bp mutation of the Ikaros or Jun DNA motifs greatly reduce or abolish this activity.

This is interesting work, with two main contributions: The SPICE pipeline (if made available to the scientific community), and the report of interaction between Ikaros and Jun. However, the distinction between DNA motifs, and the proteins actually binding and having a biological function, should be made clear consistently throughout the manuscript. The same DNA motifs can be bound by multiple factors, for instance within transcription factor families with highly homology in the DNA-binding regions of the proteins.

Some specific points:

SPICE: It is unclear if this is uploaded somewhere to be available to the scientific community.

It was unclear if Ikaros-Jun interaction was initially found from primary Jun ChIP-seq (and secondary Ikaros motif from HOCOMOCO) or from primary Ikaros CHIP-seq (and secondary Jun motif from HOCOMOCO). And - what were the two DNA motifs (primary and secondary, and their distance) from the SPICE analysis?

Authors have mostly careful considerations and statements. One additional comment is that binding does not equal function (Fig 2D), and that opening of chromatin (by any other factor(s)) can give DNA-binding factors (like Ikaros and Jun) the opportunity to bind, without functional consequence for the biological process studied.

Figure 2E: Ikaros is reported to be expressed at baseline in murine B cells, yet the Ikaros ChIP-seq in unstimulated cells had what looks to be no significant or low peaks. LPS stimulation induced strong Ikaros ChIP-seq signal. A western blot showing the Ikaros protein levels in the 3 conditions could help understand if the binding pattern is due to protein expression level induction. Similar for Jun (western in the 3 conditions), which seemed to mainly bind in the LPS+IL21 condition. Furthermore, as also suggested below, tracks showing Ikaros and Jun binding from all conditions (unstimulated, LPS only and LPS+IL21 stimulated cells), at select genomic loci, would be helpful in illustrating this difference in signal between the different cell conditions. This is relevant in regards to the point of cooperativity of binding.

ChIP-seq in mouse B cells showed that Ikaros bound strongly in LPS stimulated cells, in the (relative) absence of Jun binding (Fig. 2C). However, in EMSA (Fig 3C), there is no binding when the AP1 site is mutated, and the authors describe this as Ikaros binding site. What does the Ikaros binding look like at this genomic location in LPS (only) stimulated cells? The authors could show the same figure as in Fig 2F but show Ikaros and Jun ChIP-seq tracks at IL10 CNS9 locus from all conditions to compare binding in unstimulated, LPS and LPS+IL21 cells.

Also: How does this reconcile with the luciferase assay in Fig 4E, where LPS (only) stimulation is used, which in Fig 2E only/mainly induced Ikaros, and not Jun ChIP-seq signal (while EMSA indicate Ikaros cannot bind the site alone, but can enhance Jun-dependent binding).

Comment on statements in results section: The luciferase assays in B and T cells do not demonstrate the role of the proteins Ikaros or Jun directly (page 10, lines 208 and surrounding text). The assay measures an effect of the DNA sequences (implying binding of some transcription factor(s)), but does not identify which protein factors bind there.

Lastly, the authors only discuss Ikaros (using the term IKZF1 which is the gene symbol for the Ikaros protein). There are other Ikaros family members that have high homology and that are reported to bind similar DNA sequences (for instance Aiolos and Helios), which are expressed in B-cells and T-cells. A discussion of this is of relevance, as these are different proteins, although belonging to the same family (the Ikaros-family) of transcription factors. For instance, western for Aiolos and Helios will likely detect Aiolos in the B cells used, and Helios in the T cells used.

Reviewer #1 (Public Review):

In this manuscript, the authors explored the benefits of intermittent fasting on the cardiac physiology through a multi-omics approach and compared different fasting times (IF12; IF 16 and EOD) for a duration of 6 months. Combining the RNA-sequencing, proteomics and phosphor-proteomics analysis, the authors have made an interesting observation that different fasting times would lead to different changes that could be important for the cardiac physiology. Moreover, the changes observed at transcriptional level are different from protein level, suggesting a post-transcriptional regulation mechanism. Using western blot, the authors have confirmed the key signaling pathways, including AMPK, IRS pathway to be significantly altered upon intermittent fasting for 16hrs. Lastly, as a proof of concept for better cardiac function, the animals were challenged with dobutamine and echocardiography was performed to show the mice subjected to intermittent fasting have better cardiac systolic function.

The impact of intermittent fasting on cardiovascular health has been well characterized in several studies. This report appears to be the first one utilizing a multi-omics approach and provided an interesting dataset at transcriptome, proteome and phosphor-proteome levels, and would serve as a valuable data resource for the field. I have the following concerns:

Major concerns:

1) The rationale for choosing the intermittent fasting pattern and timing<br /> While the 16:8 intermittent fasting is relatively standard, what is the rationale to test IF 12 hours? As a 4 hour fasting difference might not cause dramatic changes in transcriptome and proteome. Also, what is the rationale to perform 6 months study? The dobutamine stress test is not a terminal procedure, have the authors examined the cardiac function prior to 6 months to see whether there is a difference?

2) Lack of validation study<br /> One interesting observation from this study is the changes of transcriptome does not reflect all the changes at protein level and there is a differential gene expression pattern in IF12, IF16 and EOD. If this is the case, the authors should select a few important targets and provide both mRNA and protein level analysis, as a proof of concept for the bioinformatics analysis accuracy.

3) Poor western blot image quality<br /> The quality of the western blot has several issues: a. the change of pAMPK/AMPK appears to be a decrease of total AMPK instead of change at p-AMPK level. Same with GSK3a/b. There appears to be an increase of total GSK3a/b. The AKT should also be blotted and quantified at phosphorylation level. The western blot should be clearly labeled, for the ones with double bands, including GSK3a/b, the author should clearly label which is GSK3a and which is GSK3b. For the IRS with non-specific band, the author should point out IRS-1 band itself.

Reviewer #1 (Public Review):

The manuscript focused on roles of a key fatty-acid synthesis enzyme, acetyl-coA-carboxylase 1 (ACC1), in the metabolism, gene regulation and homeostasis of invariant natural killer T (NKT_ cells and impact on these T cells' roles during asthma pathogenesis. The authors presented data showing that the acetyl-coA-carboxylase 1 enzyme regulates the expression of PPARg then the function of NKT cells including the secretion of Th2-type cytokines to impact on asthma pathogenesis. The results are clearcut and data were logically presented.

Reviewer #1 (Public Review):

Summary: A well-executed series of experiments that will likely be of immense interest to (a) vector-borne disease researchers and (b) gram-negative sepsis/bacteremia researchers. The study uses comparative transcriptomics to begin probing what makes Peromyscus leucopus a unique host for numerous pathogens. Most issues with the paper are trivial, relating to descriptions of statistical cutoffs. While the paper does not provide mechanistic insight into how P. leucopus restrains its immune response to LPS or other microbial invaders, it is likely that this paper will be frequently consulted by researchers trying to understand that phenomenon.

Strengths:

o Use of outbred M. musculus is a commendable choice for the studies here.<br /> o Excellent decision by the authors to use their published dataset (with appropriate statistical normalization) to improve their statistical power to examine sex-biased gene expression. Is it possible to go one step further and briefly incorporate their prior BALB/c data to see how the BALB/c compare to the outbred mice. This could perhaps be just a PCA plot to see if they cluster with the outbred mice and/or Peromyscus, or are separate.<br /> o The correlations and ratios used to try to understand immune cell dynamics are clever and likely reflect interesting biology, but caution should be used when interpreting these indirect measures. As there are no tools for cell separation in P. leucopus, the authors should continue to include these data to stimulate ideas in the field, but readers should understand the "conclusions" are hypotheses due to the nature of the bulk RNAseq.

Weaknesses:

o Supplemental Table 1 only lists genes that passed the authors statistical thresholds. The full list of genes detected in their analysis should be included with read counts, statistics, etc. as supplemental information<br /> o While P. leucopus is a critical reservoir for B. burgdorferi, caution should be taken in directly connecting the data presented here and the Lyme disease spirochete. While it's possible that P. leucopus have a universal mechanism for limiting inflammation in response to PAMPs, B. burgdorferi lack LPS and so it is also possible the mechanisms that enable LPS tolerance and B. burgdorferi tolerance may be highly divergent.<br /> o Statistical significance is binary and p-values should not be used as the primary comparator of groups (e.g. once a p-value crosses the deigned threshold for significance, the magnitude of that p-value no longer provides biological information). For instance, in comparing GO-terms, the reason for using of high p-value cutoffs ("None of these were up-regulated gene GO terms with p values < 1011 for M. musculus.") to compare species is unclear. If the authors wish to compare effect sizes, comparing enrichment between terms that pass a cutoff would likely be the better choice. Similarly, comparing DEG expression by p-value cutoff and effect size is more meaningful than analyses based on exclusively on p-value: "Of the top 100 DEGs for each species by ascending FDR p value." Description in later figures (e.g. Figure 4) is favored.<br /> o The ability to use of CD45 to normalize data is unclear. Authors should elaborate both on the use of the method and provide some data how the data change when they are normalized. For instance, do correlations between untreated Mus and Peromyscus gene expression improve? The authors seem to imply this should be a standard for interspecies comparison and so it would be helpful to either provide data to support that or, if applicable, use of the technique in literature should be referenced.<br /> o Regarding the ISG data-is a possible conclusion not that Peromyscus don't upregulate the antiviral response because it's already so high in untreated rodents? It seems untreated Peromyscus have ISG expression roughly equivalent to the LPS mice for some of the genes. This could be compared more clearly if genes were displayed as bar plots/box and whisker plots rather than in scatter plots. It is unclear why the linear regression is the key point here rather than normalized differences in expression.<br /> o Some sections of the discussion are under supported:<br />  The claim that low inflammation contributes to increased lifespan is stated both in the introduction and discussion. Is there justification to support this? Do aged pathogen-free mice show more inflammation than aged Peromyscus?<br />  The claim that reduced Peromyscus responsiveness could lead to increased susceptibility to infection is prominently proposed but not supported by any of the literature cited.<br />  References to B. burgdorferi, which do not have LPS, in the discussion need to ensure that the reader understands this and the potential that responses could be very different.

Reviewer #1 (Public Review):

The article by Reversade and colleagues reports new mutations in the PYCR1 in a progeroid disease associated with premature skin aging. Using human cell culture and a newly generated mouse model of PYCR1deficiency they identify a role for this factor in maintaining dermal homeostasis and ECM production. I have some minor concerns about the role of PYCR1 in fibroblast survival vs function and the quantification of western blots.

Reviewer #1 (Public Review):

Summary:

In this excellent manuscript by Egan et al., the authors very carefully dissect the roles of inflammasome components in restricting Salmonella Typhimurium (STm) replication in human macrophages. They show that caspase-1 is essential to mediating inflammasome responses and that caspase-4 contributes to bacterial restriction at later time points. The authors show very clear roles for the host proteins that mediate terminal lysis, gasdermin D and ninjurin-1. The unique finding in this study is that in the absence of inflammasome responses, Salmonella hypereplicates within the cytosol of macrophages. These findings suggest that caspase-1 and possibly caspase-4 play roles in restricting the replication of Salmonella in the cytosol as well as in the Salmonella containing vacuole.

Strengths:

1) The genetic and biochemical approaches have shown for the first time in human macrophages that the caspase-1-GSDMD-NINJ1 axis is very important for restricting intracellular STm replication. In addition, they demonstrate a later role for Casp4 in control of intracellular bacterial replication.

2) In addition, they show that in macrophages deficient in the caspase-1-GSDMD-NINJ1 axis that STm are found replicating in the cytosol, which is a novel finding. The electron microscopy is convincing that STm are in the cytosol.

3) The authors go on to use a chloroquine resistance assay to show that inflammasome signaling also restricts STm within SCVs in human macrophages.

4) Finally, they show that the Type 3 Secretion System encoded on Salmonella Pathogenicity Island 1 contributes to STm's cytosolic access in human macrophages.

Weaknesses:

1) Their results with human macrophages suggest that there are differences between murine and human macrophages in inflammasome-mediated restriction of STm growth. For example, Thurston et al. showed that in murine macrophages that inflammasome activation controls the replication of mutant STm that aberrantly invades the cytosol, but only slightly limits replication of WT STm. In contrast, here the authors found that primed human macrophages rely on caspase-1, gasdermin D and ninjurin-1 to restrict WT STm. I wonder if the priming of the human macrophages in this study could account for the differences in these studies. Along those lines, do the authors see the same results presented in this study in the absence of priming the macrophages with Pam3CSK4. I think that determining whether the control of intracellular STm replication is dependent on priming is very important. Another difference with the Thurston et al. paper is the way that the STm inoculum was prepared - stationary phase bacteria that were opsonized. Could this also account for differences between the two studies rather than differences between murine and human macrophages in inflammasome-dependent control of STm?<br /> 2) The authors show that the pore-forming proteins GSDMD and Ninj1 contribute to control of STm replication in human macrophages. Is it possible that leakage of gentamicin from the media contributes to this control?

3) One major question that remains to be answered is whether casp-1 plays a direct role in the intracellular localization of STm. If the authors quantify the percentage of vacuolar vs. cytosolic bacteria at early time points in WT and casp-1 KO macrophages, would that be the same in the presence and absence of casp-1? If so, then this would suggest that there is a basal level of bacterial-dependent lysis of the SCV and in WT macrophages the presence of cytosolic PAMPS trigger cell death and bacteria can't replicate in the cytosol. However, in the inflammasome KO macrophages, the host cell remains alive and bacteria can replicate in the cytosol.

Reviewer #1 (Public Review):

The manuscript by Sun and colleagues followed their previous findings on the tumor-suppressive role of PDLIM2 in lung cancer. They further investigated various mechanisms, including epigenetic modification, copy number variation, and LOH, that led to the decreased expression of PDLIM2 in human lung cancer. Next, they used a nanoparticle-based approach to specifically restore the expression in mouse lung tumors. They showed that over-expression of PDLIM2 in lung cancer repressed its progression in vivo. Also, this treatment could synergize with chemotherapy and checkpoint inhibitor anti-PD-1. Overall, the results were quite promising and convincing, using a treatment combination that would appear to have the potential for clinical implementation.

Reviewer #1 (Public Review):

Xie and Colleagues propose here to investigate the mechanism by which exercise inhibits bone metastasis progression. The authors describe that osteocyte, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing sEVs with tumor suppressor microRNAs. Furthermore, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Interestingly, exercise preconditioning effectively suppressed bone metastasis progression.

Reviewer #1 (Public Review):

In this manuscript, the authors demonstrated that YAP/TAZ promotes P-body formation in a series of cancer cell lines. YAP/TAZ modulates the transcription of multiple P-body-related genes, especially repressing the transcription of the tumor suppressor proline-rich nuclear receptor coactivator 1 (PNRC1) through cooperation with the NuRD complex. PNRC1 functions as a critical repressor in YAP-induced biogenesis of P-bodies and tumorigenesis in colorectal cancer (CRC). Reexpression of PNRC1 or disruption of P-bodies attenuated the protumorigenic effects of YAP. Overall, these findings are interesting and the study was well conducted.

Major concerns:

1. RNAseq data indicated that Yap has the capacity to suppress the expression of numerous genes. In addition to PNRC1, could there be additional Yap targeting factors involved in Yap-mediated the formation of P-bodies?<br /> 2. It is still not clear how PNRC1 regulates P-bodies. Knockdown of PNRC1 prevented the reduction of P-bodies caused by Yap knockdown. How do the genes related to P-bodies that are positively regulated by Yap, such as SAMD4A, AJUBA, and WTIP, change in this scenario? Given that the expression of Yap can differ considerably among various cell types, is it possible for P-bodies to be present in tumor cells lacking Yap expression?<br /> 3. The authors demonstrated that CHD4 can bind to Yap target genes, such as CTGF, AJUBA, SAMD4A (Figure 4 - Figure Supplement 1D). Does the NuRD complex repress the expression of these genes? the NuRD complex could prevent the formation of P-bodies?<br /> 4. YAP/TAZ promotes the formation of P-bodies which contradicts the previous study's conclusion (PMID: 34516278). Please address these inconsistent findings.

Reviewer #1 (Public Review):

This is a technically sound paper focused on a useful resource around the DRGP phenotypes which the authors have curated, pooled, and provided a user-friendly website. This is aimed to be a crowd-sourced resource for this in the future.

The authors should make sure they coordinate as well as possible with the NC datasets and community and broader fly community. It looks reasonable to me but I am not from that community.

I have only one major concern which in a more traditional review setting I would be flagging to the editor to insist the authors did on resubmission. I also have some scene setting and coordination suggestions and some minor textual / analysis considerations.

The major concern is that the authors do not comment on the distribution of the phenotypes; it is assumed it is a continuous metric and well-behaved - broad gaussian. This is likely to be more true of means and medians per line than individual measurements, but not guaranteed, and there could easily be categorical data in the future. The application of ANOVA tests (of the "covariates") is for example fragile for this.

The simplest recommendation is in the interface to ensure there is an inverse normalisation (rank and then project on a gaussian) function, and also to comment on this for the existing phenotypes in the analysis (presumably the authors are happy). An alternative is to offer a kruskal test (almost the same thing) on covariates, but note PLINK will also work most robustly on a normalised dataset.

Minor points:<br /> On the introduction, I think the authors would find the extensive set of human GWAS/PheWAS resources useful; widespread examples include the GWAS Catalog, Open Targets PheWAS, MR-base, and the FinnGen portal. The GWAS Catalog also has summary statistics submission guidelines, and I think where possible meta-data harmonisation should be similar (not a big thing). Of course, DRGP has a very different structure (line and individuals) and of course, raw data can be freely shown, so this is not a one-to-one mapping.

For some authors coming from a human genetics background, they will be interpreting correlations of phenotypes more in the genetic variant space (eg LD score regression), rather than a more straightforward correlation between DRGP lines of different individuals. I would encourage explaining this difference somewhere.

This leads to an interesting point that the inbred nature of the DRGP allows for both traditional genetic approaches and leveraging the inbred replication; there is something about looking at phenotype correlations through both these lenses, but this is for another paper I suspect that this harmonised pool of data can help.

I was surprised the authors did not crunch the number of transcript/gene expression phenotypes and have them in. Is this because this was better done in other datasets? Or too big and annoying on normalisation? I'd explain the rationale to leave these out.

I think 25% FDR is dangerously close to "random chance of being wrong". I'd just redo this section at a higher FDR, even if it makes the results less 'exciting'. This is not the point of the paper anyway.

I didn't buy the extreme line piece as being informative. Something has to be on the top and bottom of the ranks; the phenotypes are an opportunity for collection and probably have known (as you show) and cryptic correlations. I think you don't need this section at all for the paper and worry it gives an idea of "super normals" or "true wild types" which ... I just don't think is helpful.

I'd say "well-established inversion genotypes and symbiot levels" rather than generic covariates. Covariates could mean anything. You have specific "covariates" which might actually be the causal thing.

I wouldn't use the adjective tedious about curation. It's a bit of a value judgement and probably places the role of curation in the wrong way. Time-consuming due to lack of standards and best practice?

Reviewer #1 (Public Review):

General comments:<br /> This paper investigates the pH-specific enzymatic activity of mouse acidic mammalian chitinase (AMCase) and aims to elucidate its function's underlying mechanisms. The authors employ a comprehensive approach, including hydrolysis assays, X-ray crystallography, theoretical calculations of pKa values, and molecular dynamics simulations to observe the behavior of mouse AMCase and explore the structural features influencing its pH-dependent activity.

The study's key findings include determining kinetic parameters (Kcat and Km) under a broad range of pH conditions, spanning from strong acid to neutral. The results reveal pH-dependent changes in enzymatic activity, suggesting that mouse AMCase employs different mechanisms for protonation of the catalytic glutamic acid residue and the neighboring two aspartic acids at the catalytic motif under distinct pH conditions.

The novelty of this research lies in the observation of structural rearrangements and the identification of pH-dependent mechanisms in mouse AMCase, offering a unique perspective on its enzymatic activity compared to other enzymes. By investigating the distinct protonation mechanisms and their relationship to pH, the authors reveal the adaptive nature of mouse AMCase, highlighting its ability to adjust its catalytic behavior in response to varying pH conditions. These insights contribute to our understanding of the pH-specific enzymatic activity of mouse AMCase and provide valuable information about its adaptation to different physiological conditions.

Overall, the study enhances our understanding of the pH-dependent activity and catalytic properties of mouse AMCase and sheds light on its adaptation to different physiological pH environments.

Reviewer #1 (Public Review):

Summary:<br /> This study focuses on the defining cellular pathways critical for tRNA export from the nucleus. While a number of these pathways have been identified, the observation that the primary transport receptors identified thus far (Los1 and Msn5) are not essential and that cells are viable even when both the genes are deleted supports the idea that there are as yet unidentified mediators of tRNA export from the nucleus. This study implicates the helicase Dbp5 in one of these parallel pathways arguing that Dbp5 works in a pathway that is independent of Los1 and/or Msn5. The authors present genetic data to support this conclusion. At least one result suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathway to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway. The authors provide solid evidence that Dbp5 interacts with tRNA directly and that the addition of the factor Gle1 together with the previously identified co-factor InsP6 can trigger helicase activity and release of tRNA. The combination of in vivo studies and biochemistry provides evidence to consider how Dbp5 contributes to the export of tRNA and more broadly adds to the conversation about how coding and non-coding RNA export from the nucleus might be coordinated to control cell physiology.

Strengths and weaknesses:<br /> A major strength of this manuscript is the multi-pronged approach to explore a potential role for the helicase Dbp5 in one of the multiple export pathways for tRNA from the nucleus.

The obvious missing experiment here with respect to genetics is the test of whether deletion of the MSN5 gene in the cells, which combines deletion of LOS1 and the dbp5_R423A allele, shown in Figure 1D would be lethal. This key experiment would lend substance to the argument that Dbp5 functions in a tRNA export pathway that is parallel to the Los1 and Msn5 pathways.

While some of the binding assays show rather modest band shifts (Figure 4B for example), the data in Figure 4A showing that there is no binding detected unless a non-hydrolyzable ATP analogue is employed, argues for specificity in nucleic acid binding. The question that does arise is whether the binding is specific for tRNA.

With the exception of the binding studies, which also employ a mixture of yeast tRNAs, this study relies primarily on a single tRNA species to come to the conclusions drawn. Many other studies have used multiple tRNAs to explore whether pathways characterized are generalizable to other tRNAs.

The authors provide evidence of a model where the helicase Dbp5 plays a role in tRNA export from the nucleus. Further evidence is required to determine whether Dbp5 could function in the same pathway as the previously defined tRNA export receptors, Los1 and Msn5. There are genetic tests that could be performed to explore this question. Some of the biochemistry presented would show when Los1 is absent that the interaction of Dbp5 with tRNA decreases, which could support a model where Dbp5 plays a role in coordination with Los1.

This work allows insight into key questions which still remain about the multiple pathways that are required for tRNA trafficking as well as how transport pathways for coding and non-coding RNAs might be coordinated. These questions are important as many of these pathways may be regulated in response to cellular conditions or during development and defining the fundamental pathways will be critical to understanding these dynamic processes.

Reviewer #1 (Public Review):

The authors present a study focused on addressing the key challenge in drug discovery, which is the optimization of absorption and affinity properties of small molecules through in silico methods. They propose active learning as a strategy for optimizing these properties and describe the development of two novel active learning batch selection methods. The methods are tested on various public datasets with different optimization goals and sizes, and new affinity datasets are curated to provide up-to-date experimental information. The authors claim that their active learning methods outperform existing batch selection methods, potentially reducing the number of experiments required to achieve the same model performance. They also emphasize the general applicability of their methods, including compatibility with popular packages like DeepChem.

Strengths:

Relevance and Importance: The study addresses a significant challenge in the field of drug discovery, highlighting the importance of optimizing the absorption and affinity properties of small molecules through in silico methods. This topic is of great interest to researchers and pharmaceutical industries.

Novelty: The development of two novel active learning batch selection methods is a commendable contribution. The study also adds value by curating new affinity datasets that provide chronological information on state-of-the-art experimental strategies.

Comprehensive Evaluation: Testing the proposed methods on multiple public datasets with varying optimization goals and sizes enhances the credibility and generalizability of the findings. The focus on comparing the performance of the new methods against existing batch selection methods further strengthens the evaluation.

Weaknesses:

Lack of Technical Details: The feedback lacks specific technical details regarding the developed active learning batch selection methods. Information such as the underlying algorithms, implementation specifics, and key design choices should be provided to enable readers to understand and evaluate the methods thoroughly.

Evaluation Metrics: The feedback does not mention the specific evaluation metrics used to assess the performance of the proposed methods. The authors should clarify the criteria employed to compare their methods against existing batch selection methods and demonstrate the statistical significance of the observed improvements.

Reproducibility: While the authors claim that their methods can be used with any package, including DeepChem, no mention is made of providing the necessary code or resources to reproduce the experiments. Including code repositories or detailed instructions would enhance the reproducibility and practical utility of the study.

Suggestions for Improvement:

Elaborate on the Methodology: Provide an in-depth explanation of the two active learning batch selection methods, including algorithmic details, implementation considerations, and any specific assumptions made. This will enable readers to better comprehend and evaluate the proposed techniques.

Clarify Evaluation Metrics: Clearly specify the evaluation metrics employed in the study to measure the performance of the active learning methods. Additionally, conduct statistical tests to establish the significance of the improvements observed over existing batch selection methods.

Enhance Reproducibility: To facilitate the reproducibility of the study, consider sharing the code, data, and resources necessary for readers to replicate the experiments. This will allow researchers in the field to validate and build upon your work more effectively.

Conclusion:

The authors' study on active learning methods for optimizing drug discovery presents an important and relevant contribution to the field. The proposed batch selection methods and curated affinity datasets hold promise for improving the efficiency of drug discovery processes. However, to strengthen the study, it is crucial to provide more technical details, clarify evaluation metrics, and enhance reproducibility by sharing code and resources. Addressing these limitations will further enhance the value and impact of the research.

Reviewer #1 (Public Review):

The authors developed an extension to the pairwise sequentially Markov coalecent model that allows to simultaneously analyze multiple types of polymorphism data. In this paper, they focus on SNPs and DNA methylation data. Since methylation markers mutate at a much faster rate than SNPs, this potentially gives the method better power to infer size history in the recent past. Additionally, they explored a model where there are both local and regional epimutational processes.

Integrating additional types of heritable markers into SMC is a nice idea which I like in principle. However, a major caveat to this approach seems to be a strong dependence on knowing the epimutation rate. In Fig. 6 it is seen that, when the epimutation rate is known, inferences do indeed look better; but this is not necessarily true when the rate is not known. A roughly similar pattern emerges in Supp. Figs. 4-7; in general, results when the rates have to be estimated don't seem that much better than when focusing on SNPs alone. This carries over to the real data analysis too: the interpretation in Fig. 7 appears to hinge on whether the rates are known or estimated, and the estimated rates differ by a large amount from earlier published ones.

Overall, this is an interesting research direction, and I think the method may hold more promise as we get more and better epigenetic data, and in particular better knowledge of the epigenetic mutational process. At the same time, I would be careful about placing too much emphasis on new findings that emerge solely by switching to SNP+SMP analysis.

Reviewer #1 (Public Review):

Summary:<br /> The precise mechanism of how tetraspanin proteins engage in the generation of discs is still an open question in the field of photoreceptor biology. This question is of significance as the lack of photoreceptor discs or defects in disc morphogenesis due to mutations in tetraspanin proteins is a known cause of vision loss in humans. The authors of this study combine TEM and mouse models to tease out the role of tetraspanin proteins, peripherin, and Rom1 in the genesis of the photoreceptor discs. They show that the absence of Rom1 leads to an increase in peripherin and changes in disc morphology. Further rise in peripherin alleviates some of the defects observed in Rom1 knockout animals leading to the conclusion that peripherin can substitute for the absence of Rom1.

Strengths:<br /> A mouse model of Rom1 generated by the McInnes group in 2000 predicted a role for Rom1 in rim closure. They also showed enlarged discs in the absence of Rom1. This study confirmed this finding and showed the compensatory changes in peripherin, maintaining the total levels of tetraspanin proteins. Lack of Rom1 leads to excessive open disks demonstrated by darkly stained tannic acid-accessible areas in TEM. Interestingly, increased peripherin expression can rescue some morphological defects, including maintaining normal disc diameters and incisures. Overall, these observations lead authors to propose a model that ROM1 can be replaced by peripherin.

Weaknesses:<br /> The compensatory increase in peripherin and morphological rescue in the absence of ROM1 is expected, given the previous work from authors showing i) absence of peripherin showing increased ROM1 and ii) "Eliminating Rom1 also increased levels of Prph2/RRCT: mean Prph2/RRCT levels in P30 Prph2+/R retinas were 34% of WT, while levels in Prph2+/R/Rom1−/− retinas were 59% of WT" from Conley, 2019. The current study provides a comprehensive quantitative analysis. However, the mechanism behind the mechanism is unclear and warrants discussion.

Photoreceptor morphology appears better when peripherin is overexpressed. Is there a rescue of rod function (assessed by ERG or equivalent measures) in peripherin OE/Rom1-/- mice? Given the extensive work in this area and the implications the authors allude to at the end, it is important to investigate this aspect.

Reviewer #1 (Public Review):

In this study, the authors investigated the role of MAM and the Notch signaling pathway in the onset of the atrophic phenotype in both in vivo and in vitro models. The rationale used to obtain the data is one of the main strengths of the study. Already from the reading, the reasoning scheme used by the authors in setting up the study and evaluating the data obtained is clear. Using both cellular and mouse models in vivo consolidates the data obtained. The authors also methodologically described all the choices made in the supplementary section. A weakness, on the other hand, is the failure to include averages and statistical data in the results that would give a quantifiable idea of the data obtained. To complete the picture, the authors could also investigate the possible involvement of the intrinsic apoptosis pathway as well as describe probable metabolic shifts to muscle cells in atrophic conditions. The rationale used by the authors to obtain the result is linear. The data obtained are useful for understanding the onset and characterization of the atrophic phenotype under disuse and microgravity conditions. The methods used are in line with those used in the field and can be a starting point for other studies. The cellular models are well described in the materials and methods section. The selected mouse models followed a logical rationale and were in line with the intended aim.

Reviewer #1 (Public Review):

Summary:

Sender et al describe a model to estimate what fraction of DNA becomes cell-free DNA in plasma. This is of great interest to the community, as the amount of DNA from a certain tissue (for example, a tumor) that becomes available for detection in the blood has important implications for disease detection.

However, the authors' methods do not consider important variables related to cell-free DNA shedding and storage, and their results may thus be inaccurate. At this stage of the paper, the methods section lacks important detail. Thus, it is difficult to fully assess the manuscript and its results.

Strengths:

The question asked by the authors has potentially important implications for disease diagnosis. Understanding how genomic DNA degrades in the human circulation can guide towards ways to enrich for DNA of interest or may lead to unexpected methods of conserving cell-free DNA. Thus, the question "how much genomic DNA becomes cfDNA" is of great interest to the scientific and medical community. Once the weaknesses of the manuscript are addressed, I believe this manuscript has the potential to be a widely used resource.

Weaknesses:

There are two major weaknesses in how the analysis is presented. First, the methods lack detail. Second, the analysis does not consider key variables in their model.

Issues pertaining to the methods section.<br /> The current manuscript builds a flux model, mostly taking values and results from three previous studies:

1- The amount of cellular turnover by cell type, taken from Sender & Milo, 2021<br /> 2- The fractions of various tissues that contribute DNA to the plasma, taken from Moss et al, 2018 and Loyfer et al, 2023

My expertise lies in cell-free DNA, and so I will limit my comments to the manuscripts in (2).

Paper by Loyfer et al (additional context):

Loyfer et al is a recent landmark paper that presents a computational method for deconvoluting tissues of origin based on methylation profiles of flow-sorted cell types. Thus, the manuscript provides a well-curated methylation dataset of sorted cell-types. The majority of this manuscript describes the methylation patterns and features of the reference methylomes (bulk, sorted cell types), with a smaller portion devoted to cell-free DNA tissue of origin deconvolution.

I believe the data the authors are retrieving from the Loyfer study are from the 23 healthy plasma cfDNA methylomes analyzed in the study, and not the re-analysis of the 52 COVID-19 samples from Cheng et al (MED 2021).

Paper by Moss et al (additional context):

Moss et al is another landmark paper that predates the Loyfer et al manuscript. The technology used in this study (methylation arrays) is outdated but is an incredible resource for the community. This paper evaluates cfDNA tissues of origin in health and different disease scenarios. Again, I assume the current manuscript only pulled data from healthy patients, although I cannot be sure as it is not described in the methods section.

This manuscript:

The current manuscript takes (I think) the total cfDNA concentration from males and females from the Moss et al manuscript (pooled cfDNA; 2 young male groups, 2 old male groups, 2 young female groups, 2 old female groups, Supplementary Dataset; "total_cfDNA_conc" tab). I believe this is the data used as total cfDNA concentration. It would be beneficial for all readers if the authors clarified this point.

The tissues of origin, in the supplemental dataset ("fraction" tab), presents the data from 8 cell types (erythrocytes, monocytes/macrophages, megakaryocytes, granulocytes, hepatocytes, endothelial cells, lymphocytes, other). The fractions in the spreadsheet do not match the Loyfer or Moss manuscripts for healthy individuals. Thus, I do not know what values the supplementary dataset represents. I also don't know what the deconvolution values are used for the flux model.

The integration of these two methods lack detail. Are the authors here using yields (ie, cfDNA concentrations) from Moss et al, and tissue fractions from Loyfer et al? If so, why? There are more samples in the Loyfer manuscript, so why are the samples from Moss et al. being used? The authors are also selectively ignoring cell-types that are present in healthy individuals (Neurons from Moss et al, 2018). Why?

Appraisal:

At this stage of the manuscript, I think additional evidence and analysis is required to confirm the results in the manuscript.

Impact:

Once the authors present additional analysis to substantiate their results, this manuscript will be highly impactful on the community. The field of liquid biopsies (non-invasive diagnostics) has the potential to revolutionize the medical field (and has already in certain areas, such as prenatal diagnostics). Yet, there is a lack of basic science questions in the field. This manuscript is an important step forward in asking more "basic science" questions that seek to answer a fundamental biological question.

Reviewer #1 (Public Review):

The association of vitamin D supplementation in reducing Asthma risk is well studied, although the mechanistic basis for this remains unanswered. In the presented study, Kilic and co-authors aim to dissect the pathway of Vitamin D mediated amelioration of allergic airway inflammation. They use initial leads from bioinformatic approaches, which they then associate with results from a clinical trial (VDAART) and then validate them using experimental approaches in murine models. The authors identify a role of VDR in inducing the expression of the key regulator Ikzf3, which possibly suppresses the IL-2/STAT5 axis, consequently blunting the Th2 response and mitigating allergic airway inflammation.

Strengths:<br /> The major strength of the paper lies in its interdisciplinary approach, right from hypothesis generation, and linkage with clinical data, as well as in the use of extensive ex vivo experiments and in vivo approaches using knock-out mice.

The study presents some interesting findings including an inducible baseline absence/minimal expression of VDR in lymphocytes, which could have physiological implications and needs to be explored in future studies.

Weaknesses:<br /> The core message of the study relies on the role of vitamin D and its receptor in suppressing the Th2 response. However, there is scope for further dissection of relevant pathophysiological parameters in the in vivo experiments, which would enable stronger translation to allergic airway diseases like Asthma.

To a large extent, the authors have been successful in validating their results, although a few inferences could be reinforced with additional techniques, or emphasised in the discussion section (possibly utilising the ideas and speculative section offered by the journal).

The study inferences also need to be read in the context of the different sub-phenotypes and endotypes of Asthma, where the Th2 response may not be predominant. Moreover, the authors have referenced vitamin D doses for the murine models from the VDAART trials and performed the experiments in the second generation of animals. While this is appreciated, the risk of hypervitaminosis-D cannot be ignored, in view of its lipid solubility. Possibly comparison and justification of the doses used in murine experiments from previous literature, as well as the incorporation of an emphasised discussion about the side effects and toxicity of Vitamin D, is an important aspect to consider.

In no way do the above considerations undermine the importance of this elegant study which justifies trials for vitamin D supplementation and its effects on Asthma. The work possesses tremendous potential.

Reviewer #1 (Public Review):

Hwang et al., report that LRRC23 is required for RS3 head assembly and sperm motility, and the truncating LRRC23 is associated with asthenozoospermia in humans. They identified an LRRC23 variant in a consanguineous Pakistani family with infertile males diagnosed as asthenozoospermia and found this variant leads to early termination of LRRC23 translation with loss of 136 amino acids at the C-terminus. They generated Lrrc23 mutant mice that mimic the predicted outcome in human patients and found the truncated LRRC23 specifically disorganizes RS3 and the junctional structure between RS2 and RS3 in the sperm axoneme, which causes sperm motility defects and male infertility. These dates try to elucidate the pathogenicity of LRRC23 in asthenozoospermia. The conclusions of this paper are mostly well supported by data, but many aspects of data analyses and data interpretations need to be improved.

1) The pathogenesis of truncating LRRC23 in asthenozoospermia needs to be further considered. The molecular mechanism of LRRC23 demonstrated in mice should be tested in patients with the LRRC23 variant. As it may be difficult to determine the structures of RS3 in the infertile male sperm, the LRRC23 localization should be observed in the sperm from patients with the LRRC23 variant.<br /> 2) The absence of the RS3 head in LRRC23Δ/Δ mouse sperm is not sufficient to support the specific localization of LRRC23 in RS3 head. Although LRRC23 might bind to RS head protein RSPH9, the authors state that "RSPH9 is a head component of RS1 and RS2 like in C. reinhardtii (Gui et al, 2021), but not of RS3" as the protein level and the localization of RSPH9 is not altered in LRRC23Δ/Δ sperm. Thus, the specific localization of LRRC23 in RS3 head should be further confirmed.<br /> 3) The interaction between LRRC23 and RSPH9 needs to be defined. AlphaFold models could help determine the likelihood of a direct interaction. In addition, the structure of the 96-nm modular repeats of axonemes from the flagella of human respiratory cilia has been determined (PMID: 37258679), and the localization of LRRC23 in RS could be further predicted.<br /> 4) The ortholog of the RSP15 may also be predicted or confirmed by using the reported structure in human respiratory cilia (PMID: 37258679). Whether the LRCC34 in RS2 is LRRC34?

Reviewer #1 (Public Review):

Reinforcement mechanisms play a central role in learning structured behaviors, and recent studies in the songbird have shown that reinforcement learning is also integral to the imitation of the internally motivated singing behavior of songbirds. In this study, Roeser, Teoh et al. investigate the role of the lateral habenula in this process. The lateral habenula is thought to signal unexpected aversive outcomes, like reward omission, and inhibit dopaminergic neurons in the ventral tegmental area (VTA) via direct synaptic projections. Thus, the lateral habenula could logically play a key role in the trial-and-error learning of song by signaling worse performance outcomes (as evaluated by comparing to a memory of the tutor song) as birds practice copying their father's song.

The authors show that both the anatomical and functional connectivity of the lateral habenula in songbirds resembles what has been described in other vertebrates, including in afferent inputs from the ventral pallidum and efferent projections to the VTA that suppresses activity of putative dopaminergic neurons. Additionally, they show the lateral habenula circuits appear to be integrated with circuits known to be important for learning song, including receiving input from an auditory region, AIV, thought to be important in relaying song evaluation signals and providing inputs to VTA that overlap with neurons projecting to areas of the striatum essential for vocal learning (VTA-Area X neurons). They conclude that lesions of the lateral habenula early in song development do not disrupt a bird's ability to accurately imitate the song of their tutor but result in either the retention or development of unusual vocalizations that have qualities observed in the songs of zebra finches that have been experimentally raised without having access to a song tutor. The analysis of the adult song behavior is particularly compelling and provides novel approaches for identifying outlier vocalizations. Lastly, the authors show that birds will include these isolate-like syllables during courtship behaviors and that lesions of the lateral habenula do lead to disruptions in adult birds.

The conclusions stemming from the analysis of habenula connectivity require stronger support, and incomplete evidence is provided to link lesions of the lateral habenula to the observed disruptions in song learning.

This study has several strengths. First, the goal of understanding the role of the lateral habenula in natural learning of a complex behavior, like birdsong, is a valuable research avenue that can ultimately better link how natural learning of intrinsically rewarded behaviors may (or may not) harness similar learning mechanisms that have been well delineated in laboratory trained and externally reinforced behaviors. Second, the computational approaches brought to bear on the analysis of song, including variational autoencoders to help define the range of control song syllables from abnormal song syllables and anomaly scores, help provide a good framework for examining and conveying disruptions in behavior that might be associated with lesions of the lateral habenula. Lastly, the manuscript is well-written and clearly presented, and the authors do acknowledge some of the weaknesses mentioned below.

The major weakness of the article is that the authors do not verify the completeness (i.e., how much of the lateral habenula is lesioned in individual animals) or the extent (if neuronal regions adjacent to the lateral habenula neural are also lesioned) of their lesions. It is argued that this is not possible because of the timeframe (long survival times) of the experiments. However, there are standard ways of addressing this technical hurdle. One simple approach would be to first examine the correlation in the number of retrogradely labeled neurons in LHb, VP, and Area X following injections of tracer into VTA. For convergent anatomical pathways, there is typically a strong positive correlation across input circuits. Therefore, given the number of retrogradely labeled neurons in VP and Area X following VTA injections, one can make reasonable predictions for how many retrogradely labeled neurons would be expected in LHb. Using tracer injections at the end of the experiments and quantification of the retrograde labeling would allow the authors to reasonably estimate the completeness of their lesions.

This unfortunate problem with the design of the experiments significantly weakens any interpretations for the role of the lateral habenula in song learning. This is particularly important because the lateral habenula is a small area that has several adjacent brain structures that could also play significant roles in song development, most of which have not been well studied in this context. These include the medial habenula, the thalamic nuclei DMP and UVA, and forebrain axons from RA, as well as axons flowing into, out of, and interconnecting the structures previously mentioned. Additional tracer injections with different color tracers could be used to provide reasonable assurance that these other adjacent circuits are still intact at the end of each lesion experiment.

There are two weaknesses with the assessment of the functional connectivity of the lateral habenula. First, the anatomical tracing experiments are not particularly compelling. Very little data is shown and there is no quantification of any of the results. In the inset for retrograde labeling of VP-LHb and VP-VTA neurons, it is unclear that neurons of either population are shown in that image. Likewise, terminals from LHb in VTA are very sparse and it is not clear how well they overlap with VTA-X neurons which are intermingled with dopaminergic neurons projecting to other areas of the brain. The images shown seem out of focus and blurry. Although the electrophysiological experiments provide better assurance of these pathways, the sample sizes in these neurophysiology experiments seem preliminary. Stronger evidence in both regards would provide better assurance of LHb circuitry.

The interpretations and theoretical implications of these results are unclear. This is in part because it is not possible to fully tie behavioral outcomes specifically to lesions of the lateral habenula, but also because, albeit interesting, the behavioral results are somewhat confusing. The developmental lesions did not impact the ability of zebra finches to learn how to copy the song of their tutor over development, indicating, in a strict sense, this circuit is not needed for vocal imitation of a social model. However, birds clearly exhibit unusual song syllables that they throw into their song bouts, even when singing in courtship displays. What this may reflect is not addressed in this study. It could be that lesions disrupt a bird's ability to prune away poor syllables over development, and/or that lesions result in birds being unable to suppress unwanted vocal behaviors during performances. Analysis of song over development could provide insights into these possibilities and help provide a better understanding of what the lateral habenula contributes to the song-learning process.

Reviewer #1 (Public Review):

Summary: This impressive study by Bandet and Winship uses 2-photon imaging in awake-behaving mice to examine long-term changes in neural activity and functional connectivity after focal ischemic stroke. The authors discover that there are long-lasting perturbations in neural activity and functional connectivity, specifically within peri-infarct cortex but not more distant cortical regions. Overall I thought the study provided important new findings that were supported by compelling data.

Strengths: This is a technically challenging study to perform and the authors show high-quality data. The manuscript was well-written, and the figures were clearly presented. I really like the analytic tools they applied, which were rigorous and provided some novel insights regarding neural activity patterns during movement or rest. The discovery of long-lasting impairments in neural activity/functional connectivity is an important one as it is important for future stroke studies to recognize what problems need to be rectified in the post-stroke brain, even many weeks after injury. They also suggest there is a much more nuanced relationship between macroscopic changes in somatosensory maps and single-cell activity. Overall, I think this is a well-executed study whose primary conclusions were justified by the data presented.

Weaknesses: I found very little in the way of weaknesses. The statistics were notably conservative and are appropriate.

Reviewer #1 (Public Review):

Existing literature suggests that brain structures implicated in memory such as the hippocampus, and reward/punishment processing such as the striatal regions are also engaged in learning and value-based decision-making. However, how the contributions of these regions to learning and value-based decision-making change over time, particularly in children where these neural systems show protracted maturation was not studied systematically. This is the question the authors are aiming to address in this work in which children 6-to-7-years-old were recruited for a neuroimaging study that involves taking structural scans from this cohort to investigate how they correlate with changes in the way children approach a reinforcement learning task in which they learn to identify the better shape between 2 options through trial-and-error.

Particular strengths of the paper are longitudinally following up a cohort of small children and engaging them in a value-based decision-making task so that the relationship between neural maturation and improvements in reinforcement learning can be studied reliably. Towards this end, the authors make use of well-established computational modelling approaches to extract key parameters such as learning rates (which designate the speed of learning from expected versus actual outcomes) or choice stochasticity (which designate the inherent variation in people's decisions and the tendency to explore between the options) from children's choices so that their structural neural correlates can be established. As a part of this endeavour, the authors rely on methodological choices which do not warrant much criticism. Their data visualization choices are particularly spot-on and highly informative about the details of the raw data.

Also considering the importance of the hippocampal system in human memory, the key contribution of the paper is that the volumetric increases in hippocampus size between 2 assessment points correlated selectively with the delayed, but not immediate, learning score which refers to the learning condition in which the outcome feedback is given to the children after a 5-seconds delay. Although the authors also demonstrate evidence to suggest that changes in the striatal volume are also implicated in learning performance, this was more general as associations were found for both immediate and delayed feedback conditions. Thus, the paper makes an important contribution to the fields of developmental and decision neuroscience. An important question arising from the authors' findings could be that, whether the hippocampus maintains this selective role in value-based learning during the course of neuronal development, for example, whether a similar association would be found in children 8-to-9 years old. A better understanding of how these developmental trajectories map onto changes in learning and decision-making can inform fields outside neuroscience, for example tailoring educational approaches onto neural development pathways to boost learning efficiency in young children.

Reviewer #1 (Public Review):

The aim of this study is to test the overarching hypothesis that plasticity in BNST CRF neurons drives distinct behavioral responses to unpredictable threat in males and females. The manuscript provides evidence for a possible sex-specific role for CRF-expressing neurons in the BNST in unpredictable aversive conditioning and subsequent hypervigilance across sexes. As the authors note, this is an important question given the high prevalence of sex differences in stress-related disorders, like PTSD, and the role of hypervigilance and avoidance behaviors in these conditions. The study includes in vivo manipulation, bulk calcium imaging, and cellular resolution calcium imaging, which yield important insights into cell-type specific activity patterns. However, it is difficult to generate an overall conclusion from this manuscript, given that many of the results are inconsistent across sexes and across tests and there is an overall lack of converging evidence. For example, partial conditioning yields increased startle in males but not females, yet, CRF KO only increases startle response in males after full conditioning, not partial, and CRF neurons show similar activity patterns between partial and full conditioning across sexes. Further, while the study includes a KO of CRF, it does not directly address the stated aim of assessing whether plasticity in CRF neurons drives the subsequent behavioral<br /> effects unpredictable threat.

A major strength of this manuscript is the inclusion of both males and females and attention to possible behavioral and neurobiological differences between them throughout. However, to properly assess sex-differences, sex should be included as a factor in ANOVA (e.g. for freezing, startle, and feeding data in Figure 1) to assess whether there is a significant main effect or interaction with sex. If sex is not a statistically significant factor, both sexes should be combined for subsequent analyses. See, Garcia-Sifuentes and Maney, eLife 2021 https://elifesciences.org/articles/70817. There are additional cases where t-tests are used to compare groups when repeated measures ANOVAs would be more appropriate and rigorous.

Additionally, it's unclear whether the two sexes are equally responsive to the shock during conditioning and if this is underlying some of the differences in behavioral and neuronal effects observed. There are some reports that suggest shock sensitivity differs across sexes in rodents, and thus, using a standard shock intensity for both males and females may be confounding effects in this study.

The data does not rule out that BNST CRF activity is not purely tracking the mobility state of the animal, given that the differences in activity also track with differences in freezing behavior. The data shows an inverse relationship between activity and freezing. This may explain a paradox in the data which is why males show a greater suppression of BNST activity after partial conditioning than full conditioning, if that activity is suspected to drive the increased anxiety-like response. Perhaps it reflects that activity is significantly suppressed at the end of the conditioning session because animals are likely to be continuously freezing after repeated shock presentations in that context. It would also explain why there is less of a suppression in activity over the course of the recall session, because there is less freezing as well during recall compared with conditioning.

A mechanistic hypothesis linking BNST CRF neurons, the behavioral effects observed after fear conditioning, and manipulation of CRF itself are not clearly addressed here.

Reviewer #1 (Public Review):

This work by Gonzalez-Segarra et al. greatly extends previous research from the same group that identified ISNs as a key player in balancing nutrition and water ingestion. Using well-balanced sets of exploratory anatomical analyses and rigorous functional experiments, the authors identify and compile various peptidergic circuits that modulate nutrient and/or water ingestion. The findings are convincing and the experiments rigorous.

Strengths:<br /> - The authors complement anatomically-reconstructed and functionally-validated neuronal connectivity with extensive and intensive morphological and synaptic reconstruction.

- Neurons and genes involved in specific components of feeding control are undoubtedly challenging, because numerous neurons and circuits redundantly and reciprocally regulate the same components of feeding behavior. This work dissociates how multiple, parallel and interconnected, peptidergic circuits (dilp3, CCHa2, CCAP) modulate sucrose and water ingestion, in tandem and in parallel.

- The authors address some of the incongruencies/discrepancies in current literature (IPCs) and try to provide explanations, rather than ignoring inconsistent findings.

Weaknesses:<br /> - The authors have addressed several weaknesses of the paper in the revised text.

Reviewer #1 (Public Review):

Summary:<br /> In this study, the authors provide a new computational platform called Vermouth to automate topology generation, a crucial step that any biomolecular simulation starts with. Given a wide arrange of chemical structures that need to be simulated, varying qualities of structural models as inputs obtained from various sources, and diverse force fields and molecular dynamics engines employed for simulations, automation of this fundamental step is challenging, especially for complex systems and in case that there is a need to conduct high-throughput simulations in the application of computer-aided drug design (CADD). To overcome this challenge, the authors develop a programming library composed of components that carry out various types of fundamental functionalities that are commonly encountered in topological generation. These components are intended to be general for any type of molecules and not to depend on any specific force field and MD engines. To demonstrate the applicability of this library, the authors employ those components to re-assemble a pipeline called Martinize2 used in topology generation for simulations with a widely used coarse-grained model (CG) MARTINI. This pipeline can fully recapitulate the functionality of its original version Martinize but exhibit greatly enhanced generality, as confirmed by the ability of the pipeline to faithfully generate topologies for two high-complexity benchmarking sets of proteins.

Strengths:<br /> The main strength of this work is the use of concepts and algorithms associated with induced subgraph in graph theory to automate several key but non-trivial steps of topology generation such as the identification of monomer residue units (MRU), the repair of input structures with missing atoms, the mapping of topologies between different resolutions, and the generation of parameters needed for describing interactions between MRUs.

Weaknesses:<br /> Although the Vermouth library appears promising as a general tool for topology generation, there is insufficient information in the current manuscript and a lack of documentation that may allow users to easily apply this library. More detailed explanation of various classes such as Processor, Molecule, Mapping, ForceField etc. that are mentioned is still needed, including inputs, output and associated operations of these classes. Some simple demonstration of application of these classes would be of great help to users. The formats of internal databases used to describe reference structures and force fields may also need to be clarified. This is particularly important when the Vermouth needs to be adapted for other AA/CG force fields and other MD engines.

The successful automation of the Vermouth relies on the reference structures that need to be pre-determined. In case of the study of 43 small ligands, the reference structures and corresponding mapping to MARTINI-compatible representations for all these ligands have been already defined in the M3 force field and added into the Vermouth library. However, the authors need to comment on the scenario where significantly more ligands need to be considered and other force fields need to be used as CG representations with a lack of reference structures and mapping schemes.

Reviewer #1 (Public Review):

The authors investigated the function of BATF in hepatic lipid metabolism. They found BATF alleviated high-fat diet (HFD)-induced hepatic steatosis. In addition, BATF could inhibit programmed cell death protein (PD)1 expression induced by HFD. By using over expression and transcriptional activity analysis, this study confirmed that BATF regulates fat accumulation by inhibiting PD1 expression and promoting energy metabolism. Then, they found PD1 antibodies alleviated hepatic lipid deposition. These data identified the regulatory role of BATF in hepatic lipid metabolism and that PD1 is a target for alleviation of NAFLD. The conclusions of this manuscript are supported by the data.

Reviewer #1 (Public Review):

A typical path from preprocessed data to findings in systems neuroscience often includes set of analyses that often share common components. For example, an investigator might want to generate plots that relating one time series (e.g., a set of spike times) to another (measurements of a behavioral parameter such as pupil diameter or running speed). In most cases, each individual scientist writes their own code to carry out these analyses, and thus the same basic analysis is coded repeatedly. This is problematic for several reasons, including the inefficiency of different people writing the same code over and over again.

This paper presents Pynapple, a python package that aims to address those problems.

Strengths:

The authors have identified a key need in the community - well written analysis routines that carry out a core set of functions and can import data from multiple formats. In addition, they recognized that there are some common elements of many analyses, particularly those involving timeseries, and their object-oriented architecture takes advantage of those commonalities to simplify the overall analysis process.

The package is separated into a core set of applications and another with more advanced applications, with the goal of both providing a streamlined base for analyses and allowing for implementations/inclusion of more experimental approaches.

Weaknesses:

The revised version of the paper does a very good job of addressing previous concerns. It would be slightly more accurate in the Highlights section to say "A lightweight and standalone package facilitating long-term backward compatibility" but this is a very minor issue.

Ideally in the evening, before sleep, do some activity or activities that turn off the mind. You want to relax and stop thinking so much.

Interestingly enough, forgiveness, or the act of forgiving makes relaxing easy. So, if you have someone, or even yourself, to forgive... Do this right before going to sleep :)

Apparently, cold shower for roughly 3-4 minutes (rather than a hot shower) before sleep are helpful for sleep, as it decreases the core body temperature.

When you wake up, get sunlight in. Andrew Huberman also advocates for that. It tells the brain and body to wake up. It creates cortisol.

Can be combined with movement/exercise as well which also increases sleep quality. (Movement should not to be too late, however.)

Consistency is key when it comes to sleep.

Always go to bed at the same time and wake up at the same time.

Apparently, some Magnesiums can help with deep sleep.

Author takes 400mg.

It is important to block blue light in the evening. Blue light sends signals to your body to be awake.

One of the things to optimize sleep is to take care of meal timing. Author eats: - Breakfast at 8 - Lunch at noon (12) - Dinner between 5 and 6.30

Discipline and consistency is important here.

Essential is to eat dinner 3+ hours before you go to sleep.

Food increases core body temperature which negatively impacts sleep.

Reviewer #1 (Public Review):

This manuscript addresses the important and understudied issue of circuit-level mechanisms supporting habituation, particularly in pursuit of the possible role of increases in the activity of inhibitory neurons in suppressing behavioral output during long-term habituation. The authors make use of many of the striking advantages of the larval zebrafish to perform whole brain, single neuronal calcium imaging during repeated sensory exposure, and high throughput screening of pharmacological agents in freely moving, habituating larvae. Notably, several blockers/antagonists of GABAA(C) receptors completely suppress habituation of the O-bend escape response to dark flashes, suggesting a key role for GABAergic transmission in this form of habituation. Other substances are identified that strikingly enhance habituation, including melatonin, although here the suggested mechanistic insight is less specific. To add to these findings, a number of functional clusters of neurons are identified in the larval brain that have divergent activity through habituation, with many clusters exhibiting suppression of different degrees, in line with adaptive filtration during habituation, and a single cluster that potentiates during habituation. Further assessment reveals that all of these clusters include GABAergic inhibitory neurons and excitatory neurons, so we cannot take away the simple interpretation that the potentiating cluster of neurons is inhibitory and therefore exerts an influence on the other adapting (depressing) clusters to produce habituation. Rather, a variety of interpretations remain in play.

Overall, there is great potential in the approach that has been used here to gain insight into circuit-level mechanisms of habituation. There are many experiments performed by the authors that cannot be achieved currently in other vertebrate systems, so the manuscript serves as a potential methodological platform that can be used to support a rich array of future work. While there are several key observations that one can take away from this manuscript, a clear interpretation of the role of GABAergic inhibitory neurons in habituation has not been established. This potential feature of habituation is emphasized throughout, particularly in the introduction and discussion sections, meaning that one is obliged as a reader to interrogate whether the results as they currently stand really do demonstrate a role for GABAergic inhibition in habituation. Currently, the key piece of evidence that may support this conclusion is that picrotoxin, which acts to block some classes of GABA receptors, prevents habituation. However, there are interpretations of this finding that do not specifically require a role for modified GABAergic inhibition. For instance, by lowering GABAergic inhibition, an overall increase in neural activity will occur within the brain, in this case below a level that could cause a seizure. That increase in activity may simply prevent learning by massively increasing neural noise and therefore either preventing synaptic plasticity or, more likely, causing indiscriminate synaptic strengthening and weakening that occludes information storage. Sensory processing itself could also be disrupted, for instance by altering the selectivity of receptive fields. Alternatively, it could be that the increase in neural activity produced by the blockade of inhibition simply drives more behavioral output, meaning that more excitatory synaptic adaptation is required to suppress that output. The authors propose two specific working models of the ways in which GABAergic inhibition could be implemented in habituation. An alternative model, in which GABAergic neurons are not themselves modified but act as a key intermediary between Hebbian assemblies of excitatory neurons that are modified to support memory and output neurons, is not explored. As yet, these or other models in which inhibition is not required for habituation, have not been fully tested.

This manuscript describes a really substantial body of work that provides evidence of functional clusters of neurons with divergent responses to repeated sensory input and an array of pharmacological agents that can influence the rate of a fundamentally important form of learning.

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to compare, from testis tissues at different ages from mice in vivo and after culture, multiple aspects of Leydig cells. These aspects included mRNA levels, proliferation, apoptosis, steroid levels, protein levels, etc. A lot of work was put into this manuscript in terms of experiments, systems, and approaches. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation.

Second review:

The authors should be commended for substantial improvement in their manuscript for resubmission.

Sleep is the absolute foundation for top-performance.

Measure it. Track it. Optimize it.

After that, results WILL go into the sky.

Reviewer #1 (Public Review):

The authors managed to show the broad botanical landscape and not only the main crops. This unique achievement is based on decades of establishing an excellent collection of a full comparative seed collection of the current flora. This allows the identification of species that usually are not identifiable. The authors were able to compare the crops that were grown there and identify the contribution of the Roman period with that of the Arab one. This excellent study is a landmark in how such studies should be done. The list of identified species will be used for many other studies on this subject.

Reviewer #1 (Public Review):

The paper proposes a novel approach, named ModCRE, which utilizes structure-based learning to predict the DNA binding preferences of transcription factors (TFs). The authors integrate both experimental knowledge of the structures of TF-DNA complexes and large amounts of high-throughput TF-DNA interaction data. Additionally, the authors have developed a server that automatically produces these characteristics for other TFs and their complexes with co-factors.

Strengths: The paper's integration of experimental knowledge and high-throughput data to develop statistical knowledge-based potentials to score the binding capability of TFs in cis-regulatory elements is a powerful strategy. The proposed approach can be applied to more than 80% of TF sequences, making it a general method for characterizing binding preferences.

Weaknesses: The paper is difficult to follow, as it contains many technical details and implementation details. The method applied is not always clear, and the paper focuses on implementation rather than the message. The results indicate that the nearest neighbors approach in Figure 4 outperforms the proposed method in many cases, and the proposed method seems to perform better only when similarity with the target is low. The same applies in Fig. 5 when using normalized ranked scores.

It appears that the authors have successfully developed a structure-based learning approach for predicting DNA binding preferences of transcription factors. However, the paper's technical language and implementation focus make it challenging to follow at times.

It seems the authors have successfully achieved most of their aims in improving predictions for TF-DNA interaction, and the results support their conclusions.

This work has the potential to significantly impact the field of TF-DNA binding and gene regulation, particularly for those interested in predicting PWMs for TFs with limited or unreliable experimental data.

Reviewer #1 (Public Review):

Summary:<br /> Rai1 encodes the transcription factor retinoic acid-induced 1 (RAI1), which regulates expression of factors involved in neuronal development and synaptic transmission. Rai1 haploinsufficiency leads to the monogenic disorder Smith-Magenis syndrome (SMS), which is associated with excessive feeding, obesity and intellectual disability. Consistent with findings in human subjects, Rai1+/- mice and mice with conditional deletion of Rai1 in Sim+ neurons, which are abundant in the paraventricular nucleus (PVN), exhibit hyperphagia, obesity and increased adiposity. Furthermore, RAI1-deficient mice exhibit reduced expression of brain-derived neurotrophic factor (BDNF), a satiety factor essential for the central control of energy balance. Notably, overexpression of BDNF in PVN of RAI1-deficient mice mitigated their obesity, implicating this neurotrophin in the metabolic dysfunction these animals exhibit. In this follow up study, Javed et al. interrogated the necessity of RAI1 in BDNF+ neurons promoting metabolic health.

Consistent with previous reports, the authors observed reduced BDNF expression in the hypothalamus of Rai1+/- mice. Moreover, proteomics analysis indicated impairment in neurotrophin signaling in the mutants. Selective deletion of Rai1 in BDNF+ neurons in the brain during development resulted in increased body weight, fat mass and reduced locomotor activity and energy expenditure without changes in food intake. There was also a robust effect on glycemic control, with mutants exhibiting glucose intolerance. Selective depletion of RAI1 in BDNF+ neurons in PVN in adult mice also resulted in increased body weight, reduced locomotor activity, and glucose intolerance without affecting food intake. Blunting RAI1 activity also leads to increases and decreases in the inhibitory tone and intrinsic excitability, respectively, of BDNF+ neurons in the PVN.

Strengths:<br /> Overall, the experiments are well designed and multidisciplinary approaches are employed to demonstrate that RAI1 deficits in BDNF+ neurons diminish hypothalamic BDNF signaling and produce metabolic dysfunction. The most significant advance relative to previous reports is the finding from electrophysiological studies showing that blunting RAI1 activity leads to increases and decreases the inhibitory tone and intrinsic excitability, respectively, of BDNF+ neurons in the PVN. Furthermore, that intact RAI1 function is required in BDNF+ neurons for the regulation of glucose homeostasis.

Weaknesses:<br /> Some of the data need to be reconciled with previous findings by others. For example, the authors report that more than 50% of BDNF+ neurons in PVN also express pTrkB whereas about 20% of pTrkB+ cells contain BDNF, raising the possibility that autocrine mechanisms might be at play. This is in conflict with a previous study by An et al, (2015) showing that these cell populations are largely non-overlapping in the PVN.

Another issue that deserves more in depth discussion is that diminished BDNF function appears to play a minor part driving deficits in energy balance regulation. Accordingly, both global central depletion of Rai1 in BDNF+ neurons during development and deletion of Rai1 in BDNF+ neurons in the adult PVN elicited modest effects on body weight (less than 18% increase) and did not affect food intake. This contrasts with mice with selective Bdnf deletion in the adult PVN, which are hyperphagic and dramatically obese (90% heavier than controls). Therefore, the results suggest that deficits in RAI1 in PVN or the whole brain only moderately affect BDNF actions influencing energy homeostasis and that other signaling cascades and neuronal populations play a more prominent role driving the phenotypes observed in Rai1+/- mice, which are hyperphagic and 95% heavier than controls. The results from the proteomic analysis of hypothalamic tissue of Rai1 mutant mice and controls could be useful in generating alternative hypotheses.

Depleting RAI1 in BDNF+ neurons had a robust effect compromising glycemic control. However, as the approach does not necessarily impact BDNF exclusively, there should be a larger discussion of alternative mechanisms.

Reviewer #1 (Public Review):

Summary:<br /> This paper reported a protocol of using human-induced pluripotent stem cells to generate cells expressing microglia-enriched genes and responding to LPS by drastic upregulation of proinflammatory cytokines. Upon subretinal transplantation in mice, hiPSC-derived cells integrated into the host retina and maintained retinal homeostasis, while they responded to RPE injury by migration, proliferation and phagocytosis. The findings revealed the potential of using hiPSC-derived cell transplantation for microglia replacement as a therapeutic strategy for retinal diseases.

Strengths:<br /> The paper demonstrates a method of consistently generating a significant quantity of hiPSC-derived microglia-like cells for in vitro study or for in vivo transplantation. RNAseq analysis offers an opportunity for comprehensive transcriptome profiling of the derived cells. It is impressive that following transplantation, these cells integrated into the retina well, migrated to the corresponding layers, adopted microglia-like morphologies, and survived long term without generating apparent harm. The work has laid a foundation for future utilization of hiPSC-derived microglia in lab and clinical applications.

Weaknesses:<br /> 1. The primary weakness of the paper concerns its conclusion of having generated "homogenous mature microglia", partly based on the RNAseq analysis. However, the comparison of gene profiles was carried out only between "hiPSC-derived mature microglia" and the proliferating myeloid progenitors. While the transcriptome profiles revealed a trend of enrichment of microglia-like gene expression in "hiPSC-derived mature microglia" compared to proliferating myeloid progenitors, this is not sufficient to claim they are "mature microglia". It is important that one carries out a comparative analysis of the RNAseq data with those of primary human microglia, which may be done by leveraging the public database. To convincingly claim these cells are mature microglia, questions need to be addressed including how similar the molecular signatures of these cells are compared with the fully differentiated primary microglia cell or if they remain progenitor-like or take on mosaic properties, and how they distinguish from macrophages.

2. While the authors attempted to demonstrate the functional property of "hiPSC-derived mature microglia" in culture, they used LPS challenge, which is an inappropriate assay. This is because human microglia respond poorly to LPS alone but need to be activated by a combination of LPS with other factors, such as IFNγ. Their data that "hiPSC-derived mature microglia" showed robust responses to LPS indeed implicates that these cells do not behave like mature human microglia.

3. The resolution of Figs. 4 - 6 is so low that even some of the text and labels are hardly readable. Based on the morphology shown in Fig. 4 and the statement in line 147, these hiPSC-derived "cells altered their morphology to a rounded shape within an hour of incubation and rapidly internalized the fluorescent-labeled particles". This is a peculiar response. Usually, microglia do not respond to fluorescent-labeled zymosan by turning into a rounded shaped within an hour when they internalize them. Such a behavior usually implicates weak phagocytotic capacity.

4. Data presented in Fig. 5 are not very convincing to support that transplanted cells were immunopositive for "human CD11b (Fig.5C), as well as microglia signature markers P2ry12 and TMEM119 (Fig.5D)" (line 167). The resolution and magnification of Fig. 5D is too low to tell the colocalization of tdT and human microglial marker immunolabeling. In the flat-mount images (C, I), hCD11b immunolabeling is not visible in the GCL or barely visible in the IPL. This should be discussed.

5. Microglia respond to injury by becoming active and lose their expression of the resting state microglial marker, such as P2ry12, which is used in Fig. 6 for detection of migrated microglia. To confirm that these cells indeed respond to injury like native microglia, one should check for activated microglial markers and induction of pro-inflammatory cytokines in the sodium iodate-injury model.

Reviewer #1 (Public Review):

The inferior colliculus (IC) is the central auditory system's major hub. It integrates ascending brainstem signals to provide acoustic information to the auditory thalamus. The superficial layers of the IC ("shell" IC regions as defined in the current manuscript) also receive a massive descending projection from the auditory cortex. This auditory cortico-collicular pathway has long fascinated the hearing field, as it may provide a route to funnel "high-level" cortical signals and impart behavioral salience upon an otherwise behaviorally agnostic midbrain circuit.

Accordingly, IC neurons can respond differently to the same sound depending on whether animals engage in a behavioral task (Ryan and Miller 1977; Ryan et al., 1984; Slee & David, 2015; Saderi et al., 2021; De Franceschi & Barkat, 2021). Many studies also report a rich variety of non-auditory responses in the IC, far beyond the simple acoustic responses one expects to find in a "low-level" region (Sakurai, 1990; Metzger et al., 2006; Porter et al., 2007). A tacit assumption is that the behaviorally relevant activity of IC neurons is inherited from the auditory cortico-collicular pathway. However, this assumption has never been tested, owing to two main limitations of past studies:

1) Prior studies could not confirm if data were obtained from IC neurons that receive monosynaptic input from the auditory cortex.

2) Many studies have tested how auditory cortical inactivation impacts IC neuron activity; the consequence of cortical silencing is sometimes quite modest. However, all prior inactivation studies were conducted in anesthetized or passively listening animals. These conditions may not fully engage the auditory cortico-collicular pathway. Moreover, the extent of cortical inactivation in prior studies was sometimes ambiguous, which complicates interpreting modest or negative results.

Here, the authors' goal is to directly test if auditory cortex is necessary for behaviorally relevant activity in IC neurons. They conclude that surprisingly, task relevant activity in cortico-recipient IC neuron persists in absence of auditory cortico-collicular transmission. To this end, a major strength of the paper is that the authors combine a sound-detection behavior with clever approaches that unambiguously overcome the limitations of past studies.

First, the authors inject a transsynaptic virus into the auditory cortex, thereby expressing a genetically encoded calcium indicator in the auditory cortex's postsynaptic targets in the IC. This powerful approach enables 2-photon Ca2+ imaging from IC neurons that unambiguously receive monosynaptic input from auditory cortex. Thus, any effect of cortical silencing should be maximally observable in this neuronal population. Second, they abrogate auditory cortico-collicular transmission using lesions of auditory cortex. This "sledgehammer" approach is arguably the most direct test of whether cortico-recipient IC neurons will continue to encode task-relevant information in absence of descending feedback. Indeed, their method circumvents the known limitations of more modern optogenetic or chemogenetic silencing, e.g. variable efficacy.

I also see three weaknesses which limit what we can learn from the authors' hard work, at least in the current form. I want to emphasize that these issues do not reflect any fatal flaw of the approach. Rather, I believe that their datasets likely contain the treasure-trove of knowledge required to completely support their claims.

1. The conclusion of this paper requires the following assumption to be true: That the difference in neural activity between Hit and Miss trials reflects "information beyond the physical attributes of sound." The data presentation complicates asserting this assumption. Specifically, they average fluorescence transients of all Hit and all Miss trials in their detection task. Yet, Figure 3B shows that mice's d' depends on sound level, and since this is a detection task the smaller d' at low SPLs presumably reflects lower Hit rates (and thus higher Miss rates). As currently written, it is not clear if fluorescence traces for Hits arise from trials where the sound cue was played at a higher sound level than on Miss trials. Thus, the difference in neural activity on Hit and Miss trials could indeed reflect mice's behavior (licking or not licking). But in principle could also be explained by higher sound-evoked spike rates on Hit compared to Miss trials, simply due to louder click sounds. Indeed, the amplitude and decay tau of their indicator GCaMP6f is non-linearly dependent on the number and rate of spikes (Chen et al., 2013), so this isn't an unreasonable concern.

2. The authors' central claim effectively rests upon two analyses in Figures 5 and 6. The spectral clustering algorithm of Figure 5 identifies 10 separate activity patterns in IC neurons of control and lesioned mice; most of these clusters show distinct activity on averaged Hit and Miss trials. They conclude that although the proportions of neurons from control and lesioned mice in certain clusters deviates from an expected 50/50 split, neurons from lesioned mice are still represented in all clusters. A significant issue here is that in addition to averaging all Hits and Miss trials together, the data from control and lesioned mice are lumped for the clustering. There is no direct comparison of neural activity between the two groups, so the reader must rely on interpreting a row of pie charts to assess the conclusion. It's unclear how similar task relevant activity is between control and lesioned mice; we don't even have a ballpark estimate of how auditory cortex does or does not contribute to task relevant activity. Although ideally the authors would have approached this by repeatedly imaging the same IC neurons before and after lesioning auditory cortex, this within-subjects design may be unfeasible if lesions interfere with task retention. Nevertheless, they have recordings from hundreds to thousands of neurons across two groups, so even a small effect should be observable in a between-groups comparison.

3. In Figure 6, the authors show that logistic regression models predict whether the trial is a Hit or Miss from their fluorescence data. Classification accuracy peaks rapidly following sound presentation, implying substantial information regarding mice's actions. The authors further show that classification accuracy is reduced, but still above chance in mice with auditory cortical lesions. The authors conclude from this analysis task relevant activity persists in absence of auditory cortex. In principle I do not disagree with their conclusion.

The weakness here is in the details. First, the reduction in classification accuracy of lesioned mice suggests that auditory cortex does nevertheless transmit some task relevant information, however minor it may be. I feel that as written, their narrative does not adequately highlight this finding. Rather one could argue that their results suggest redundant sources of task-relevant activity converging in the IC. Secondly, the authors conclude that decoding accuracy is impaired more in partially compared to fully lesioned mice. They admit that this conclusion is at face value counterintuitive, and provide compelling mechanistic arguments in the Discussion. However, aside from shaded 95% CIs, we have no estimate of variance in decoding accuracy across sessions or subjects for either control or lesioned mice. Thus we don't know if the small sample sizes of partial (n = 3) and full lesion (n = 4) groups adequately sample from the underlying population. Their result of Figure 6B may reflect spurious sampling from tail ends of the distributions, rather than a true non-monotonic effect of lesion size on task relevant activity in IC.

Reviewer #1 (Public Review):

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to the loss of innervation of skeletal muscles, caused by the dysfunction and eventual death of lower motor neurons. A variety of approaches have been taken to treat this disease. With the exception of three drugs that modestly slow progression, most therapeutics have failed to provide benefit. Replacing lost motor neurons in the spinal cord with healthy cells is plagued by a number of challenges, including the toxic environment, inhibitory cues that prevent axon outgrowth to the periphery, and proper targeting of the axons to the correct muscle groups. These challenges seem to be well beyond our current technological approaches. Avoiding these challenges altogether, Bryson et al. seek to transplant the replacement motor neurons into the peripheral nerves, closer to their targets. The current manuscript addresses some of the challenges that will need to be overcome, such as immune rejection of the allograft and optimizing maturation of the neuromuscular junction.

Bryson et al. begin by examining the survival of mESC-derived motor neurons allografted into SOD1 mice. The motor neurons, made on a 129S1/SvImJ, were transplanted into the tibial nerve of SOD1 mice on a C57BL/6J background. Without immunosuppression, most cells were lost between 14 and 35 days, suggesting an immune response had eliminated them. Tacrolimus prevented cell loss, but it also inhibited innervation of the muscle. It also uncovered the tumorigenic potential of contaminating pluripotent cells. In contrast, immunosuppression using H57-597, an antibody targeting T-cell receptor beta, prevented graft rejection while permitting some innervation of muscle. Pretreatment of the cells with mitomycin-C eliminated pluripotent cells, preventing tumor formation. The authors noted that this combination only innervated ~10% of endplates, likely due to the fact that the implanted motor neurons are not active.

The authors then began the process of optimizing the cells themselves, using measurements taken in late-stage SOD1 mice. Fast-firing and slow-firing populations of neurons were first compared. Using optical stimulation, these two cell types appeared to be similar. The authors opted to use slow-firing neurons in the subsequent experiments. Recognizing that neuromuscular junction (NMJ) innervation and maintenance are dependent on motor neuron activity, implantable optical stimulators were also evaluated. 14 days after transplanting the cells, optical stimulation training was initiated for one hour each day. This training led to a nearly 13-fold increase in force generation, although this still remained well below the force generated by electrical stimulation. The enhanced innervation also prevented the atrophy of muscle fibers caused by denervation.

Overall, the data for the function of the implanted cells are convincing. The dCALMS technique that the authors have developed is quite interesting and will likely be applicable to analyze muscles for other therapeutics. The identification of calcineurin inhibitors as inhibitors of reinnervation will also be important for the development of other cell-based therapeutics for ALS.

However, there are some issues that should be addressed. These include some common misconceptions about ALS. While ALS is split into familial and sporadic forms based on the presence or absence of a family history of the disease, mutations in the known ALS-associated genes are found in both forms. The authors also state that exercise programs are likely to accelerate degeneration in ALS. This is incorrect. Moderate exercise is part of the current guidelines for treating ALS, and mouse studies have demonstrated a therapeutic effect of moderate exercise. Regarding the experimental design, there are some important details missing. The animals do not appear to have been operated on at the same age, and the criteria for when to perform the operation were not described. A similar problem exists for when the animals were determined to reach endpoint. The authors also do not seem to address a potential pitfall of this approach: acceleration of the disease process. Indeed, some of the data comparing the ipsilateral side to the contralateral side suggest that the implantation of the cells and/or the light source increase the denervation of the muscle. Finally, there is a fairly large difference between the motor output provided by optical stimulation relative to electrical stimulation. It is currently unclear what level needs to be reached to provide an effective response in the intact animal. Thus, it is difficult to determine if the level of reinnervation that this study has achieved will be sufficient to improve a patient's quality of life.

Reviewer #1 (Public Review):

This is a key paper examining the evolution of an important structure (pillars) in the shell architecture of organo-phosphatic brachiopods. The advantages of these structures are adequately discussed and the evolution of the pillars is described and illustrated. There is much that is of fundamental significance here in understanding the ecology and evolution of these groups as a whole.

1) In several places the biological control on the development of the pillars is noted. This is explained in terms of their relationship to the growth and evolution of epithelial cells. It would be useful and make the paper more understandable if this link was mentioned early on in the paper and developed during the narrative.

2) The Cambrian Explosion is mentioned a number of times. Are these changes driven by the Cambrian Explosion, i.e. the expansion of major new body plans, or are the changes merely coincident with the long duration of the 'Explosion'?

3) I have no doubt the process is one of adaptive innovation but it would be useful to expand on this. Why is it adaptive?

4) Are pillars present in living Lingula?

Reviewer #1 (Public Review):

The present study examines whether one can identify kinematic signatures of different motor strategies in both humans and non-human primates (NHP). The Critical Stability Task (CST) requires a participant to control a cursor with complex dynamics based on hand motion. The manuscript includes datasets on performance of NHPs collected from a previous study, as well as new data on humans performing the same task. Further human experiments and optimal control models highlight how different strategies lead to different patterns of hand motion. Finally, classifiers were developed to predict which strategy individuals were using on a given trial. There are several strengths to this manuscript. I think the CST task provides a useful behavioural task to explore the neural basis of voluntary control. While reaching is an important basic motor skill, there is much to learn by looking at other motor actions to address many fundamental issues on the neural basis of voluntary control. I also think the comparison between human and NHP performance is important as there is a common concern that NHPs can be overtrained in performing motor tasks leading to differences in their performance as compared to humans. The present study highlights that there are clear similarities in motor strategies of humans and NHPs. While the results are promising, I would suggest that the actual use of these paradigms and techniques likely need some improvement/refinement. Notably, the threshold or technique to identify which strategy an individual is using on a given trial needs to be more stringent given the substantial overlap in hand kinematics between different strategies.

The most important goal of this study is to set up future studies to examine how changes in motor strategies impact neural processing. I have a few concerns that I think need to be considered. First, a classifier was developed to identify whether a trial reflected Position Control with success deemed to be a probability of >70% by the classifier. In contrast, a probability of <30% was considered successfully predicting Velocity Control (Uncertain bandwidth middle 40%). While this may be viewed as acceptable for purposes of quantifying behaviour, I'm not sure this is strict enough for interpreting neural data. Figure 7A displays the OFC Model results for the two strategies and demonstrates substantial overlap for RMS of Cursory Position and Velocity at the lowest range of values. In this region, individual trials for humans and NHP are commonly identified as reflecting Position Control by the classifier although this region clearly also falls within the range expected for Velocity Control, just a lower density of trials. The problem is that neural data is messy enough, but having trials being incorrectly labelled will make it even messier when trying to quantify differences in neural processing between strategies. A further challenge is that trials cannot be averaged as the patterns of kinematics are so different from trial-to-trial. One option is to just move up the threshold from >70%/<30% to levels where you have a higher confidence that performance only reflects one of the two strategies (perhaps 95/5% level). Another approach would be to identify the 95% confidence boundary for a given strategy and only classify a trial as reflecting a given strategy when it is inside its 95% boundary, but outside the other strategies 95% boundary (or some other level separation). A higher threshold would hopefully also deal with the challenge of individuals switching strategies within a trial. Admittedly, this more stringent separation will likely drop the number of trials prohibitively, but there is a clear trade-off between number of trials and clean data. For the future, a tweak to the task could be to lengthen the trial as this would certainly increase separation between the two conditions.

While the paradigm creates interesting behavioural differences, it is not clear to me what one would expect to observe neurally in different brain regions beyond paralleling kinematic differences in performance. Perhaps this could be discussed. One extension of the present task would be to add some trials where visual disturbances are applied near the end of the trial. The prediction is that there would be differences in the kinematics of these motor corrections for different motor strategies. One could then explore differences in neural processing across brain regions to identify regions that simply reflect sensory feedback (no differences in the neural response after the disturbance), versus those involved in different motor strategies (differences in neural responses after the disturbance).

It seems like a mix of lambda values are presented in Figure 5 and beyond. There needs to be some sort of analysis to verify that all strategies were equally used across lambda levels. Otherwise, apparent differences between control strategies may simply reflect changes in the difficulty of the task. It would also be useful to know if there were any trends across time? Strategies used for blocks of trials or one used early when learning and then changing later.

Figure 2 highlights key features of performance as a function of task difficulty. Lines 187 to 191 highlight similarities in motor performance between humans and NHPs. However, there is a curious difference in hand/cursor Gain for Monkey J. Any insight as to the basis for this difference?

Reviewer #1 (Public Review):

This paper falls in a long tradition of studies on the costs of reproduction in birds and its contribution to understanding individual variation in life histories. Unfortunately, the meta-analyses only confirm what we know already, and the simulations based on the outcome of the meta-analysis have shortcomings that prevent the inferences on optimal clutch size, in contrast to the claims made in the paper.

There was no information that I could find on the effect sizes used in the meta-analyses other than a figure listing the species included. In fact, there is more information on studies that were not included. This made it impossible to evaluate the data-set. This is a serious omission, because it is not uncommon for there to be serious errors in meta-analysis data sets. Moreover, in the long run the main contribution of a meta-analysis is to build a data set that can be included in further studies.

The main finding of the meta-analysis of the brood size manipulation studies is that the survival costs of enlarging brood size are modest, as previously reported by Santos & Nakagawa on what I suspect to be mostly the same data set. The paper does a very poor job of critically discussing whether we should take this at face value or whether instead there may be short-comings in the general experimental approach. A major reason why survival cost estimates are barely significantly different from zero may well be that parents do not fully adjust their parental effort to the manipulated brood size, either because of time/energy constraints, because it is too costly and therefore not optimal, or because parents do not register increased offspring needs. Whatever the reason, as a consequence, there is usually a strong effect of brood size manipulation on offspring growth and thereby presumably their fitness prospects. In the simulations (Fig.4), the consequences of the survival costs of reproduction for optimal clutch size were investigated without considering brood size manipulation effects on the offspring. Effects on offspring are briefly acknowledged in the discussion, but otherwise ignored. Assuming that the survival costs of reproduction are indeed difficult to discern because the offspring bear the brunt of the increase in brood size, a simulation that ignores the latter effect is unlikely to yield any insight in optimal clutch size. It is not clear therefore what we learn from these calculations.

There are other reasons why brood size manipulations may not reveal the costs of reproduction animals would incur when opting for a larger brood size than they produced spontaneously themselves. Firstly, the manipulations do not affect the effort incurred in laying eggs (which also biases your comparison with natural variation in clutch size). Secondly, the studies by Boonekamp et al on Jackdaws found that while there was no effect of brood size manipulation on parental survival after one year of manipulation, there was a strong effect when the same individuals were manipulated in the same direction in multiple years. This could be taken to mean that costs are not immediate but delayed, explaining why single year manipulations generally show little effect on survival. It would also mean that most estimates of the fitness costs of manipulated brood size are not fit for purpose, because typically restricted to survival over a single year.

Details of how the analyses were carried out were opaque in places, but as I understood the analysis of the brood size manipulation studies, manipulation was coded as a covariate, with negative values for brood size reductions and positive values for brood size enlargements (and then variably scaled or not to control brood or clutch size). This approach implicitly assumes that the trade-off between current brood size (manipulation) and parental survival is linear, which contrasts with the general expectation that this trade-off is not linear. This assumption reduces the value of the analysis, and contrasts with the approach of Santos & Nakagawa.

The observational study selection is not complete and apparently no attempt was made to make it complete. This is a missed opportunity - it would be interesting to learn more about interspecific variation in the association between natural variation in clutch size and parental survival.

Reviewer #1 (Public Review):

The authors present a scRNAseq study describing the transcriptomes of the tendon enthesis during postnatal development. This is an important topic that has major implication for the care of common clinical problems such as rotator cuff repair. The results are a valuable addition to the literature, providing a descriptive data set reinforcing other, more comprehensive studies. There are weaknesses, however, in the scRNAseq analyses.

1.The authors should provide additional rationale for the PCA analysis shown in Fig 1d. It is uncommon to use PCA for histomorphologic parameters. These results do not convincingly demonstrate that P7 is as a critical developmental timepoint.

2. According to the methods, it appears that the entire humeral head-supraspinatus tendon was used for cell isolation for scRNAseq. This results in the inclusion of cells from a variety of tissues, including bone, growth plate, enthesis and tendon. As such, only a very small percentage of cells in the analysis came from the enthesis. Inclusion of such a wide range of cells makes interpretation of enthesis cells difficult.

3. The differentiation/pseudotime analysis described in Fig 3 is difficult to follow. This map includes cell transcriptomes from vastly different tissues. Presumably, embedded in these maps are trajectories for osteoblast differentiation, chondrocyte differentiation, tenocyte differentiation, etc. With so many layers of overlapping information, it is difficult to (algorithmically) deduce a differentiation path of a particular cell type.

4. The authors uses the term "function" throughout the paper (e.g., "functional definition of fibrocartilage subpopulations"). However, this is a descriptive scRNAseq study, and "function" can therefore only theoretically be inferred from the algorithms used to analyze the data. A functional role for any of the identified pathways or processes can only be defined with gain- and/or loss-of-function studies.

5. "C2 highly expressed biomineralization-related genes (Clec3a, Tnn, Acan)". The three example genes are not related to biomineralization.

6. The functional characterization of the three enthesis cell clusters is not convincing. For example, activation of metabolism-related processes can mean a lot of things (including changes in differentiation), yet the authors interpret it very specifically as "role in postnatal fibrochondrocyte formation and growth".

7. The pseudotime analysis of the enthesis cell clusters is not convincing. The three clusters are quite close and overlapping on the UMAP. Furthermore, the authors focus on Tnn as a novel and unique gene, yet the expression pattern shown in Fig 5g implies even expression of this gene across all three clusters.

8. The TC1 markers (Ly6a, Dlk3, Clec3b) imply a non-tendon-specific cell population. Perhaps a tendon progenitor pool or an endothelial cell phenotype is more appropriate.

9. Pseudotime analyses assume that your data set includes cells from progenitor through mature cell populations. It is unclear that the timepoints studied here included cells from early progenitor states.

10. The CellChat analysis is difficult to follow, as the authors included 18 cell types. The number of possible interactions among so many cell types is enormous, and deducing valid connections between any two cell types in this case should be justified. Is the algorithm robust to so many possible interactions?