Reviewer #2 (Public Review):

Summary:<br /> This is an unusual, but interesting approach to link the "taste" of plants and plant extracts to their therapeutic use in ancient Graeco-Roman culture. The authors used a panel of 11 trained tasters to test ~700 different medicinal plants and describe them in terms of 22 "taste" descriptors. They correlated these descriptors with the plant's medical use as reported in the De Materia Medica (DMM 1st Century, CE). Correcting for some of the plants' evolutionary phylogenetic relationships, the authors found that taste descriptors along with intensity measures were correlated with the "versatility" and/or a specific therapeutic use of the medicine. For example, simple but intense tastes were correlated with versatility of a medicine. Specific intense tastes were linked to versatility while others were not; intense bitter, starchy, musky, sweet, cooling and soapy were associated with versatility, but sour and woody were negatively associated. Also some specific tastes could be associated with specific uses - both positive and negative associations. Some of these findings make sense immediately, but others are somewhat surprising, and the authors propose some links between taste and medicinal use (both historical and modern use) in the discussion. The authors state that this study allows for a re-evaluation of pre-scientific knowledge, pointing toward a central role for taste in medicine.

Strengths:<br /> The real strength of this study is the novelty of this approach - using modern day tasters to evaluate ancient medicinal plants to understand the potential relationships between taste and therapeutic use, lending some support to the idea that the "taste" of a medicine is linked to its effectiveness as a treatment.

Weaknesses:<br /> Because of the limitations of time and the type of botanicals being tested, there is an inherent difficulty in assessing taste intensity. However, because these botanicals are tested by multiple panelists and sometimes tested repeatedly by individual panelists, this helps support the author's analyses.

Reviewer #2 (Public Review):

Erk2 is an essential element of the MAP kinase signaling cascade and directly controls cell proliferation, migration, and survival. Therefore, it is one of the most important drug targets for cancer therapy. The catalytic subunit of Erk2 has a bilobal architecture, with the small lobe harboring the nucleotide-binding pocket and the large lobe harboring the substrate-binding cleft. Several studies by the Ahn group revealed that the catalytic domain hops between (at least) two conformational states: active (R) and inactive (L), which exchange in the millisecond time scale based on the chemical shift mapping. The R state is a signature of the double phosphorylated Erk2 (2P-Erk2), while the L state has been associated with the unphosphorylated kinase (0P-Erk2). Interestingly, the X-ray structures reveal only minimal differences between these two states, a feature that led to the conclusion that active and inactive states are structurally similar but dynamically very different. The Ahn group also found that ATP-competitive inhibitors can steer the populations of Erk2 either toward the R or the L state, depending on their chemical nature. The latter opens up the possibility of modulating the activity of this kinase by changing the chemistry of the ATP-competitive inhibitor. To prove this point, the authors present a set of nineteen compounds with diverse chemical substituents. From their combined NMR and HDX-Mass Spec analyses, fourteen inhibitors drive the kinase toward the R state, while four compounds keep the kinase hopping between the R and L states. Based on these data, the authors rationalize the effects of these inhibitors and the importance of the nature of the substituents on the central scaffold to steer the kinase activity. While all these inhibitors target the ATP binding pocket, they display diverse structural and dynamic effects on the kinase, selecting a specific structural state. Although the inhibited kinase is no longer able to phosphorylate substrates, it can initiate signaling events functioning as scaffolds for other proteins. Therefore, by changing the chemistry of the inhibitors it may be possible to affect the MAP cascade in a predictable manner. This concept, recently introduced as proof of principle, finds here its significance and practical implications. The design of the next-generation inhibitors must be taken into account for these design principles.<br /> The research is well executed, and the data support the author's conclusions.

Reviewer #2 (Public Review):

In this manuscript the authors present and characterize LOVdeg, a modified version of the blue-light sensitive AsLOV2 protein, which functions as a light-inducible degron in Escherichia coli. Light has been shown to be a powerful inducer in biological systems as it is often orthogonal and can be controlled in both space and time. Many optogenetic systems target regulation of transcription, however in this manuscript the authors target protein degradation to control protein levels in bacteria. This is an important advance in bacteria, as inducible protein degradation systems in bacteria have lagged behind eukaryotic systems due to protein targeting in bacteria being primarily dependent on primary amino acid sequence and thus more difficult to engineer. In this manuscript, the authors exploit the fact that the J-alpha helix of AsLOV2, which unwinds into a disordered domain in response to blue light, contains an E-A-A amino acid sequence which is very similar to the C-terminal L-A-A sequence in the SsrA tag which is targeted by the unfoldases ClpA and ClpX. They truncate AsLOV2 to create AsLOV2(543) and combine this truncation with a mutation that stabilizes the dark state to generate AsLOV2*(543) which, when fused to the C-terminus of mCherry, confers light-induced degradation. The authors do not verify the mechanism of degradation due to LOVdeg, but evidence from deletion mutants contained in the supplemental material hints that there is a ClpA dominated mechanism. The LOVdeg is able to target mCherry for protein degradation across different phases of bacterial growth, which is important for regulating processes at stationary phase and a potential additional advantage over transcriptional repression systems. They demonstrate modularity of this LOVdeg by using it to degrade the LacI repressor, CRISPRa activation through degradation of MCP-SoxS, and the AcrB protein which is part of the AcrAB-TolC multidrug efflux pump. In all cases, measurement of the effect of the LOVdeg is indirect as the authors measure reduction in LacI repression, reduction in CRISPRa activation, and drug resistance rather than directly measuring protein levels. Nevertheless the evidence is convincing, although seemingly less effective than in the case of mCherry degradation, although it is hard to compare due to the different endpoints being measured. The authors further modify LOVdeg to contain a known photocycle mutation that slows its reversion time in the dark, so that LOVdeg is more sensitive to short pulses of light which could be useful in low light conditions or for very light sensitive organisms. They also demonstrate that combining LOVdeg with a blue-light transcriptional repression system (EL222) can decrease protein levels an additional 23-fold (relative to 7-fold with LOVdeg alone). Finally, the authors apply LOVdeg to a metabolic engineering task, namely reducing expression of octanoic acid by regulating the enzyme CpFatB1, an acyl-ACP thioesterase. The authors show that tagging CpFatB1 with LOVdeg allows light induced reduction in octanoic acid titer over a 24 hour fermentation. In particular, by comparing control of CpFatB1 with EL222 transcriptional repression alone, LOVdeg, or both the authors show that light-induced protein degradation is more effective than light-induced transcriptional repression. The authors suggest that this is because transcriptional repression is not effective when cells are at stationary phase (and thus there is no protein dilution due to cell division). Overall, the authors have generated a modular, light-activated degron tag for use in Escherichia coli that is likely to be a useful tool in the synthetic biology and metabolic engineering toolkit.

Reviewer #2 (Public Review):

In this study, the authors hypothesized that the balance of mTOR complexes in Sertoli cells may also play a significant role in age-dependent changes in the sperm epigenome. To test this hypothesis, the authors use transgenic mice with manipulated activity of mTOR complexes in Sertoli cells. These results suggest that the mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.

The authors attempt to demonstrate that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. The authors have effectively met their research objectives, and their conclusions are supported by the data presented.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors work to extend their previous observation that galectin-9 interacts with arabinogalactans of Mtb in their EMBO reports 2021 manuscript. Here they provide evidence that the CARD2 domain of galectin-9 can inhibit the growth of Mtb in culture. In addition, antibodies that also bind to AG appear to inhibit Mtb growth in culture. These data indicate that independent of the common cell-associated responses to galectin-9 and antibodies, the interaction of these proteins with AG of mycobacteria may have consequences for bacterial growth.

Strengths:

The authors provided several lines of evidence in culture media that the introduction of galectin-9 proteins and antibodies inhibits the growth rate of Mtb.

Weaknesses:

In light of other observations that cleaved galectin-9 levels in the plasma is a biomarker for severe infection (Padilla A et al Biomolecules 2021 and Iwasaki-Hozumi H et al. Biomoleucles 2021) it is difficult to reconcile the author's interpretation that the elevated gal-9 in Active TB patients (Figure 1E) contributes to the maintenance of latent infection in humans. The authors should consider incorporating these observations in the interpretation of their own results.

The anti-AG titers were measured only in individuals with active TB (Figure 3C), generally thought to be a less protective immunological state. The speculation that individuals with anti-AG titers have some protection is not founded. Further only 2 mAbs were tested to demonstrate restriction of Mtb in culture. It is possible that clones of different affinities for AG present within a patient's polyclonal AG-antibody responses may or may not display a direct growth restriction pressure on Mtb in culture. The authors should soften the claims about the presence of AG-titers in TB patients being indicative of protection.

Reviewer #2 (Public Review):

Summary:

The authors generated a DNA methylation score in cord blood for detecting exposure to cigarette smoke during pregnancy. They then asked if it could be used to predict height, weight, BMI, adiposity, and WHR throughout early childhood.

Strengths:

The study included two cohorts of European ancestry and one of South Asian ancestry.

Weaknesses:

1. The number of mothers who self-reported any smoking was very low, much lower than in the general population and practically non-existent in the South Asian population. As a result, all analyses appeared to have been underpowered. It is possibly for this reason that the authors chose to generate their DNA methylation model using previously published summary statistics. The resulting score is not of great value in itself due to the low-powered dataset used to estimate covariance between CpG sites. In fact, a score was generated for a much larger, better-powered dataset several years ago (Reese, EHP, 2017, PMID 27323799).

2. The conclusion that "even minimal smoking exposure in South Asian mothers who were not active smokers showed a DNAm signature of small body size and low birthweight in newborns" is not warranted because no analyses were performed to show that the association between DNA methylation and birth size/weight was driven by maternal smoking.

3. Although it was likely that some mothers were exposed to second-hand smoke and/or pollution, data on this was either non-existent or not included in this study. Including this would have allowed a more novel investigation of the effects of smoke exposure on the pregnancies of non-smoking mothers.

4. One of the European cohorts and half of the South Asian cohort had DNA methylation measured on only 2500 CpG sites. This set of sites included only 125 sites previously linked to prenatal smoking. The resulting model of prenatal smoking was small (only 11 CpG sites). It is possible that a large model may have been more powerful.

5. The health outcomes investigated are potentially interesting but there are other possibly more important outcomes of interest such as birth complications, asthma, and intellectual impairment which are known to be associated with prenatal smoking.

Reviewer #2 (Public Review):

Summary:<br /> Peled et al identified HER2 mutations in connection with resistance to the anti-HER2 antibody Pertuzumab-mediated therapy. After constructing a yeast display library of Pertuzumab variants with 3.86×1011 sequences for targeted screening of variant combinations in chosen 6 out of 14 residues, the authors performed experimental screening to obtain the clones that bind to HER2 WT and/or mutants (S310Y and S310F), and then combined new variations to obtain antibodies with a broad spectrum binding to both WT and two HER2 mutants. These are interesting studies of clinical impact and translational potential.

Strengths:<br /> 1. Deep computational analyses of large datasets of clinical data provide useful information about HER2 mutations and their potential relevance to antibody therapy resistance.

2. There is valuable information analyzing the residues within or near the interface between the antigen HER2 and the Pertuzumab antibody (heavy chain). The experimental antibody library screening obtained 90+ clones from 3.86×1011 sequences for further functional validation.

Weaknesses:<br /> 1. There is a lack of assessment for antibody variant functions in cancer cell phenotypes in vitro (proliferation, cell death, motility) or in vivo (tumor growth and animal survival). The only assay was the western blotting of phosphopho-HER3 in Figure 4. However, HER2 levels and phosphor-HER2 were not analyzed.

2. There is a misleading impression from the title of computational engineering of a therapeutic antibody and the statement in the abstract "we designed a multi-specific version of Pertuzumab that retains original function while also bindings these HER2 variants" for a few reasons:<br /> a. The primary method used for variant antibody identification for HER2 mutant binding is rather traditional experimental screening based on yeast display instead of the computational design of a multi-specific version of Pertuzumab.<br /> b. There is insufficient or lack of computational power in the antibody design or prioritization in choosing variant residues for the library construction of 3.86×1011 sequences. It seems random combinations from 6 residues out of 4 groups with 20 amino acid options.<br /> c. The final version of the tri-binding variant is a combination of screened antibody clones instead of computation design from scratch.<br /> d. There is incomplete experimental evidence about the therapeutic values of newly obtained antibody clones.

3. Figures can be improved with better labeling and organization. Some essential pieces of data such as Supplementary Figure 1B on HER2 mutations in S310 that abrogated its binding to Pertuzumab should be placed in the main figures.

4. It is recommended to provide a clear rationale or flowchart overview into the main Figure 1. Figure 2A can be combined with Figure 1 to the list of targeted residues.

5. The quality of Figures such as Figure 2B-C flow data needs to be improved.

Reviewer #2 (Public Review):

Ma X. et al proposed that A. muciniphila was a key strain that promotes the proliferation and differentiation of intestinal stem cells by acting on the Wnt/b-catenin signaling pathway. They used various models, such as the piglet model, mouse model, and intestinal organoids to address how A. muciniphila and B. fragilis offer protection against ETEC infection. They showed that FMT with fecal samples, A. muciniphila or B. fragilis protected piglets and/or mice from ETEC infection, and this protection is manifested as reduced intestinal inflammation/bacterial colonization, increased tight junction/Muc2 proteins, as well as proper Treg/Th17 cells. Additionally, they demonstrated that A. muciniphila protected basal-out and/or apical-out intestinal organoids against ETEC infection via Wnt signaling. While a large body of work has been performed in this study, there are quite a few questions to be addressed.

Major comments:

- The similar protective effect of FMT with fecal samples, A. muciniphila or B. fragilis is perhaps not that surprising, considering that FMT likely restores microbiota-mediated colonization resistance against ETEC infection. While FMT with fecal samples increases SCFAs, it is unclear whether/how FMT with A. muciniphila or B. fragilis alter the microbiota composition/abundance as well as metabolites in the current models in a way that offers protection.

- Does ETEC infection in piglets/mice cause histological damage in the intestines? These data should be shown.

- Line 447, "ETEC adheres to intestinal epithelial cells". However, there is no data showing the adherence (or invasion) of ETEC to intestinal epithelial cells, irrespective of piglets/mouse/organoids.

- In both basal-out and apical-out intestinal organoid models, A. muciniphila protects organoids against ETEC infection. Did ETEC enter into intestinal epithelial cells at all after only one hour of infection? Is the protection through certain A. muciniphila metabolites?

Reviewer #2 (Public Review):

In this manuscript, Tiedje and colleagues longitudinally track changes in parasite numbers across four time points as a way of assessing the effect of malaria control interventions in Ghana. Some of the study results have been reported previously, and in this publication, the authors focus on age-stratification of the results. Malaria prevalence was lower in all age groups after IRS. Follow-up with SMC, however, maintained lower parasite prevalence in the targeted age group but not the population as a whole. Additionally, they observe that diversity measures rebounds more slowly than prevalence measures. Overall, I found these results clear, convincing, and well-presented. They add to a growing literature that demonstrates the relevance of asymptomatic reservoirs.

There is growing interest in developing an expanded toolkit for genomic epidemiology in malaria, and detecting changes in transmission intensity is one major application. As the authors summarize, there is no one-size-fits-all approach, and the Bayesian MOIvar estimate developed here has the potential to complement currently used methods. I find its extension to a calculation of absolute parasite numbers appealing as this could serve as both a conceptually straightforward and biologically meaningful metric. However, I am not fully convinced the current implementation will be applied meaningfully across additional studies.

1. I find the term "census population size" problematic as the groups being analyzed (hosts grouped by age at a single time point) do not delineate distinct parasite populations. Separate parasite lineages are not moving through time within these host bins. Rather, there is a single parasite population that is stochastically divided across hosts at each time point. I find this distinction important for interpreting the results and remaining mindful that the 2,000 samples at each time point comprise a subsample of the true population. Instead of "census population size", I suggest simplifying it to "census count" or "parasite lineage count".

It would be fascinating to use the obtained results to model absolute parasite numbers at the whole population level (taking into account, for instance, the age structure of the population), and I do hope this group takes that on at some point even if it remains outside the scope of this paper. Such work could enable calculations of absolute---rather than relative---fitness and help us further understand parasite distributions across hosts.

2. I'm uncertain how to contextualize the diversity results without taking into account the total number of samples analyzed in each group. Because of this, I would like a further explanation as to why the authors consider absolute parasite count more relevant than the combined MOI distribution itself (which would have sample count as a denominator). It seems to me that the "per host" component is needed to compare across age groups and time points---let alone different studies.

3. Thinking about the applicability of this approach to other studies, I would be interested in a larger treatment of how overlapping DBLa repertoires would impact MOIvar estimates. Is there a definable upper bound above which the method is unreliable? Alternatively, can repertoire overlap be incorporated into the MOI estimator?

Smaller comments:<br /> - Figure 1 provides confidence intervals for the prevalence estimates, but these aren't carried through on the other plots (and Figure 5 has lost CIs for both metrics). The relationship between prevalence and diversity is one of the interesting points in this paper, and it would be helpful to have CIs for both metrics when they are directly compared.

Reviewer #2 (Public Review):

Summary:<br /> In the manuscript by Luo et al, the authors investigated the nature and function of TRAIL-HS binding for the regulation of TRAIL-mediated apoptosis in cancer cells. The authors discovered that TRAIL binds to 12mer HS and identified the amino acid residues critical for the binding. The authors further nicely showed that 12mer HS binds to TRAIL homotrimer and larger HS can further promote the formation of larger TRAIL oligomers. Structural analyses were conducted to characterize the binding of TRAIL/HS complexes. At functional level, the authors demonstrated that HS promotes the cell surface binding of TRAIL to enhance TRAIL-mediated apoptosis in a variety of cancer cells. Moreover, the ability of TRAIL to induce apoptosis is correlated with cell surface HS level. Lastly, the authors showed that HS forms complex with TRAIL and its receptor DR5 and promotes DR5 internalization.

Strengths:<br /> Overall, this is a nicely executed study providing both mechanistic and functional insight for TRAIL-mediated apoptosis. It conducted detailed characterization on the direct binding between HS and TRAIL and provided solid evidence supporting the role of such interaction for the regulation of TRAIL-induced apoptosis. The experiments were well-designed with proper controls included. The data interpretation is accurate. The manuscript was clearly written and easy to follow by general readers.

Weaknesses:<br /> There is no major weakness identified from this study. As the authors pointed out, the current relationship between cell surface HS level and sensitivity to TRAIL-mediated apoptosis is still correlative and will need further investigation in the future.

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1.

The authors have sufficiently addressed prior comments and added new experiments to provide details on possible mechanisms.

Reviewer #2 (Public Review):

Summary:

In their manuscript, the authors address the question of whether the inversion polymorphism in D. melanogaster can be explained by sexually antagonistic selection. They designed a new simulation tool to perform computer simulations, which confirmed their hypothesis. They also show a tradeoff between male reproduction and survival. Furthermore, some inversions display sex-specific survival.

Strengths:<br /> It is an interesting idea on how chromosomal inversions may be maintained

Weaknesses:<br /> General points:<br /> The manuscript lacks clarity of writing. It is impossible to fully grasp what the authors did in this study and how they reached their conclusions. Therefore, I will highlight some cases that I found problematic.<br /> Although this is an interesting idea, it clearly cannot explain the apparent influence of seasonal and clinal variation on inversion frequencies.

Specific points:<br /> The simulations are highly specific and make very strong assumptions, which are not well-justified.

Reviewer #2 (Public Review):

Ehring et al. analyze contributions of Dispatched, Scube2, serum lipoproteins and Sonic Hedgehog lipid modifications to the generation of different Shh release forms. Hedgehog proteins are anchored in cellular membranes by N-terminal palmitate and C-terminal cholesterol modifications, yet spread through tissues and are released into the circulation. How Hedgehog proteins can be released, and in which form, remains controversial. The authors systematically dissect contributions of several previously identified factors, and present evidence that Disp, Scube2 and lipoproteins concertedly act to release a novel Shh variant that is cholesterol-modified but not palmitoylated. The results provide new insights into the function of Disp and Scube2 in Hedgehog release. The findings concerning the function of lipoproteins and cholesterol in Hedgehog release are largely confirmatory (PMID 23554573, 20685986). However, in light of the multitude of competing models for Hedgehog release, the present study is a valuable contribution that provides further insights into the relevance of lipoproteins in this process.

A novel and surprising finding of the present study is the differential removal of Shh N- or C-terminal lipid anchors depending on the presence of HDL and/or Disp. In particular, the identification of a non-palmitoylated but cholesterol-modified Shh variant that associates with lipoproteins is potentially important. The authors use RP-HPLC and defined controls to assess the properties of processed Shh forms, but their precise molecular identify remains to be defined. A caveat is the strong reliance on over-expression of Shh in a single cell line. The authors detect Shh variants that are released independently of Disp and Scube2 in secretion assays, which however are excluded from interpretation as experimental artifacts. Thus, it would be important to demonstrate key findings in cells that secrete Shh endogenously.

Reviewer #2 (Public Review):

By mapping the sites of the Mcm2-7 replicative helicase loading across the budding yeast genome using high-resolution chromatin endogenous cleavage or ChEC, Bedalov and colleagues find that these markers for origins of DNA replication are much more broadly distributed than previously appreciated. Interestingly, this is consistent with early reconstituted biochemical studies that showed that the ACS was not essential for helicase loading in vitro (e.g. Remus et al., 2009, PMID: 19896182). To accomplish this, they combined the results of 12 independent assays to gain exceptionally deep coverage of Mcm2-7 binding sites. By comparing these sites to previous studies mapping ssDNA generated during replication initiation, they provide evidence that at least a fraction of the 1600 most robustly Mcm2-7-bound sequences act as origins. A weakness of the paper is that the group-based (as opposed to analyzing individual Mcm2-7 binding sites) nature of the analysis prevents the authors from concluding that all of the 1,600 sites mentioned in the title act as origins. The authors also show that the location of Mcm2-7 location after loading are highly similar in the top 500 binding sites, although the mobile nature of loaded Mcm2-7 double hexamers prevents any conclusions about the location of initial loading. Interestingly, by comparing subsets of the Mcm2-7 binding sites, they find that there is a propensity of at least a subset of these sites to be nucleosome depleted, to overlap with at least a partial match to the ACS sequence (found at all of the most well-characterized budding yeast origins), and a GC-skew centered around the site of Mcm loading. Each of these characteristics is related to previously characterized S. cerevisiae origins of replication.

Overall, this manuscript greatly broadens the number of sites that are capable of loading Mcm2-7 in budding yeast cells and shows that a subset of these additional sites act as replication origins. Although these studies show that the sequence specificity of S. cerevisiae replication origins still sets it apart from metazoan origins, the ability to license and initiate replication from sites with increasing sequence divergence suggests a previously unappreciated versatility.

Reviewer #2 (Public Review):

This is a thorough and convincing body of work that represents an incremental but significant improvement on iterations of this method of CRISPR-based Sterile Insect Technique ('pgSIT'). In this version, compared to previous, the authors target more genes than previously, in order to induce both female inviability (targeting the genes intersex and doublesex, compared to fem-myo previously) and male sterility (targeting a beta-tubulin, as previously in the release generation.

The characterization of the lines is extensive and this data will be useful to the field. However, what is lacking is some context as to how this formulation compares to the previous iteration. Mention is made of the possible advantage of removing most females, compared to just making them flightless (as previously) but there is no direct comparison, either experimental, or theoretical i.e. imputing the life history traits into a model. For me this is a weakness, yet easily addressed. In a similar vein, much is made in alluding to the 'safety concerns of gene drive' and how this is a more palatable half-way house, just because it has CRISPR component within it; it is not. It would be much more sensible, and more informative, to compare this pgSIT technology to RIDL (release of insects carrying a dominant lethal), which is essentially a transgene-based version of the Sterile Insect Technique, as is the work presented here.

The authors achieve impressive results and show that these strains, under a scenario of high levels of release ratios compared to WT, could achieve significant local suppression of mosquito populations. The sensitivity analysis that examines the effect of changing different biological or release parameters is well performed and very informative.

The authors are honest in acknowledging that there are still challenges in bringing this to field release, namely in developing sexing strains and optimizing release strategies.

Reviewer #2 (Public Review):

In this work, Vollenweider et al. examine the effectiveness of using natural products, specifically molecules that chelate iron, to treat infectious agents. Through the purification of 320 environmental isolates, 25 potential candidates were identified from natural products based on inhibition assays and were further screened. The structural information and chemical composition were determined.

The paper is well-structured and thorough; targeting virulence factors in this manner is a great idea. My enthusiasm is dampened by the mediocre effects of the compounds. The lack of a dose-response curve in the survivability assays suggests a limited scope for these molecules. While it is encouraging that the best survivability occurred at the lowest toxicity level, it opens questions as to how effective such molecules can be. Either the reduction in mortality was offset by using higher concentrations, which was not observed in the compound-alone test, or there is no dose-response curve. The latter would suggest to me that the variation in survivability is not due to the addition of siderophores.

I would also like to see how these molecules compare to other iron-chelating molecules. Desferoxamine is a bacteria-derived siderophore that is FDA-approved. However, it is not used to treat infections. Would the author consider comparing their candidate molecules to well-studied molecules? This also raises questions about the novelty of this work; I think the authors could rephrase the discussion to better reflect that bioprospecting for iron-chelating molecules has previously occurred and been successful.

Finally, I am concerned about the few mutations reported in the resistance study. Looking at the SI, it appears that very few mutations were seen. It is unclear what filtering the authors used to arrive at such a low number of mutations. Even filtering against mutations that were selected by adaptation to the media, it seems low that only a handful of clones had distinct mutations.

This paper has a lot of strengths. The workflow is logical and well-executed; the only significant weakness is the effect of the molecules and the lack of an explanation for a dose-response curve in the survivability assay, especially when compared to the data reported in Figure 3. As the authors describe in lines 214-217.

Reviewer #2 (Public Review):

Summary:<br /> Dr. Khulman and colleagues present a very interesting experimental and mathematical modeling work on IS608 transposition. The system has a number of unique advantages that create experimental possibilities utilized here to investigate transposition dynamics. This kind of approach is badly missing from the field and I believe the experiments and modeling work shown here and the type of results that can be derived are groundbreaking and certainly deserve high visibility.

Strengths:<br /> The attempt to measure and model transposition dynamics in cells.

Weaknesses:<br /> - Lack of controls using an active site mutant of TnpB.<br /> - Lack of control in RecA- cells as transposon restoration is proposed to be dependent on homologous recombination.<br /> - Lack of consideration of the levels of ωRNA present.

Reviewer #2 (Public Review):

Hagihara et al. conducted a study investigating the correlation between decreased brain pH, increased brain lactate, and poor working memory. They found altered brain pH and lactate levels in animal models of neuropsychiatric and neurodegenerative disorders. Their study suggests that poor working memory performance may predict higher brain lactate levels.

However, the study has some significant limitations. One major concern is that the authors examined whole-brain pH and lactate levels, which might not fully represent the complexity of disease states. Different brain regions and cell types may have distinct protein and metabolite profiles, leading to diverse disease outcomes. For instance, certain brain regions like the hippocampus and nucleus accumbens exhibit opposite protein/signaling pathways in neuropsychiatric disease models.

Moreover, the memory tests used in the study are specific to certain brain regions, but the authors did not measure lactate levels in those regions. Without making lactate measurements in brain-regions and cell types involved in these diseases, any conclusions regarding the role of lactate in CNS diseases is premature.

Additionally, evidence suggests that exogenous treatment with lactate has positive effects, such as antidepressant effects in multiple disease models (Carrard et al., 2018, Carrard et al., 2021, Karnib et al., 2019, Shaif et al., 2018). It also promotes learning, memory formation, neurogenesis, and synaptic plasticity (Suzuki et al., 2011, Yang et al., 2014, Weitian et al., 2015, Dong et al., 2017, El Hayek et al. 2019, Wang et al., 2019, Lu et al., 2019, Lev-Vachnish et a.l, 2019, Descalzi G et al., 2019, Herrera-López et al., 2020, Ikeda et al., 2021, Zhou et al., 2021,Roumes et al., 2021, Frame et al., 2023, Akter et al., 2023).

In conclusion, the relevance of total brain pH and lactate levels as indicators of the observed correlations is controversial, and evidence points towards lactate having more positive rather than negative effects. It is important that the authors perform studies looking at brain-region-specific concentrations of lactate and that they modulate lactate levels (decrease) in animal models of disease to validate their conclusions. It is also important to consider the above-mentioned studies before concluding that "altered brain pH and lactate levels are rather involved in the underlying pathophysiology of some patients with neuropsychiatric disorders" and that "lactate can serve as a potential therapeutic target for neuropsychiatric disorders".

Reviewer #2 (Public Review):

The study is novel and valuable to the field and provides new and important insights into the role of lipid mediators in VML injuries. By expanding our understanding of the mechanisms that regulate muscle regeneration following VML injuries, the study has the potential to guide the development of novel therapeutic interventions that promote tissue repair and recovery. The data presented in the manuscript is of good quality. The findings and conclusions are supported by a variety of different analyses (e.g., gene expression, histology, flow cytometry).

Despite the strengths of the study, some limitations are identified. Specifically, the impact of maresin 1 on macrophage phenotypes (M1/M2) could have been explored in more detail using histological or protein expression analysis. Moreover, additional data are needed to substantiate the claims about increased muscle regeneration. Lastly, the study does not address myofiber innervation, myofiber-type transitions, or motor unit remodeling.

Reviewer #2 (Public Review):

In this study, the goal is to leverage the power of Bayesian inference to estimate online the probability that any given arbitrarily chosen strategy is being used by the decision-maker. By computing the trial-by-trial MAP and variance of the posterior distribution for each candidate strategy, the authors can not only see which strategy is primarily being used at every given time during the task and when strategy changes occur but also detect when the target rule of a learning task becomes the front-running strategy, i.e., when successful learning occurs.

Strengths:<br /> 1. The proposed approach adds to recent methods for capturing the dynamics of decision-making at finer temporal resolution (trials) (Roy et al., 2021; Ashwood et al., 2022) but it is novel and differs from these in that it is suited especially well for analyzing when learning occurs, or when a rule switches and learning must recommence, and it does not necessitate large numbers of trials.

2. The manuscript starts with a validation of the approach using synthetic data and then is applied to datasets of trial-based two-alternative forced choice tasks ranging from rodent to non-human primate to human, providing solid evidence of its utility.

3. Compared to classic procedures for identifying when an animal has learned a contingency which typically needs to be conservative in favor of better accuracy, this method retrieves signs of learning happening earlier (~30 trials earlier on average). This is achieved by identifying the moment (trial) when the posterior probability of the correct "target" rule surpasses the probability of all other strategies. Having greater temporal precision in detecting when learning happens may have a very significant impact on studies of the neural mechanisms of learning.

4. This approach seems amenable to testing many different strategies depending on the purpose of the analysis. In the manuscript, the authors test target versus non-target strategies (correct versus incorrect) and also in another version of the analysis, they test what they call "exploratory" strategies.

5. One of the main appeals of this method is its apparent computational simplicity. It necessitates only updating on every trial the parameters of a beta distribution (prior distribution for a given strategy) with the evidence that the behavior on trial was either consistent or inconsistent with the strategy. Two scalars, the mode of the posterior (MAP) and the inverse of the variance, are all that are required for identifying the decision criterion (highest MAP and if tied lowest variance) and the learning criterion (first trial where MAP for target strategy is higher than chance).

Weaknesses:<br /> 1. It seems like a limitation of this approach is that the candidate strategies to arbitrate between must be known ex-ante. It is not clear how this approach could be applied to uncover latent strategies that are not mixtures of the strategies selected.

2. Different strategies may be indistinguishable from each other and thus it may not be possible to distinguish between them. Similarly, the fact that two strategies seem to be competing for the highest MAP doesn't necessarily mean that those are correct strategies and perhaps interchangeable as the manuscript seems to suggest.

3. The decay parameter is a necessary component to make the strategy selection non-stationary and accommodate data sets where the rules are changing throughout the task. However, the choice of the decay parameter value bounds does not seem very principled. Having this parameter as a free-parameter adds a flexibility that seems to have significant effects on when the strategy switch is detected and how stable the detected switch is.

4. This method is a useful approach for arbitrating between strategies and describing the behavior with a temporal precision that may prove important for studies attempting to tie these precise events to changes in neural activity. However, it seems limited in its explanatory power. In its current form, this method does not provide a prediction of the probability to transition from one strategy to another. And, because the MAP of different strategies may be close at any given moment, it is hard to imagine using this approach to tease out the different "mental states" that represent each strategy being at play.

Reviewer #2 (Public Review):

Working memory is not error free. Behavioral reports of items held in working memory display several types of bias, including contraction bias and serial dependence. Recent work from Akrami and colleagues demonstrates that inactivating rodent PPC reduces both forms of bias, raising the possibility of a common cause.

In the present study, Boboeva, Pezotta, Clopath, and Akrami introduce circuit and descriptive variants of a model in which the contents of working memory can be replaced samples from recent sensory history. This volatility manifests as contraction bias and serial dependence in simulated behavior, parsimoniously explaining both sources of bias. The authors validate their model by showing that it can recapitulate previously published and novel behavioral results in rodents and neurotypical and atypical humans.

Both the modeling and the experimental work is rigorous, providing convincing evidence that a model of working memory in which reports sometimes sample past experience can produce both contraction bias and serial dependence, and that this model is consistent with behavioral observations across rodents and humans in the parametric working memory (PWM) task.

These efforts constitute an admirable initial validation of the proposed model, and the authors present several novel predictions that will allow for further tests in future experiments. First, the authors note that their circuit model predicts a bimodal error distribution in delayed estimation paradigms (due to noisy sampling of sensory history on a subset of trials) that merges into a unimodal distribution when recent sensory history and the current to-be-reported stimulus have very similar values (Fig. 5c). Analysis of extent delayed estimation datasets (e.g., https://osf.io/jmkc9/) or new experiments will provide the opportunity for a straightforward test of this hypothesis.

Second, the bulk of the modeling efforts presented here are devoted to a circuit-level description of how putative posterior parietal cortex (PPC) and working-memory (WM) related networks may interact to produce such volatility and biases in memory. This effort is extremely useful because it allows the model to be constrained by neural observations and manipulations in addition to behavior, and the authors begin this line of inquiry here (by showing that the circuit model can account for effects of optogenetic inactivation of rodent PPC). As the authors note, population electrophysiology in PPC and WM-related areas and single-trial analyses will play an important role in the ultimate validation of this model.

Finally, it is noteworthy that, in the spirit of moving away from an overreliance on p-values (e.g., Amrhein et al., PeerJ 2017), the authors eschew conventional hypothesis testing when reporting their experimental results. The p-values aren't missed, in large part thanks to excellent visualizations and apparently large effect sizes. It's unclear how well this approach would generalize to other questions and datasets; nevertheless, this study provides an interesting data point in the ongoing conversation around reproducibility and rigor.

Reviewer #2 (Public Review):

Schmid et al present a lovely study looking at the effect of passive auditory exposure on learning a categorization task.<br /> The authors utilize a two-alternative choice task where mice have to discriminate between upward and downward moving frequency sweeps. Once mice learn to discriminate easy stimuli, the task is made psychometric and additional intermediate stimuli are introduced (as is standard in the literature). The authors introduce an additional two groups of animals, one that was passively exposed to the task stimuli before any behavioral shaping, and one that had passive exposure interleaved with learning. The major behavioral finding is that passive exposure to sounds improves learning speed. The authors show this in a number of ways through linear fits to the learning curves. Additionally, by breaking down performance based on the "extreme" vs "psychometric" stimuli, the authors show that passive exposure can influence responses to sounds that were not present during the initial training period. One limitation here is that the presented analysis is somewhat simplistic, does not include any detailed psychometric analysis (bias, lapse rates etc), and primarily focuses on learning speed. Ultimately though, the behavioral results are interesting and seem supported by the data.

To investigate the neural mechanisms that may underlie their behavioral findings, the authors turn to a family of artificial neural network models and evaluate the consequences of different learning algorithms and schedules, network architectures, and stimulus distributions, on the learning outcomes. The authors work through five different architectures that fail to recapitulate the primary behavior findings before settling on a final model, utilizing a combination of supervised and unsupervised learning, that was capable of reproducing the key aspects of the experiments. Ultimately, the behavioral results presented are consistent with network models that build latent representations of task-relevant features that are determined by statistical properties of the input distribution.

Reviewer #2 (Public Review):

This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however the biological insight gained is rather limited.

Reviewer #2 (Public Review):

The authors attempt to address a long-standing controversy in the study of the neural correlates of visual awareness, namely whether neurons in prefrontal cortex are necessarily involved in conscious perception. Several leading theories of consciousness propose a necessary role for (at least some sub-regions of) PFC in basic perceptual awareness (e.g., global neuronal workspace theory, higher order theories), while several other leading theories posit that much of the previously reported PFC contributions to perceptual awareness may have been confounded by task-based cognition that co-varied between the aware and unaware reports (e.g., recurrent processing theory, integrated information theory). By employing intracranial EEG in human patients and a threshold detection task on low-contrast visual stimuli, the authors assessed the timing and location of neural populations in PFC that are differentially activated by stimuli that are consciously perceived vs. not perceived. Overall, the reported results support the view that certain regions of PFC do contribute to visual awareness, but at time-points earlier than traditionally predicted by GNWT and HOTs.

Major strengths of this paper include the straightforward visual threshold detection task including the careful calibration of the stimuli and the separate set of healthy control subjects used for validation of the behavioral and eye tracking results, the high quality of the neural data in six epilepsy patients, the clear patterns of differential high gamma activity and temporal generalization of decoding for seen versus unseen stimuli, and the authors' interpretation of these results within the larger research literature on this topic. This study appears to have been carefully conducted, the data were analyzed appropriately, and the overall conclusions seem warranted given the main patterns of results.

Weaknesses include the saccadic reaction time results and the potential flaws in the design of the reporting task. As the authors acknowledge, this is not a "no report" paradigm, rather, it's a paradigm aimed at balancing the post-perceptual cognitive and motor requirements between the seen and unseen trials. On each trial, subjects/patients either perceived the stimulus or not, and had to briefly maintain this "yes/no" judgment until a fixation cross changed color, and the color change indicated how to respond (saccade to the left or right). Differences in saccadic RTs (measured from the time of the fixation color change to moving the eyes to the left or right response square) were evident between the seen and unseen trials (faster for seen). In the discussion, the authors summarize several alternative explanations of the saccade results and limitations of their report paradigm that will help guide future research.

The current results help advance our understanding of the contribution of PFC to visual awareness. These results, when situated within the larger context of the rapidly developing literature on this topic provide converging evidence that some sub-regions of PFC contribute to visual awareness, but at latencies earlier than originally predicted by proponents of, especially, global neuronal workspace theory. Three recent studies that used "no report paradigms", but with clearly visible stimuli, reported very similar results in PFC (Vishne et al., 2023; Broday-Dvir et al., 2023; Cogitate et al., 2023). The current study uses a report paradigm, but with consciously seen vs. unseen conditions, to fill the gap left by these previous studies, i.e., it remained unclear whether the PFC results from the previous studies were related to conscious or unconscious processing. Taken as a whole, evidence appears to be converging for a limited and early-in-time (200-300ms) contribution of PFC to visual awareness, after task and motor confounds are minimized.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediators of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from post-mortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the pre-symptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied with the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function. This is an expected result, given the fairly focal approach of ephrinB2 knockdown in C3-C5 spinal segments.

The major limitation of the study is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many other that comprise the ventral gray matter, have not been investigated in this study. Nonetheless, there is convincing evidence to suggest astrocytes play a significant role, as compared to oligodendrocytes in promoting ALS pathogenesis.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript by Martin-Flores et al. examines the role of DKK3 in Alzheimer's disease, focusing on the regulation of synaptic number. Using human AD brain databases and tissue samples, the authors demonstrate increased levels of DKK3 protein and mRNA in the brains of AD patients. DKK3 is expressed in excitatory neurons in WT mouse brains and accumulates at atrophic neurites around amyloid plaques in AD mouse brains. Interestingly, the secretion of DKK3 appears to be regulated by NMDAR antagonists, as well as chemical LTD. Through gain and loss of function studies, the authors reveal that DKK3 regulates the number of both excitatory and inhibitory synapses with distinct downstream pathways. Finally, the authors investigate the contribution of DKK3 to synaptic changes in AD and find that DKK3 loss of function rescues both excitatory and inhibitory synaptic defects, resulting in improved memory function in J20 mice.

Strengths:<br /> Overall, the data is clearly presented and deals with the novel roles of DKK3 in controlling excitatory and inhibitory synapses. The finding that shRNA expression of DKK3 in AD model mice rescues synaptic phenotypes and memory impairment is potentially interesting and may provide a new strategy for AD treatment.

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

Reviewer #3 (Public Review):

Summary:<br /> Onck and co-workers present in this work the identification of binding partners and sites of polyPR on various nuclear transport components and elucidate how polyPR might potentially influence the transport process. It's interesting to note that some interaction sites on transport components also serve as their inherent/functional binding sites (Figure 3). The difference in the effects between short polyPR (PR7) and long polyPR (PR50) is also evident, although the authors might need to clarify the mechanisms better. Overall, I find this manuscript well organized and concisely written, and it would greatly enhance our understanding of the toxicity induced by polyPR.

Strengths:<br /> The 1-bead per atom force field model used in the study is well-tuned for studying the interactions between polyPR and proteins, as the essential cation-pi interactions (between Arg and Phe/Tyr/Trp) was included using a 8-6 LJ model.

Weaknesses:<br /> To cite the author's response: "At the moment, accurately capturing the binding of NCT components to their native binding targets and the competition with polyPR are best resolved by all-atom molecular dynamics simulations, which come with significant computational demands. This level of detail and computation-intensive analyses is beyond the scope of the current study."

Reviewer #2 (Public Review):

Summary:<br /> Chang et al. investigate neuronal activity firing patterns across various cortical regions in an interesting context-dependent tactile vs visual detection task, developed previously by the authors (Chevee et al., 2021; doi: 10.1016/j.neuron.2021.11.013). The authors report the important involvement of a medial frontal cortical region (MM, probably a similar location to wM2 as described in Esmaeili et al., 2021 & 2022; doi: 10.1016/j.neuron.2021.05.005; doi: 10.1371/journal.pbio.3001667) in mice for determining task rules.

Strengths:<br /> The experiments appear to have been well carried out and the data well analysed. The manuscript clearly describes the motivation for the analyses and reaches clear and well-justified conclusions. I find the manuscript interesting and exciting!

Weaknesses:<br /> I did not find any major weaknesses.

Reviewer #2 (Public Review):

Summary:<br /> The authors' long-term goals are to understand the utility of precisely phased cortex stimulation regimes on recovery of function after spinal cord injury (SCI). In prior work, the authors explored the effects of contralesion cortex stimulation. Here, they explore ipsilesion cortex stimulation in which the corticospinal fibers that cross at the pyramidal decussation are spared. The authors explore the effects of such stimulation in intact rats and rats with a hemisection lesion at the thoracic level ipsilateral to the stimulated cortex. The appropriately phased microstimulation enhances contralateral flexion and ipsilateral extension, presumably through lumbar spinal cord crossed-extension interneuron systems. This microstimulation improves weight bearing in the ipsilesion hindlimb soon after injury, before any normal recovery of function would be seen. The contralateral homologous cortex can be lesioned in intact rats without impacting the microstimulation effect on flexion and extension during gait. In two rats ipsilateral flexion responses are noted, but these are not clearly demonstrated to be independent of the contralateral homologous cortex remaining intact.

Strengths:<br /> This paper adds to prior data on cortical microstimulation by the laboratory in interesting ways. First, the strong effects of the spared crossed fibers from the ipsi-lesional cortex in parts of the ipsi-lesion leg's step cycle and weight support function are solidly demonstrated. This raises the interesting possibility that stimulating the contra-lesion cortex as reported previously may execute some of its effects through callosal coordination with the ipsi-lesion cortex tested here. This is not fully discussed by the authors but may represent a significant aspect of these data. The authors demonstrate solidly that ablation of the contra-lesional cortex does not impede the effects reported here. I believe this has not been shown for the contra-lesional cortex microstimulation effects reported earlier, but I may be wrong.

Effects and neuroprosthetic control of these effects are explored well in the ipsi-lesion cortex tests here.

Weaknesses:<br /> Some data is based on very few rats. For example (N=2) for ipsilateral flexion effects of microstimulation. N=3 for homologous cortex ablation, and only ipsi extension is tested it seems. There is no explicit demonstration that the ipsilateral flexion effects in only 2 rats reported can survive the contra-lateral cortex ablation.

Some improvements in clarity and precision of descriptions are needed, as well as fuller definitions of terms and algorithms.

Likely Impacts:<br /> This data adds in significant ways to prior work by the authors, and an understanding of how phased stimulation in cortical neuroprosthetics may aid in recovery of function after SCI, especially if a few ambiguities in writing and interpretation are fully resolved.

Reviewer #2 (Public Review):

Summary:<br /> This is a fundamental and elegant study showing the role of BMP signaling in cerebellar development. This is an important question because there are multiple diseases, including aggressive childhood cancers, which involve granule cell precursors. Thus understanding of the factors that govern the formation of the granule cell layer is important both from a basic science and a disease perspective.

Overall, the manuscript is clear and well-written. The figures are extremely clear, wonderfully informative, and overall quite beautiful.

Figures 1-3 show the experimental design and report how BMP activity is altered over development in both the chick and the human developing cerebellum. Both data are very impressive and convincing.

They then go on to modulate BMP activity in the developing chick, using a complex electroporation paradigm that allows them to label cells with GFP as well as with cell-specific reporters of BMP activity levels. They bidirectionally modulate BMP levels and then can look at both cell-specific and non-specific alterations in the formation of the external and internal granule cell layer, across different developmental timepoints. These are really elegant and rigorous experiments, as they look at both sagittal and transverse sections to collect this data. This makes the data extremely compelling. With these rigorous techniques, they show that BMP signaling serves more than one function across development: it is involved in the initial tangential migration from the rhombic lip, but at a later time, both up- and down-regulation of BMP activity reduces the density of amplifying cells in the external granule cell layer.

Strengths:<br /> Overall, I think the paper is interesting and important and the data is strong. The use of both chick and human tissue strengthens the findings. They are extremely rigorous, analyzing data from multiple planes at multiple ages, which also really strengthens their findings. The dual electroporation approach is extremely elegant, providing beautiful visual representations of their findings.

Weaknesses:<br /> I find no significant weaknesses.

Reviewer #2 (Public Review):

Summary:

H2S is a gas that is toxic to many animals and causes avoidance in animals such as C. elegans. The authors show that H2S increases the frequency of turning and the speed of locomotion. The response was shown to be modulated by a number of neurons and signaling pathways as well as by ambient oxygen concentrations. The long-term adaptation involved gene expression changes that may be related to iron homeostasis as well as the homeostasis of mitochondria.

Strengths:

Overall, the authors provide many pieces that will be important for solving how H2S signals through neuronal circuits to change gene expression and physiological programs. The experiments rely mostly on a behavioral assay that measures the increase of locomotion speed upon exposure to H2S. This assay is then combined with manipulations of environmental factors, different wild-type strains, and mutants. The mutants analyzed were obtained as candidates from the literature and from transcriptional profiling that the authors carried out in worms that were exposed to H2S. These studies imply several genetic signaling pathways, some neurons, and metabolism-related factors in the response to H2S. Hence the data provided should be useful for the field.

Weaknesses:

On the other hand, many important aspects of the underlying mechanisms remain unsolved and the reader is left with many loose ends. For example, it is not clear how H2S is actually sensed, how sensory neurons are activated and signal to downstream circuits, and what the role of ciliated and RMG neurons is in this circuit. It remains unclear how signals lead to gene expression and physiological changes such as metabolic rewiring. Solving all this would clearly be beyond the scope of a single manuscript. Yet, the manuscript also does not focus on understanding one of these central aspects and rather is all over the place, which makes it harder to understand for readouts that are not in this core field. Multiple additional methods and approaches exist to dig deeper into these mechanisms in the future, such as neuronal calcium imaging, optogenetics, and metabolic analysis. To generate a story that will be interesting to a broad readership substantial additional experimentation would be required. Further, in the current manuscript, it is often difficult to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved in terms of documentation, narration/explanation, and visualization.

Reviewer #2 (Public Review):

Summary:<br /> Authors hypothesized that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. They test this hypothesis in a mouse model of TBI induced by controlled cortical impact in wild-type and kcnt1 knock-out (KO) mice. The authors performed a series of experiments including behavioral assessment, electrographic recordings in vivo and in vitro, western blotting, and immunofluorescence imaging with the goal of investigating the contributory role of KNa1.1 channel to post-traumatic epileptogenesis.

Strengths:<br /> The hypothesis is innovative, focusing on the specific role of the KNa1.1 channel in the development of PTE post-TBI. The use of a comprehensive set of techniques, including EEG, whole cell patch clamp, western blot, immunofluorescence imaging, and behavioral assessments, provides a diverse data set. The study makes initial steps in correlating specific molecular changes with functional outcomes in TBI models, offering potential pathways for therapeutic intervention.

Weaknesses:<br /> 1) The study presents interesting findings on early changes in protein expression and electrophysiological properties following TBI. However, I would like to draw attention to the timeline of EEG and cellular assessments that require further clarification or consideration. The patch clamp recordings and other assays were conducted within 14 days post-TBI, while EEG recordings with or without PTZ testing were performed at 3 months post-injury. This temporal gap leaves a period where changes in electrophysiological properties and PTE status are not accounted for. Since epileptogenesis post-TBI involves a dynamic process spanning from hyperacute and acute phases to chronic development, capturing these changes continuously or at least at more frequent intervals (on and off bi-weekly) could provide a more comprehensive understanding of this progression. In the current study design, the one-week duration of EEG recordings at the 3-month timepoint raises the possibility that some seizures might have occurred undetected between the early post-injury phase and the EEG recording period. This gap could potentially affect the interpretation of results, especially when correlating early post-injury cellular changes with later seizure activity and thresholds and hence is a significant limitation to data interpretation. Experiments using western blots, immunofluorescence, and patch clamp were done at an early timepoint hence the relevance of these datasets to PTE status outcome is not established.

2) While referencing Nichols et al., 2015, to justify the 14-day timeline for characterizing seizures is understandable, it is important to consider differences in animal models (juvenile rats in Nichols vs. adult mice in the present study) which might influence the generalizability of the findings.

3) Behavior: Authors performed behavioral assays using the rotarod technique evaluating the hanging time of mice with different severity of TBI (mild, moderate, severe). The purpose of the testing is explained as 'to sort out an appropriate TBI model'. The authors also measured mortality rates 'to attain a stable model'. It is not clear what is assumed by the terms 'appropriate' or 'stable' model. Furthermore, the relevance of this to post-traumatic epileptogenesis is unclear. Additionally, the mortality in the Sham group within 2 weeks of craniectomy is not explained.

4) Seizure assessment: authors report seizure severity in PTZ-induced seizures but no mention about the severity of spontaneous seizures between different TBI severity modalities. When characterizing the PTZ-induced seizures, the mild TBI group does not have generalized seizures. Does this mean that al all 6 tested animals in the mild TBI group had exclusively focal seizures? What about the spontaneous seizure occurrence: were all seizures generalized or were any focal too? Did that differ between mild, moderate, and severe TBI?

5) Experiments with KCNT1 KO mice: in all experiments with a mutant mouse line, authors only used them in TBI group. Without the Sham group, it is difficult to discern whether any observed changes in seizure susceptibility in the KCNT1 KO TBI group are due to gene deletion, the TBI, or a combination of both. This group would provide a crucial comparison point to isolate the effects of the KCNT1 knockout from those of TBI. This limits the ability to make comprehensive conclusions about the role of the KCNT1 gene in seizure susceptibility following TBI.

6) While the current study showed interesting data about the KNa1.1 changes early after TBI, the study design and disconnect between early and late electrophysiology experiments timeline, does not establish a correlative or causative link between KNa1.1 and post-traumatic epileptogenesis since it remained unresolved whether KCNT1 KO mice developed no PTE (or less severe/ less frequent seizures at 3 months) compared with WT mice and what are the seizure properties of KCNT1 KO Sham mice compared to WT TBI and Sham groups. The hypothesis was that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. The part about 'promotes the occurrence of PTE' was not established.

7) NeuN is not the best marker of neurons in the context of TBI since TBI affects its expression patterns which will influence the interpretation of co-localization results. Unlike NeuN, Nissl staining is less likely to be affected by factors that alter protein expression, such as TBI. Therefore, it can be a more stable marker for identifying neurons in injured brain tissue.

8) Statistics: Authors report only SEM, which shows the precision of the mean and it will decrease as the sample size increases and does not reflect the data variability.

Reviewer #2 (Public Review):

In this study, Leighton et al performed remarkable experiments by combining in-vivo patch-clamp recording with two-photon dendritic Ca2+ imaging. The voltage-clamp mode is a major improvement over the pioneer versions of this combinatorial experiment that has led to major breakthroughs in the neuroscience field for visualizing and understanding synaptic input activities in single cells in-vivo (sharp electrodes: Svoboda et al, Nature 1997, Helmchen et al, Nature Neurosci 1999; whole-cell current-clamp: Jia et al, Nature 2010, Chen et al, Nature 2011. I suggest that these papers would be cited). This is because in voltage-clamp mode, despite the full control of membrane voltage in-vivo not being realistic, is nevertheless most effective in preventing back-propagation action potentials, which would severely confound the measurement of individual synaptically-induced Ca2+ influx events. Furthermore, clamping the cell body at a strongly depolarized potential (here the authors did -30mV) also facilitates the detection of synaptically-induced Ca2+ influx. As a result, the authors successfully recorded high-quality Ca2+ imaging data that can be used for precise analysis. To date, even in view of the rapid progress of voltage-sensitive indicators and relevant imaging technologies in recent years, this very old 'art' of combining single-cell electrophysiology and two-photon imaging (ordinary, raster-scanned, video-rate imaging) of Ca2+ signals still enables measurements of the best-level precision.

On the other hand, the interpretation of data in this study is a bit narrow-minded and lacks a comprehensive picture. Some suggestions to improve the manuscript are as follows:

1. The authors made a segregation of 'spine synapse' and 'shaft synapse' based solely on the two-photon images in-vivo. However, caution shall be taken here, because the optical resolution under in-vivo imaging conditions like this cannot reliably tell apart whether a bright spot within or partially overlapping a segment of the dendrite is a spine on top of (or below) it. Therefore, what the authors consider as a 'shaft synapse' (by detecting Ca2+ hotspots) has an unknown probability of being just a spine on top or below the dendrite. If there is other imaging data of higher axial resolution to validate or calibrate, the authors shall take some further considerations or analysis to check the consistency of their data, as the authors do need such a segregation between spine and shaft synapses to show how they evolve over the brain development stages.

2. The use of terminology 'bursts of spontaneous inputs' for describing voltage-clamp data seems improper. Conventionally, 'burst' refers to suprathreshold spike firing events, but here, the authors use 'burst' to refer to inward synaptic currents collected at the cell body. Not every excitatory synaptic input (or ensemble of inputs) activation will lead to spike firing under naturalistic conditions, therefore, these two concepts are not equivalent. It is recommended to use 'barrage of inputs' instead of 'burst of inputs'. Imagine a full picture of the entire dendritic tree, the fact that the authors could always capture spontaneous Ca2+ events here and there within a few pieces of dendrites within an arbitrary field-of-view suggests that, the whole dendritic tree must have many more such events going on as a barrage while the author's patch electrode picks up the summed current flow from the whole dendritic tree.

3. Following the above issue, an analysis of the temporal correlation between synaptic (not segregating 'spine' or 'shaft') Ca2+ events and EPSCs is absent. Again, the authors drew arbitrary time windows to clump the events for statistical analysis. However, the demonstrated example data already shows that the onset times of individual synaptic Ca2+ events do not necessarily align with the beginning of a 'barrage' inward current event.

4. The authors claim that "these observations indicate that the activity patterns investigated here are not or only slightly affected by low-level anesthesia". It would be nice to show some of the recordings in this work without any anesthesia to support this claim.

Reviewer #2 (Public Review):

Summary: The study by Jaime-Tobon & Moser is a truly major effort to bridge the gap between classical observations on how auditory neurons respond to sounds and the synaptic basis of these phenomena. The so-called spiral ganglion neurons (SGNs) are the primary auditory neurons connecting the brain with hair cells in the cochlea. They all respond to sounds increasing their firing rates, but also present multiple heterogeneities. For instance, some present a low threshold to sound intensity, whereas others have high threshold. This property inversely correlates with the spontaneous rate, i.e., the rate at which each neuron fires in the absence of any acoustic input. These characteristics, along with others, have been studied by many reports over the years. However, the mechanisms that allow the hair cells-SGN synapses to drive these behaviors are not fully understood.

Strengths:<br /> The level of experimental complexity described in this manuscript is unparalleled, producing data that is hardly found elsewhere. The authors provide strong proof for heterogeneity in transmitter release thresholds at individual synapses and they do so in extremely complex experimental settings. In addition, the authors found other specific differences such as in synaptic latency and max EPSCs. A reasonable effort is put into bridging these observations with those extensively reported in in vivo SGNs recordings. Similarities are many and differences are not particularly worrying as experimental conditions cannot be perfectly matched, despite the authors' efforts in minimizing them.

Weaknesses:<br /> Some concern surges in relation to mismatches with previous reports of IHC-SGN synapses function. EPSCs at these synapses present a peculiar distribution of amplitudes, shapes, and rates. These characteristics are well-established and some do not seem to be paralleled in this study. Here, amplitude distributions are drastically shifted to smaller values, and rates of events are very low, all compared with previous evidence. The reasons for these discrepancies are unclear. The rate at which spontaneous EPSCs appear is an especially sensitive matter. A great part of the conclusions relies on the definition of which of the SGNs (or should say synapses) belong to the low end and which to the high end in the spectrum of spontaneous rates. The data presented by the authors seem a bit off and the criteria used to classify recordings are not well justified. The authors should clarify the origin of these differences since they do not seem to come from obvious reasons such as animal ages, recording techniques, mouse strain, or even species.

Reviewer #2 (Public Review):

Summary:

In this paper, Sang et al. set out to identify gustatory receptors involved in salt taste sensation in Drosophila melanogaster. In a two-choice assay screen of 30 Ir mutants, they identified that Ir60b is required for avoidance of high salt. In addition, they demonstrate that activation of Ir60b neurons is sufficient for gustatory avoidance using either optogenetics or TRPV1 to specifically activate Ir60b neurons. Then, using tip recordings of labellar gustatory sensory neurons and proboscis extension response behavioral assays in Ir60b mutants, the authors demonstrate that Ir60b is dispensable for labellar taste neuron responses to high salt and the suppression of proboscis extension by high salt. Since external gustatory receptor neurons (GRNs) are not implicated, they look at Poxn mutants, which lack external chemosensory sensilla but have intact pharyngeal GRNs. High salt avoidance was reduced in Poxn mutants but was still greater than Ir60b mutants, suggesting that pharyngeal gustatory sensory neurons alone are sufficient for high salt avoidance. The authors use a new behavioral assay to demonstrate that Ir60b mutants ingest a higher volume of sucrose mixed with high salt than control flies do, suggesting that the action of Ir60b is to limit high salt ingestion. Finally, they identify that Ir60b functions within a single pair of gustatory sensory neurons in the pharynx, and that these neurons respond to high salt but not bitter tastants.

Strengths:

A great strength of this paper is that it rigorously corroborates previously published studies that have implicated specific Irs in salt taste sensation. It further introduces a new role for Ir60b in limiting high salt ingestion, demonstrating that Ir60b is necessary and sufficient for high salt avoidance and convincingly tracing the action of Ir60b to a particular subset of gustatory receptor neurons. Overall the authors have achieved their aim by identifying a new gustatory receptor involved in limiting high salt ingestion. They use rigorous genetic, imaging, and behavioral studies to achieve this aim, often confirming a given conclusion with multiple experimental approaches. They have further done a great service to the field by replicating published studies and corroborating the roles of a number of other Irs in salt taste sensation. An aspect of this study that merits further investigation is how the same gustatory receptor neurons and Ir in the pharynx can be responsible for regulating the ingestion of both appetitive (sugar) and aversive tastants (high salt).

Weaknesses:

There are several weaknesses that, if addressed, could greatly improve this work.<br /> 1) The authors combine the results and discussion but provide a very limited interpretation of their results. More discussion of the results would help to highlight what this paper contributes, how the authors interpret their results, and areas for future study.<br /> 2) The authors rename previously studied populations of labellar GRNs to arbitrary letters, which makes it difficult to understand the experiments and results in some places. These GRN populations would be better referred to according to the gustatory receptors they are known to express.<br /> 3) The conclusion that GRNs responsible for high salt aversion may be inhibited by those that function in low salt attraction is not well substantiated. This conclusion seems to come from the fact that overexpression of Ir60b in salt attraction and salt aversion sensory neurons still leads to salt aversion, but there need not be any interaction between these two types of sensory neurons if they act oppositely on downstream circuits.<br /> 4) The authors rely heavily on a new Droso-X behavioral apparatus that is not sufficiently described here or in the previous paper the authors cite. This greatly limits the reader's ability to interpret the results.

Reviewer #2 (Public Review):

Summary:<br /> McSwiggen et al present a high throughput platform for SPT that allows them to identify pharmaceutics interactions with the diffusional behavior of receptors and in turn to identify potent new ligands and cellular mechanisms. The manuscript is well written, it provides a solid new mentor and a proper experimental foundation

Strengths:<br /> The method capitalizes and extends to existing high throughput toolboxes and is directly applied to multiple receptors and ligands. The outcomes are important and relevant for society. 10^6 cells and >400 ligands per is a significant achievement.

The method can detect functionally relevant changes in transcription factor dynamics and accurately differentiate the ligand/target specificity directly within the cellular environment. This will be instrumental in screening libraries of compounds to identify starting points for the development of new therapeutics. Identifying hitherto unknown networks of biochemical signaling pathways will propel the field of single-particle live cell and quantitative microscopy in the area of diagnostics. The manuscript is well-written and clearly conveys its message.

Weaknesses:<br /> There are a few elements, that if rectified would improve the claims of the manuscript.

The authors claim that they measure receptor dynamics. In essence, their readout is a variation in diffusional behavior that correlates to ligand binding. While ligand binding can result in altered dynamics or /and shift in conformational equilibrium, SPT is not recording directly protein structural dynamics, but their effect on diffusion. They should correct and elaborate on this.

L 148 What do the authors mean 'No correlation between diffusion and monomeric protein size was observed, highlighting the differences between cellular protein dynamics versus purified systems'. This is not justified by data here or literature reference. How do the authors know these are individual molecules? Intensity distributions or single bleaching steps should be presented.

Along the same lines, the data in Figs 2 and 4 show that not only the immobile fraction is increased but also that the diffusion coefficient of the fast-moving (attributed to free) is reduced. The authors mention this and show an extended Fig 5 but do not provide an explanation. How do potential transient ligand binding and the time-dependent heterogeneity in motion (see comment above) contribute to this? Also, in line 216 the authors write "with no evidence" of transient diffusive states. How do they define transient diffusive states? While there are toolboxes to directly extract the existence and abundance of these either by HMM analysis or temporal segmentation, the authors do not discuss or use them.

The authors discuss the methods for extracting kinetic information of ligand binding by diffusion. They should consider the temporal segmentation of heterogenous diffusion. There are numerous methods published in journals or BioRxiv based on analytical or deep learning tools to perform temporal segmentation. This could elevate their analysis of Kon and Koff.

Reviewer #2 (Public Review):

Summary:

In the present manuscript, Zhang et al utilize single-nuclei RNA-Seq to investigate the heterogeneity of perirenal adipose tissue. The perirenal depot is interesting because it contains both brown and white adipocytes, a subset of which undergo functional "whitening" during early development. While adipocyte thermogenic transdifferentiation has been previously reported, there remain many unanswered questions regarding this phenomenon and the mechanisms by which it is regulated.

Strengths:

The combination of UCP1-lineage tracing with the single nuclei analysis allowed the authors to identify four populations of adipocytes with differing thermogenic potential, including a "whitened" adipocyte (mPRAT-ad2) that retains the capacity to rapidly revert to a brown phenotype upon cold exposure. They also identify two populations of white adipocytes that do not undergo browning with acute cold exposure.

Anatomically distinct adipose depots display interesting functional differences, and this work contributes to our understanding of one of the few brown depots present in humans.

Weaknesses:

The most interesting aspect of this work is the identification of a highly plastic mature adipocyte population with the capacity to switch between a white and brown phenotype. The authors attempt to identify the transcriptional signature of this ad2 subpopulation, however, the limited sequencing depth of single nuclei somewhat lessens the impact of these findings. Furthermore, the lack of any form of mechanistic investigation into the regulation of mPRAT whitening limits the utility of this manuscript. However, the combination of well-executed lineage tracing with comprehensive cross-depot single-nuclei presented in this manuscript could still serve as a useful reference for the field.

Reviewer #2 (Public Review):

This study by Matsuo-Takasaki et al. reported the development of a novel suspension culture system for hiPSC maintenance using Wnt/PKC inhibitors. The authors showed elegantly that inhibition of the Wnt and PKC signaling pathways would repress spontaneous differentiation into neuroectoderm and mesendoderm in hiPSCs, thereby maintaining cell pluripotency in suspension culture. This is a solid study with substantial data to demonstrate the quality of the hiPSC maintained in the suspension culture system, including long-term maintenance in >10 passages, robust effect in multiple hiPSC lines, and a panel of conventional hiPSC QC assays. Notably, large-scale expansion of a clinical grade hiPSC using a bioreactor was also demonstrated, which highlighted the translational value of the findings here. In addition, the author demonstrated a wide range of applications for the IWR1+LY suspension culture system, including support for freezing/thawing and PBMC-iPSC generation in suspension culture format. The novel suspension culture system reported here is exciting, with significant implications in simplifying the current culture method of iPSC and upscaling iPSC manufacturing.

Another potential advantage that perhaps wasn't well discussed in the manuscript is the reported suspension culture system does not require additional ECM to provide biophysical support for iPSC, which differentiates from previous studies using hydrogel and this should further simplify the hiPSC culture protocol.

Interestingly, although several hiPSC suspension media are currently available commercially, the content of these suspension media remained proprietary, as such the signaling that represses differentiation/maintains pluripotency in hiPSC suspension culture remained unclear. This study provided clear evidence that inhibition of the Wnt/PKC pathways is critical to repress spontaneous differentiation in hiPSC suspension culture.

I have several concerns that the authors should address, in particular, it is important to benchmark the reported suspension system with the current conventional culture system (eg adherent feeder-free culture), which will be important to evaluate the usefulness of the reported suspension system. Also, the manuscript lacks a clear description of a consistent robust effect in hiPSC maintenance across multiple cell lines. There are also several minor comments that should be addressed to improve readability, including some modifications to the wording to better reflect the results and conclusions.

Reviewer #2 (Public Review):

Summary:

The authors have developed novel Ovalbumin model (OTII) peptide that can be labeled with a site-specific FlAsH dye to track agonist peptides both in vitro and in vivo. The utility of this tool could allow better tracking of activated polyclonal T cells particularly in novel systems. The authors have provided solid evidence that peptides are functional, capable of activating OTII T cells, and these peptides can undergo trogocytosis by cognate T cells only.

Strengths:<br /> -An extensive array of in vitro and in vivo studies are used to assess peptide functionality.<br /> -Nice use of cutting edge intravital imaging,<br /> -internal controls such as multiple non-cogate T cells were used to improve robustness of the results<br /> -One of the strengths is the direct labeling of the peptide, and the potential utility in other systems.

Weaknesses:<br /> -Peptide labeling specificity and efficiency is not clear. High levels of background labeling. While it was sufficient for demonstrating the system works, it may pose problems depending on the peptide sequence, and/or use at lower dose.<br /> -Only one peptide system was tested, namely OVA323-339 region.<br /> -Limited novel biological findings. This study mostly describes a new tool that may have exciting potential.

Reviewer #2 (Public Review):

Summary:

The goal of this study was to examine the role of FNDC5 in the response of the murine skeleton to either lactation or a calcium-deficient diet. The authors find that female FNDC5 KO mice are somewhat protected from bone loss and osteocyte lacunar enlargement caused by either lactation or a calcium-deficient diet. In contrast, male FNDC5 KO mice lose more bone and have a greater enlargement of osteocyte lacunae than their wild-type controls. Based on these results, the authors conclude that in males irisin protects bone from calcium deficiency but that in females it promotes calcium removal from bone for lactation.

While some of the conclusions of this study are supported by the results, it is not clear that the modest effects of FNDC5 deletion have an impact on calcium homeostasis or milk production.

Specific comments:

1. The authors sometimes refer to FNDC5 and other times to irisin when describing causes for a particular outcome. Because irisin was not measured in any of the experiments, the authors should not conclude that lack of irisin is responsible. Along these lines, is there any evidence that either lactation or a calcium-deficient diet increases the production of irisin in mice?

2. The results of the irisin-rescue experiment shown in figure 2G cannot be appropriately interpreted without normal diet controls. In addition, some evidence that the AAV8-irisin virus actually increased irisin levels in the mice would strengthen the conclusion.

3. There is insufficient evidence to support the idea that the effect of FNDC5 on bone resorption and osteocytic osteolysis is important for the transfer of calcium from bone to milk. Previous studies by others have shown that bone resorption is not required to maintain milk or serum calcium when dietary calcium is sufficient but is critical if dietary calcium is low (Endo. 156:2762-73, 2015). To support the conclusions of the current study, it would be necessary to determine whether FNDC5 is required to maintain calcium levels when lactating mice lack sufficient dietary calcium.

4. The amount of cortical bone loss due to lactation is very similar in both WT and FNDC5 KO mice. The results of the statistical analysis of the data presented in figure 1B are surprising given the very similar effect size of lactation. The key result from the 2-way ANOVA is whether there is an effect of genotype on the effect size of lactation (genotype-lactation interaction). The interaction terms were not provided. Similar concerns are noted for the results shown in figure 1G and H.

5. It is not clear what justifies the term 'primed' or 'activated' for resorption. Is there evidence that a certain level of TRAP expression lowers the threshold for osteocytic osteolysis in response to a stimulus?

Review #2 (Public Review)

In the manuscript Watanuki et al. define the metabolic profile of HSCs in stress/proliferative (myelosuppression with 5-FU), and mitochondrial inhibition and homeostatic conditions. Their conclusions are that during proliferation HSCs rely more on glycolysis (as other cell types) while HSCs in homeostatic conditions are mostly dependent on mitochondrial metabolism. Mitochondrial inhibition is used to demonstrate that blocking mitochondrial metabolism results in similar features of proliferative conditions.

The authors used state-of-the-art technologies that allow metabolic readout in a limited number of cells like rare HSCs. These applications could be of help in the field since one of the major issues in studying HSCs metabolism is the limited sensitivity of the "standard" assays, which make them not suitable for HSC studies.

Reviewer #2 (Public Review):

Summary:<br /> The work by Varadharajan et. al. explored a previously known genetic variant and its pathophysiology in the development of alcohol-associated liver injury. It provides a plausible mechanism for how varying levels of MBOAT7 could impact the lipid metabolomics of the cell, leading to a deleterious phenotype in MBOAT7 knockout. The authors further characterized the impact of the lipidomic changes and raised lysosomal biogenesis and autophagic flux as mechanisms of how MBOAT7 deletion causes the progression of ALD.

Strengths:<br /> Connecting the GWAS data on MBOAT7 variants with plausible pathophysiology greatly enhances the translational relevance of these findings. The global lipidomic profiling of ALD mice is also very informative and may lead to other discoveries related to lipid handling pathways.

Weaknesses:<br /> The rationale of why MBOAT7 metabolites are lower in heavy drinkers than in normal individuals is not well explained. MBOAT7 loss of function drives ALD, but unclear if MBOAT7 deletion also drives preference for alcohol or if alcohol inhibits MBOAT7 function. Presuming most individuals studied here were WT and expressed an appropriate level of MBOAT7?<br /> Also, the discussion of mechanisms of MBOAT7-induced dysregulation of lysosomal biogenesis/autophagy, while very interesting, seems incomplete. It is not clear how MBOAT7 an enzyme involved in membrane phospholipid remodeling increases mTOR which leads to decreased TFEB target gene transcription. Furthermore, given the significant disturbances of global lipidomic profiling in MBOAT7 knockout, many pathways are potentially affected by this deletion. Further in vivo modeling that specifically addresses these pathways (TFEB targeting, mTOR inhibitor) would help strengthen the conclusions of this paper.

Reviewer #2 (Public Review):

Summary:

While a significant portion of immunotherapy research has focused on the pivotal role of T cells in tumor immunity, their effectiveness may be limited by the suppressive nature of the tumor environment. On the other hand, myeloid cells are commonly found within tumors and can withstand these adverse conditions. However, these cells often adopt an immunosuppressive phenotype when infiltrating tumors. Therefore, manipulating myeloid cells could potentially enhance the anti-tumor potential of immunotherapy.

In this manuscript, Farhat-Younes and colleagues have demonstrated that activating the IgM receptor signaling in myeloid cells induces an oxygen burst, the secretion of Granzyme B, and the lysis of adjacent tumor cells. Furthermore, they have outlined a strategy to utilize these features to generate CAR macrophages. However, they have identified a limitation: the expression of scFv in myeloid cells induces ER stress and the degradation of misfolded proteins. To address this issue, chimeric receptors were designed based on the high-affinity FcγRI for IgG. When macrophages transfected with these receptors were exposed to tumor-binding IgG, extensive tumor cell killing, and the release of reactive oxygen species and Granzyme B were observed.

Strengths:<br /> In general, I consider this work to be significant, and the results are compelling. It emphasizes the specific considerations and requirements for successful manipulation in myeloid cells, which could further advance the field of cellular engineering for the benefit of immunotherapy

Weaknesses:

Nevertheless, there are several minor issues that should be addressed:

1- TCR fragments are commonly used to induce ER stress in non-immune cells. Therefore, it would be interesting to investigate whether TCR fragments can be expressed in myeloid cells and if they induce ER stress. Addressing this issue would support the notion that these cells lack the ER chaperones required for folding immunoglobulin variable chains.<br /> 2- It would be valuable to determine whether, after the degradation of scFv fragments by myeloid cells, they are presented on MHC-I and MHC-II.<br /> 3- Some methodological details, such as the vaccination protocol and high-resolution microscopy procedures, are missing from the text.

Reviewer #2 (Public Review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to readers.

The data, for the most part, support the conclusions but I do have concerns about some of the data and the interpretations that should be addressed.

Reviewer #2 (Public Review):

Summary:

This combined experimental-theoretical paper introduces a novel two-domain statistical thermodynamic model (primarily Equation 1) to study allostery in generic systems but focusing here on the tetracycline repressor (TetR) family of transcription factors. This model, building on a function-centric approach, accurately captures induction data, maps mutants with precision, and reveals insights into epistasis between mutations.

Strengths:

The study contributes innovative modeling, successful data fitting, and valuable insights into the interconnectivity of allosteric networks, establishing a flexible and detailed framework for investigating TetR allostery. The manuscript is generally well-structured and communicates key findings effectively.

Weaknesses:

The only minor weakness I found was that I still don't have a better sense into (a) intuition and (b) mathematical derivation of Equation 1, which is so central to the work. I would recommend that the authors provide this early on in the main text.

Reviewer #3 (Public Review):

Summary<br /> In this research advance, the authors purport to show that the unified neutral theory of biodiversity (UNTB) is not a suitable null model for exploring the relationship between macroecological quantities, and additionally that the stochastic logistic growth model (SLM) is a viable replacement. They do this by citing other studies where UNTB was unable to capture individual macroecological quantities, and then demonstrating SLM's strength at predicting the same diversity metrics. They extend this analysis to show SLM's modeling capability at multiple scales of coarse graining, in addition to its failures at predicting these metrics' variances. Finally, authors conduct a similar analysis to Madi et al. (2020) by investigating the relationship between diversity measures within a group and across coarse-grained groups (e.g. genera diversity in one family compared to diversity of families). The authors show that choosing SLM as a null model reveals some previously reported relationships to be no longer "novel", in the sense that the patterns can be adequately captured by the null model. Authors also show that relationships not captured by the null model can be recovered by adding correlations, suggesting interactions are the driving force behind them.

Strengths<br /> 1. Authors make a strong argument that UNTB is not a good null model of macroecological observables and especially relationships between them. Authors convincingly argue that a SLM is a better null since the gamma distribution it predicts is a better description of the empirical Abundance Fluctuation Distributions (AFD).<br /> 2. Authors show that the gamma distribution predicted by SLM is a good fit for the AFD's at many different scales of coarse graining, not just the OTU level as was previously demonstrated. Authors show the same distribution predicted the mean diversity and richness at all scales of coarse graining.<br /> 3. Authors convincingly demonstrate how SLM can be used to test the relevance of interactions to macroecological relationships.

Weaknesses<br /> This reviewer's concerns were convincingly addressed by the revisions.

Overall Impact<br /> The authors present a convincing argument for the use of SLM as a better non-interacting null model for macroecological quantities and relationships.

Reviewer #2 (Public Review):

Summary:

In this study, the authors investigate the effect of ACh on neuronal responses in the auditory cortex of anesthetized rats during an auditory oddball task. The paradigm consisted of two pure tones (selected from the frequency responses at each recording site) presented in a pseudo-random sequence. One tone was presented frequently (the "standard" tone) and the other infrequently (the "deviant" tone). The authors found that ACh enhances the detection of unexpected stimuli in the auditory environment by increasing or decreasing the neuronal responses to deviant and standard tones.

Strengths:

The study includes the use of appropriate and validated methodology in line with the current state-of-the-art, rigorous statistical analysis and the demonstration of the effects of acetylcholine on auditory processing.

Weaknesses:

The study was conducted in anesthetized rats, and further research is needed to determine the behavioral relevance of these findings.

Reviewer #2 (Public Review):

Summary:

This interesting study addresses the ability of Ym1 protein crystals to promote pulmonary type 2 inflammation in vivo, in mice.

Strengths:

The data are extremely high quality, clearly presented, significantly extending previous work from this group on the type 2 immunogenicity of protein crystals.

Weaknesses:

There are no major weaknesses in this study. It would be interesting to see if Ym2 crystals behave similarly to Ym1 crystals in vivo. Some additional text in the Introduction and Discussion would enrich those sections.

Reviewer #2 (Public Review):

Summary:<br /> The work fruitfully adds to the tools to study cannabinoid action and pharmacology specifically, but also this method is applicable to other drugs, in particular, if lipophilic in nature.

Strengths:<br /> The addition of chocolate flavor overcomes aversive reactions which are often experienced in pharmacological treatments, leading to possible caveats in the interpretation of the behavioral outcomes.

Weaknesses:<br /> Certainly, more THC mediated behavioral outcomes could have been tested, but the work presents a proof-of-concept study to investigate acute THC treatment.<br /> It would have been interesting if this application form is also possible for chronic treatment regimen.

Reviewer #3 (Public Review):

Summary: This paper presents evidence of recursive self-embedding in the vocalization structure of orangutans, using fine-grained acoustical analysis. It proves the existence of isochrony nested in isochrony in the motifs produced by a nonhuman vocal system.

Strengths: Very clear written, clear analysis, excellent responses to the Reviewers.

Weaknesses: Jargonous language may be reduced. A video showing the sound as it unfolds and the spectrogram (as in Fig 1A) of the long call could be useful to best exemplify the results.

Reviewer #2 (Public Review):

Summary:<br /> Morucci et al. tested the influence of linguistic prosody long-term priors in forming predictions about simple acoustic rhythmic tone sequences composed of alternating tone duration, by violating context-dependent short-term priors formed during sequence listening. Spanish and Basque participants were selected due to the different rhythmic prosody of the two languages (functor-initial vs. Functor final, respectively), despite a common cultural background. The authors found that neuromagnetic responses to casual tone omissions reflected the linguistic prosody pattern of the participant's dominant language: in Spanish speakers, omission responses were larger to short tones, whereas in Basque speakers, omission responses were larger to long tones. Source localization of these responses revealed this interaction pattern in the left auditory cortex, which the authors interpret as reflecting a perceptual bias due to acoustic cues (inherent linguistic rhythms, rather than linguistic content). Importantly, this pattern was not found when the rhythmic sequence entailed pitch, rather than duration, cues. To my knowledge, this is the first study providing neural signatures of a known behavioral effect linking ambiguous rhythmic tone sequence perceptual organization to linguistic experience.

The conclusions of the study are well supported by the data, albeit weakly by the source analysis, but I have the impression that the rationale of the study and the analyses performed may be missing an important aspect of rhythmic sequence perception, namely the involvement of entrained oscillatory activity to the perceived rhythm, particularly phase alignment to pattern onsets. This view would not change the impact of the results but add depth to their interpretation.

Strengths:<br /> 1) The choice of participants. The bilingual population of the Basque country is perfect for performing studies that need to control for cultural and socio-economic background while having profound linguistic differences. In this sense, having dominant Basque speakers as a sample equates that in Molnar et al. (2016), and thus overcomes the lack of direct behavioral evidence for a difference in rhythmic grouping across linguistic groups. Molnar et al. (2016)'s evidence on the behavioral effect is compelling, and the evidence on neural signatures provided by the present study aligns with it.

2) The experimental paradigm. It is a well-designed acoustic sequence, that considers aspects such as gap length insertion, to be able to analyze omission responses free from subsequent stimulus-driven responses, and which includes a control sequence that uses pitch instead of duration as a cue to rhythmic grouping, which provides a stronger case for the differences found between groups to be due to prosodic duration cues.

3) Data analyses. Sound, state-of-the-art methodology in the event-related field analyses at the sensor level.

Weaknesses:<br /> 1) Despite the evidence provided on neural responses, the main conclusion of the study reflects a known behavioral effect on rhythmic sequence perceptual organization driven by linguistic background (Molnar et al. 2016, particularly). Also, the authors themselves provide a good review of the literature that evidences the influence of long-term priors in neural responses related to predictive activity. Thus, in my opinion, the strength of the statements the authors make on the novelty of the findings may be a bit far-fetched in some instances.

2) Albeit the paradigm is well designed, I fail to see the grounding of the hypotheses laid by the authors as framed under the predictive coding perspective. The study assumes that responses to an omission at the beginning of a perceptual rhythmic pattern will be stronger than at the end. I feel this is unjustified. If anything, omission responses should be larger when the gap occurs at the end of the pattern, as that would be where stronger expectations are placed: if in my language a short sound occurs after a long one, and I perceptually group tone sequences of alternating tone duration accordingly, when I hear a short sound I will expect a long one following; but after a long one, I don't necessarily need to expect a short one, as something else might occur.

3) In this regard, it is my opinion that what is reflected in the data may be better accounted for (or at least, additionally) by a different neural response to an omission depending on the phase of an underlying attentional rhythm (in terms of Large and Jones rhythmic attention theory, for instance) and putative underlying entrained oscillatory neural activity (in terms of Lakatos' studies, for instance). Certainly, the fact that the aligned phase may differ depending on linguistic background is very interesting and would reflect the known behavioral effect.

4) Source localization is performed on sensor-level significant data. The lack of source-level statistics weakens the conclusions that can be extracted. Furthermore, only the source reflecting the interaction pattern is taken into account in detail as supporting their hypotheses, overlooking other sources. Also, the right IFG source activity is not depicted, but looking at whole brain maps seems even stronger than the left. To sum up, source localization data, as informative as it could be, does not strongly support the author's claims in its current state.

Reviewer #2 (Public Review):

Summary:<br /> In this study, Hauser et al investigate the role of amphibian (Xenopus laevis) mast cells in cutaneous immune responses to the ecologically important pathogen Batrachochytrium dendrobatidis (Bd) using novel methods of in vitro differentiation of bone marrow-derived mast cells and in vivo expansion of skin mast cell populations. They find that bone marrow-derived myeloid precursors cultured in the presence of recombinant X. laevis Stem Cell Factor (rSCF) differentiate into cells that display hallmark characteristics of mast cells. They inject their novel (r)SCF reagent into the skin of X. laevis and find that this stimulates the expansion of cutaneous mast cell populations in vivo. They then apply this model of cutaneous mast cell expansion in the setting of Bd infection and find that mast cell expansion attenuates the skin burden of Bd zoospores and pathologic features including epithelial thickness and improves protective mucus production and transcriptional markers of barrier function. Utilizing their prior expertise with expanding neutrophil populations in X. laevis, the authors compare mast cell expansion using (r)SCF to neutrophil expansion using recombinant colony-stimulating factor 3 (rCSF3) and find that neutrophil expansion in Bd infection leads to greater burden of zoospores and worse skin pathology.

Strengths:<br /> The authors report a novel method of expanding amphibian mast cells utilizing their custom-made rSCF reagent. They rigorously characterize expanded mast cells in vitro and in vivo using histologic, morphologic, transcriptional, and functional assays. This establishes solid footing with which to then study the role of rSCF-stimulated mast cell expansion in the Bd infection model. This appears to be the first demonstration of the exogenous use of rSCF in amphibians to expand mast cell populations and may set a foundation for future mechanistic studies of mast cells in the X. laevis model organism.

Weaknesses:<br /> The conclusions regarding the role of mast cell expansion in controlling Bd infection would be stronger with a more rigorous evaluation of the model, as there are some key gaps and remaining questions regarding the data. For example:

1. Granulocyte expansion is carefully quantified in the initial time courses of rSCF and rCSF3 injections, but similar quantification is not provided in the disease models (Figures 3E, 4G, 5D-G). A key implication of the opposing effects of mast cell vs neutrophil expansion is that mast cells may suppress neutrophil recruitment or function. Alternatively, mast cells also express notable levels of csfr3 (Figure 2) and previous work from this group (Hauser et al, Facets 2020) showed rG-CSF-stimulated peritoneal granulocytes express mast cell markers including kit and tpsab1, raising the question of what effect rCSF3 might have on mast cell populations in the skin. Considering these points, it would be helpful if both mast cells and neutrophils were quantified histologically (based on Figure 1, they can be readily distinguished by SE or Giemsa stain) in the Bd infection models.

2. Epithelial thickness and inflammation in Bd infection are reported to be reduced by rSCF treatment (Figure 3E, 5A-B) or increased by rCSF3 treatment (Figure 4G) but quantification of these critical readouts is not shown.

3. Critical time points in the Bd model are incompletely characterized. Mast cell expansion decreases zoospore burden at 21 dpi, while there is no difference at 7 dpi (Figure 3E). Conversely, neutrophil expansion increases zoospore burden at 7 dpi, but no corresponding 21 dpi data is shown for comparison (Figure 4G). Microbiota analysis is performed at a third time point,10 dpi (Figure 5D-G), making it difficult to compare with the data from the 7 dpi and 21 dpi time points. Reporting consistent readouts at these three time points is important to draw solid conclusions about the relationship of mast cell expansion to Bd infection and shifts in microbiota.

4. Although the effect of rSCF treatment on Bd zoospores is significant at 21 dpi (Figure 3E), bacterial microbiota changes at 21 dpi are not (Figure S3B-C). This discrepancy, how it relates to the bacterial microbiota changes at 10 dpi, and why 7, 10, and 21 dpi time points were chosen for these different readouts (Figure 5F-G), is not discussed.

5. The time course of rSCF or rCSF3 treatments relative to Bd infection in the experiments is not clear. Were the treatments given 12 hours prior to the final analysis point to maximize the effect? For example, in Figure 3E, were rSCF injections given at 6.5 dpi and 20.5 dpi? Or were treatments administered on day 0 of the infection model? If the latter, how do the authors explain the effects at 7 dpi or 21 dpi given mast cell and neutrophil numbers return to baseline within 24 hours after rSCF or rCSF3 treatment, respectively?

The title of the manuscript may be mildly overstated. Although Bd infection can indeed be deadly, mortality was not a readout in this study, and it is not clear from the data reported that expanding skin mast cells would ultimately prevent progression to death in Bd infections.

Reviewer #2 (Public Review):

Summary:<br /> This study focuses on the 5-HT3 serotonin receptor, a pentameric ligand-gated ion channel important in chemical neurotransmission. There are many cryo-EM structures of this receptor with diverse ligands bound, however assignment of functional states to the structures remains incomplete. The team applies voltage-clamp fluorometry to measure, at once, both changes in ion channel activity, and changes in fluorescence. Four cysteine mutants were selected for fluorophore labeling, two near the neurotransmitter site, one in the ECD vestibule, and one at the ECD-TMD junction. Agonists, partial agonists, and antagonists were all found to yield similar changes in fluorescence, a proxy for conformational change, near the neurotransmitter site. The strength of the agonist correlated to a degree with propagation of this fluorescence change beyond the local site of neurotransmitter binding. Antagonists failed to elicit a change in fluorescence in the vestibular the ECD-TMD junction sites. The VCF results further turned up evidence supporting intermediate (likely pre-active) states.

Strengths:<br /> The experiments appear rigorous, the problem the team tackles is timely and important, the writing and the figures are for the most part very clear. We sorely need approaches orthogonal to structural biology to annotate conformational states and observe conformational transitions in real membranes- this approach, and this study, get right to the heart of what is missing.

Weaknesses:<br /> The weaknesses in the study itself are overall minor, I only suggest improvements geared toward clarity. What we are still missing is application of an approach like this to annotate the conformation of the part of the receptor buried in the membrane; there is important debate about which structure represents which state, and that is not addressed in the current study.

Reviewer #2 (Public Review):

Summary:<br /> The authors have developed a comprehensive set of tools to describe dynamics within a single time-series or across multiple time-series. The motivation is to better understand interacting networks within the human brain. The time-series used here are from direct estimates of the brain's electrical activity; however the tools have been used with other metrics of brain function and would be applicable to many other fields.

Strengths:<br /> The methods described are principled, based on generative probabilistic models.<br /> This makes them compact descriptors of the complex time-frequency data.<br /> Few initial assumptions are necessary in order to reveal this compact description.<br /> The methods are well described and demonstrated within multiple peer reviewed articles.<br /> This toolbox will be a great asset to the brain imaging community.

Weaknesses:<br /> The only question I had (originally) was how to objectively/quantitatively compare different network models. This has now been addressed by the authors in the latest revision.

Reviewer #2 (Public Review):

Summary:<br /> The authors build a gene expression model based on histone post-translational modifications and find that H3K27ac is correlated with gene expression. They proceed to perturb H3K27ac at 8 gene promoters, and measure gene expression changes to test their model.

Strengths:<br /> The combination of multiple methods to model expression, along with utilizing 6 histone datasets in 13 cell types allowed the authors to build a model that correlates between 0.7-0.79 with gene expression. This group also utilized a tool they are experts in, dCas9-p300 fusions to perturb H3K27ac and monitor gene expression to test their model. Ranked correlations showed some support for the predictions after the perturbation of H3K27ac.

Weaknesses:<br /> The perturbation of only 8 genes, and the only readout being qPCR-based gene expression, as opposed to including H3K27ac, weakened their validation of the computational model. Likewise, the use of six genes that were not expressed being most activated by dCas9-p300 might weaken the correlations vs. looking at a broad range of different gene expressions as the original model was trained on.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript characterizes a chemoattractant response to human serum by pathogenic bacteria, focusing on pathogenic strains of Salmonella enterica (Se). The researchers conducted the chemotaxis assays using a micropipette injection method that allows real-time tracking of bacterial population densities. They found that clinical isolates of several Se strains present a chemoattractant response to human serum. The specific chemoattractant within the serum is identified as L-serine, a highly characterized and ubiquitous chemoattractant, that is sensed by the Tsr receptor. They further show that chemoattraction to serum is impaired with a mutant strain devoid of Tsr. X-ray crystallography is then used to determine the structure of L-serine in the Se Tsr ligand binding domain, which differs slightly from a previously determined structure of a homologous domain. They went on to identify other pathogens that have a Tsr domain through a bioinformatics approach and show that these identified species also present a chemoattractant response to serum.

Strengths and Weaknesses:<br /> This study is well executed and the experiments are clearly presented. These novel chemotaxis assays provide advantages in terms of temporal resolution and the ability to detect responses from small concentrations. That said, it is perhaps not surprising these bacteria respond to serum as it is known to contain high levels of known chemoattractants, serine certainly, but also aspartate. In fact, the bacteria are shown to respond to aspartate and the tsr mutant is still chemotactic. The authors do not adequately support their decision to focus exclusively on the Tsr receptor. Tsr is one of the chemoreceptors responsible for observed attraction to serum, but perhaps, not the receptor. Furthermore, the verification of chemotaxis to serum is a useful finding, but the work does not establish the physiological relevance of the behavior or associate it with any type of disease progression. I would expect that a majority of chemotactic bacteria would be attracted to it under some conditions. Hence the impact of this finding on the chemotaxis or medical fields is uncertain.

The authors also state that "Our inability to substantiate a structure-function relationship for NE/DHMA signaling indicates these neurotransmitters are not ligands of Tsr." Both norepinephrine (NE) and DHMA have been shown previously by other groups to be strong chemoattractants for E. coli (Ec), and this behavior was mediated by Tsr (e.g. single residue changes in the Tsr binding pocket block the response). Given the 82% sequence identity between the Se and Ec Tsr, this finding is unexpected (and potentially quite interesting). To validate this contradictory result the authors should test E. coli chemotaxis to DHMA in their assay. It may be possible that Ec responds to NE and DHMA and Se doesn't. However, currently, the data is not strong enough to rule out Tsr as a receptor to these ligands in all cases. At the very least the supporting data for Tsr being a receptor for NE/DHMA needs to be discussed.

The authors also determine a crystal structure of the Se Tsr periplasmic ligand binding domain bound to L-Ser and note that the orientation of the ligand is different than that modeled in a previously determined structure of lower resolution. I agree that the SeTsr ligand binding mode in the new structure is well-defined and unambiguous, but I think it is too strong to imply that the pose of the ligand in the previous structure is wrong. The two conformations are in fact quite similar to one another and the resolution of the older structure, is, in my view, insufficient to distinguish them. It is possible that there are real differences between the two structures. The domains do have different sequences and, moreover, the crystal forms and cryo-cooling conditions are different in each case. It's become increasingly apparent that temperature, as manifested in differential cooling conditions here, can affect ligand binding modes. It's also notable that full-length MCPs show negative cooperativity in binding ligands, which is typically lost in the isolated periplasmic domains. Hence ligand binding is sensitive to the environment of a given domain. In short, the current data is not convincing enough to say that a previous "misconception" is being corrected.

Reviewer #2 (Public Review):

Greve et al. investigated the effects of a disease-associated gamma-actin mutation (E334Q) on actin filament polymerization, association of selected actin-binding proteins, and myosin activity. Recombinant wildtype and mutant proteins expressed in sf9 cells were found to be folded and stable, and the presence of the mutation altered a number of activities. Given the location of the mutation, it is not surprising that there are changes in polymerization and interactions with actin binding proteins. Nevertheless, it is important to quantify the effects of the mutation to better understand disease etiology. Some weaknesses were identified in the paper as discussed below.

Throughout the paper, the authors report average values and the standard-error-of-the-mean (SEM) for groups of three experiments. Reporting the SEM is not appropriate or useful for so few points, as it does not reflect the distribution of the data points. When only three points are available, it would be better to just show the three different points. Otherwise, plot the average and the range of the three points.

The description and characterization of the recombinant actin is incomplete. Please show gels of purified proteins. This is especially important with this preparation since the chymotrypsin step could result in internally cleaved proteins and altered properties, as shown by Ceron et al (2022). The authors should also comment on N-terminal acetylation of actin.

The authors do not use the best technique to assess actin polymerization parameters. Although the TIRF assay is excellent for some measurements, it is not as good as the standard pyrene-actin assays that provide critical concentration, nucleation, and polymerization parameters. The authors use pyrene-actin in other parts of the paper, so it is not clear why they don't do the assays that are the standard in the actin field.

The authors' data suggest that, while the binding of cofilin-1 to both the WT and mutant actins remains similar, the major defect of the E334Q actin is that it is not as readily severed/disassembled by cofilin. What is missing is a direct measurement of the severing rate (number of breaks per second) as measured in TIRF.

Figure 4 shows that the E334Q mutation increases rather than decreases the number of filaments that spontaneously assemble in the TIRF assay, but it is unclear how reduced severing would lead to increased filament numbers, rather, the opposite would be expected. A more straightforward approach would be to perform experiments where severing leads to more nuclei and therefore enhances the net bulk assembly rate.

Figure 5 A: in the pyrene disassembly assay, where actin is diluted below its critical concentration, cofilin enhances the rate of depolymerization by generating more free ends. The E334Q mutation leads to decreased cofilin-induced severing and therefore lower depolymerization. While these data seem convincing, it would be better to present them as an XY plot and fit the data to lines for comparison of the slopes.

Figure 5 B and C: the cosedimentation data do not seem to help elucidate the underlying mechanism. While the authors report statistical significance, differences are small, especially for gel densitometry measurements where the error is high, which suggests that there may be little biological significance. Importantly, example gels from these experiments should be shown, if not the complete set included in the supplement. In B, the higher cofilin concentrations would be expected to stabilize the filaments and thus the curve should be U-shaped.

Figure 5 D: these data show that the binding of cofilin to WT and E334Q actin is approximately the same, with the mutant binding slightly more weakly. It would be clearer if the two plots were normalized to their respective plateaus since the difference in arbitrary units distracts from the conclusion of the figure. If the difference in the plateaus is meaningful, please explain.

Figure 6: It is assumed that the authors are trying to show in this figure that cofilin binds both actins approximately the same but does not sever as readily for E334Q actin. The numerous parameters measured do not directly address what the authors are actually trying to show, which presumably is that the rate of severing is lower for E334Q than WT. It is therefore puzzling why no measurement of severing events per second per micron of actin in TIRF is made, which would give a more precise account of the underlying mechanism.

Actin-activated steady-state ATPase data of the NM2A with mutant and WT actin would have been extremely useful and informative. The authors show the ability to make these types of measurements in the paper (NADH assay), and it is surprising that they are not included for assessing the myosin activity. It may be because of limited actin quantities. If this is the case, it should be indicated.

(line 310) The authors state that they "noticed increased rapid dissociation and association events for E334Q filaments" in the motility assay. This observation motivates the authors to assess actin affinities of NM2A-HMM. Although differences in rigor and AM.ADP affinities are found between mutant and wt actins, the actin attachment lifetimes (many minutes) are unlikely to be related to the rapid association and dissociation event seen in the motility assay. Rather, this jiggling is more likely to be related to a lower duty ratio of the myosins, which appears to be the conclusion reached for the myosin-V data. These points should be clarified in the text.

(line 327) The authors report that the 1/K1 value is unchanged. There are no descriptions of this experiment in the paper. I am assuming the authors measured the ATP-induced dissociation of actomyosin and determined ATP affinity (K1) from this experiment. If this is the case, they should describe the experiment and show the data, provide a second-order rate constate for ATP binding, and report the max rate of dissociation (k2). This is a kinetic experiment done frequently by this group, so the absence of these details is surprising.

Reviewer #2 (Public Review):

Summary:<br /> In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibited form is stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

Strengths:<br /> The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gbeta/gamma subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

Reviewer #2 (Public Review):

Summary:<br /> The study by Yeo and co-authors addresses a long-lasting issue about botulinum neurotoxin (BoNT) intoxication. The current view is that the toxin binds to its receptors at the axon terminus by its HCc domain and is internalized in recycled neuromediator vesicles just after the release of the neuromediators. Then, the HCn domain assists the translocation of the catalytic light chain (LC) of the toxin through the membrane of these endocytic vesicles into the cytosol of the axon terminus. There, the LC cleaves its SNARE substrate and blocks neurosecretion. However, other views involving kinetic aspects of intoxication suggest that the toxin follows the retrograde axonal transport up to the nerve cell body and then back to the nerve terminus before cleaving its substrate.

In the current study, the authors claim that the BoNT/A (isotype A of BoNT) not only progresses to the cell body but once there, follows the retrograde transport trafficking pathway in a retromer-dependent fashion, through the Golgi apparatus, until reaching the endoplasmic reticulum. Next, the LC dissociates from the HC (a process not studied here) and uses the translocon Sec61 machinery to retro-translocate into the cytosol. Only then, does the LC traffic back to the nerve terminus following the anterograde axonal transport. Once there, LC cleaves its SNARE substrate (SNAP25 in the case of BoTN/A) and blocks neurosecretion.

To reach their conclusion, Yeo and co-authors use a combination of engineered tools: a cell line able to differentiate into neurons (ReNcell VN), a reporter dual fluorescent protein derived from SNAP25, the substrate of BoNT/A (called SNAPR), the use of either native BoNT/A or a toxin to which three fragment 11 of the reporter fluorescent protein Neon Green (mNG) are fused to the N-terminus of the LC (BoNT/A-mNG11x3), and finally ReNcell VN transfected with mNG1-10 (a protein consisting of the first 10 beta strands of the mNG).

SNAPR is stably expressed all over in the ReNcell VN. SNAPR is yellow (red and green) when intact and becomes red only when cleaved by BoNT/A LC, the green tip being degraded by the cell. When the LC of BoNT/A-mNG11x3 reaches the cytosol in ReNcell VN transfected by mNG1-10, the complete mNG is reconstituted and emits a green fluorescence.

In the first experiment, the authors show that the catalytic activity of the LC appears first in the cell body of neurons where SNAPR is cleaved first. This phenomenon starts 24 hours after intoxication and progresses along the axon towards the nerve terminus during an additional 24 hours. In a second experiment, the authors intoxicate the ReNcell VN transfected by mNG1-10 using the BoNT/A-mNG11x3. The fluorescence appears also first in the soma of neurons, then diffuses in the neurites in 48 hours. The conclusion of these two experiments is that translocation occurs first in the cell body and that the LC diffuses in the cytosol of the axon in an anterograde fashion.

In the second part of the study, the authors perform a siRNA screen to identify regulators of BoNT/A intoxication. Their aim is to identify genes involved in intracellular trafficking of the toxin and translocation of the LC. Interestingly, they found positive and negative regulators of intoxication. Regulators could be regrouped according to the sequential events of intoxication. Genes affecting binding to the cell-surface receptor (SV2) and internalization. Genes involved in intracellular trafficking. Genes involved in translocation such as reduction of the disulfide bond linking the LC to the HC and refolding in the cytosol. Genes involved in signaling such as tyrosine kinases and phosphatases. All these groups of genes may be consistent with the current view of BoNT intoxication within the nerve terminus. However, two sets of genes were particularly significant to reach the main conclusion of the work and definitely constitute an original finding important to the field. One set of genes consists of those of the retromer, and the other relates to the Sec61 translocon. This should indicate that once endocytosed, the BoNT traffics from the endosomes to the Golgi apparatus, and then to the ER. Ultimately, the LC should translocate from the ER lumen to the cytosol using the Sec61 translocon. The authors further control that the SV2 receptor for the BoNT/A traffics along the axon in a retromer-dependent fashion and that BoNT/A-mNG11x3 traverses the Golgi apparatus by fusing the mNG1-10 to a Golgi resident protein.

Strengths:<br /> The findings in this work are convincing. The experiments are carefully done and are properly controlled. In the first part of the study, both the activity of the LC is monitored together with the physical presence of the toxin. In the second part of the work, the most relevant genes that came out of the siRNA screen are checked individually in the ReNcell VN / BoNT/A reporter system to confirm their role in BoNT/A trafficking and retro-translocation.

These findings are important to the fields of toxinology and medical treatment of neuromuscular diseases by BoNTs. They may explain some aspects of intoxication such as slow symptom onset, aggravation, and appearance of central effects.

Weaknesses:<br /> The findings antagonize the current view of the intoxication pathway that is sustained by a vast amount of observations. The findings are certainly valid, but their generalization as the sole mechanism of BoNT intoxication should be tempered. These observations are restricted to one particular neuronal model and engineered protein tools. Other models such as isolated nerve/muscle preparations display nerve terminus paralysis within minutes rather than days. Also, the tetanus neurotoxin (TeNT), whose mechanism of action involving axonal transport to the posterior ganglia in the spinal cord is well described, takes between 5 and 15 days. It is thus possible that different intoxication mechanisms co-exist for BoNTs or even vary depending on the type of neurons.

Although the siRNA experiments are convincing, it would be nice to reach the same observations with drugs affecting the endocytic to Golgi to ER transport (such as Retro-2, golgicide or brefeldin A) and the Sec61 retrotranslocation (such as mycolactone). Then, it would be nice to check other neuronal systems for the same observations.

Reviewer #2 (Public Review):

Summary:<br /> This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:<br /> The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:<br /> The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

1) At the lower level, we first need to understand what the individual species do and "like" (their environmental niche). That information is not presented, and the methods suggest that the representation of each species in the JSDM is likely to be extremely poor.

2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the model is not set up this way, and the authors thus do a lot of indirect exploration, rather than direct hypothesis testing.

Even when the focus is not the individual species, but rather their association, we need to formulate what the expectation is. The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one can then add the layer of interspecific associations.

The change in activity and space use can be analysed much simpler, by looking at the activity times and spatial distribution directly. It remains unclear what the contribution of the JSDM is, unless it is able to represent this activity and spatial information, and put it in a testable interaction with human disturbance.

The topic is actually rather complicated. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

Then, in the next step, one would have to formulate specific hypotheses about which species are likely to change their associations more, and which less (based e.g. on predator-prey or competitive interactions). The data and analyses presented do not answer any of these issues.

Another more substantial point is that, according to my understanding of the methods, the per-species models are very inappropriate: the predictors are only linear, and there are no statistical interactions (L374). There is no conceivable species in the world whose niche would be described by such an oversimplified model.

We have no idea of even the most basic characteristics of the per-species models: prevalences, coefficient estimates, D2 of the model, and analysis of the temporal and spatial autocorrelation of the residuals, although they form the basis for the association analysis! Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

Also, all correlations among species should be shown for the raw data and for the model residuals: how much does that actually change and can thus be explained by the niche models?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. I failed to see any advance in ecological understanding at the community level.

Reviewer #2 (Public Review):

Allison Coté et al. investigated the ordering and spatial distribution of nascent transcripts in several cells using smFISH, expansion microscopy, and live-cell imaging. They find that pre-mRNA splicing occurs post-transcriptionally at the clouds around the transcription start site, termed the transcription site proximal zone. They show that pre-mRNA may undergo continuous splicing when they pass through the zone after transcription. These data suggest a unifying model for explaining previously reported co-transcriptional splicing events and provide a direction for further study of the nature of the slow-moving zone around the transcription start site.

This paper is well-written. The findings are very important, and the data supports the conclusions well. However, some aspects of the image and description need to be clarified and revised.

1) The sentence "By distinguishing the separate fluorescent signals from probes bound to exons and introns, we could visualize splicing intermediates (represented by colocalized intron and exon spots) relative to the site of transcription (represented by bright colocalized intron and exon spots) and fully spliced products (represented by exon spots alone)." is accidentally repeated twice, one of them should be deleted.<br /> 2) The authors describe Figure 4E and 4F results in the main text as that "we performed RNA FISH simultaneously with immunofluorescence for SC35, a component of speckles, and saw that these compartmentalized pre-mRNA did indeed appear near nuclear speckles both before (Supplementary Figure 6C) and after (Figure 4E) splicing inhibition." However, no SC35 staining is shown in the Figure 4E. A similar situation happened in describing Figure 4F.

Reviewer #2 (Public Review):

Lin et al attempt to examine the role of lncRNAs in human evolution in this manuscript. They apply a suite of population genetics and functional genomics analyses that leverage existing data sets and public tools, some of which were previously built by the authors, who clearly have experience with lncRNA binding prediction. However, I worry that there is a lack of suitable methods and/or relevant controls at many points and that the interpretation is too quick to infer selection. While I don't doubt that lnc RNAs contribute to the evolution of modern humans, and certainly agree that this is a question worth asking, I think this paper would benefit from a more rigorous approach to tackling it.

I thank the authors for their revisions to the manuscript; however, I find that the bulk of my comments have not been addressed to my satisfaction. As such, I am afraid I cannot say much more than what I said last time, emphasising some of my concerns with regards to the robustness of some of the analyses presented. I appreciate the new data generated to address some questions, but think it could be better incorporated into the text - not in the discussion, but in the results.

Reviewer #2 (Public Review):

In this manuscript the authors generate an annotated brain atlas for the prairie vole, which is a widely studied organism. This species has a suite of social behaviors that are difficult or impossible to study in conventional rodents, and has attracted a large community of researchers. The atlas is impressive and will be a fantastic resource. The authors use this atlas to examine brain-wide c-fos expression in prairie voles that were paired with same sex or opposite sex vole across multiple timepoints. In some sense the design resembles PET studies done in primates that take whole brain scans after an important behavioral experience. The authors observed increased c-fos expression across a network of brain regions that largely corresponds with the previous literature. The study design captured several novel observations including that c-fos expression in some regions correlate strongly between males and females during pair bond formation and mating, suggesting synchrony in neural activity. The authors address an important caveat that c-fos provides a snapshot of neural activity and that important populations of neurons could be active and not express c-fos. Thus observed correlations are likely to be robust, but that the absence of differences (in say accumbens) may just reflect the limits of c-fos estimation of neural activity. Similarly, highly coordinated neural activity between males and females might still be driven by different mechanisms if different cell types were activated within a specific region. The creation of this resource and it's use in a well designed study is an important accomplishment.

Reviewer #2 (Public Review):

Summary:<br /> The authors describe a novel ML approach to predict binding between MHC-bound peptides and T-Cell receptors. Such approaches are particularly useful for predicting the binding of peptide sequences with low similarity when compared to existing data sets. The authors focus on improving dataset quality and optimizing model architecture to achieve a pan-specific predictive model in hopes of achieving a high-precision model for novel peptide sequences.

Strengths:<br /> Since assuring the quality of training datasets is the first major step in any ML training project, the extensive human curation, computational analysis, and enhancements made in this manuscript represent a major contribution to the field. Moreover, the systematic approach to testing redundancy reduction and data augmentation is exemplary, and will significantly help future research in the field.

The authors also highlight how their model can identify outliers and how that can be used to improve the model around known sequences, which can help the creation and optimization of future datasets for peptide binding.

The new models presented here are novel and built using paired α/β TCR sequence data to predict peptide-specific TCR binding, and have been extensively and rigorously tested.

Weaknesses:<br /> Achieving an accurate pan-specific model is an ambitious goal, and the authors have significant difficulties when trying to achieve non-random performance for the prediction of TCR binding to novel peptides. This is the most challenging task for this kind of model, but also the most desirable when applying such models to biotechnological and bioengineering projects.

The manuscript is a highly technical and extremely detailed computational work, which can make the achievements and impact of the work hard to parse for application-oriented researchers.<br /> The authors briefly mention real-world use cases for TCR specificity predictions, but do not contextualize the work into possible applications.

Reviewer #2 (Public Review):

Summary:

This paper nicely introduces WormPsyQi, an imaging analysis pipeline that effectively quantifies synaptically localized fluorescent signals in C. elegans through high-throughput automation. This toolkit is particularly valuable for the analysis of densely packed regions in 3D space, such as the nerve ring. The authors applied WormPsyQi to various aspects, including the examination of sexually dimorphic synaptic connectivity, presynaptic markers in eight head neurons, five GRASP reporters, electrical synapses, the enteric nervous system, and developmental synapse comparisons. Furthermore, they validated WormPsyQi's accuracy by comparing its results to manual analysis.

Strengths:

Overall, the experiments are well done, and their toolkit demonstrates significant potential and offers a valuable resource to the C. elegans community. This will expand the range of possibilities for studying synapses in C. elegans.

Reviewer #2 (Public Review):

Summary:<br /> Developing a mechanical model of C. elegans is difficult to do from basic principles because it moves at a low (but not very small) Reynolds number, is itself visco-elastic, and often is measured moving at a solid/liquid interface. The ElegansBot is a good first step at a kinetic model that reproduces a wide range of C. elegans motiliy behavior.

Strengths:<br /> The model is general due to its simplicity and likely useful for various undulatory movements. The model reproduces experimental movement data using realistic physical parameters (e.g. drags, forces, etc). The model is predictive (semi?) as shown in the liquid-to-solid gait transition. The model is straightforward in implementation and so likely is adaptable to modification and addition of control circuits.

Weaknesses:<br /> Since the inputs to the model are the actual shape changes in time, parameterized as angles (or curvature), the ability of the model to reproduce a realistic facsimile of C. elegans motion is not really a huge surprise.

The authors do not include some important physical parameters in the model and should explain in the text these assumptions. 1) The cuticle stiffness is significant and has been measured [1]. 2) The body of C. elegans is under high hydrostatic pressure which adds an additional stiffness [2]. 3) The visco-elasticity of C. elegans body has been measured. [3]

There is only a very brief mention of proprioception. The lack of inclusion of proprioception in the model should be mentioned and referenced in more detail in my opinion.

These are just suggested references. There may be more relevant ones available.

1. Rahimi M, Sohrabi S, Murphy CT. Novel elasticity measurements reveal C. elegans cuticle stiffens with age and in a long-lived mutant. Biophys J. 2022 Feb 15;121(4):515-524. doi: 10.1016/j.bpj.2022.01.013. Epub 2022 Jan 19. PMID: 35065051; PMCID: PMC8874029.

2. Park SJ, Goodman MB, Pruitt BL. Analysis of nematode mechanics by piezoresistive displacement clamp. Proc Natl Acad Sci U S A. 2007 Oct 30;104(44):17376-81. doi: 10.1073/pnas.0702138104. Epub 2007 Oct 25. PMID: 17962419; PMCID: PMC2077264.

3. Backholm M, Ryu WS, Dalnoki-Veress K. Viscoelastic properties of the nematode Caenorhabditis elegans, a self-similar, shear-thinning worm. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):4528-33. doi: 10.1073/pnas.1219965110. Epub 2013 Mar 4. PMID: 23460699; PMCID: PMC3607018.

Reviewer #2 (Public Review):

In this study, Hernandez-Hernandez et al developed a gender-dependent mathematical model of arterial myocytes based on a previous model and new experimental data. The ionic currents of the model and its sex difference were formulated based on patch clamp experimental data, and the model properties were compared with single cell and tissue scale experimental results. This is a study that is of importance for the modeling field as well as for experimental physiology.

Reviewer #2 (Public Review):

Summary:<br /> The authors are studying the behavioral response to pathogen exposure. They and others have previously describe the role that the G-protein coupled receptors in the nervous system plays in detecting pathogens, and initiating behavioral patterns (e.g. avoidance/learned avoidance) that minimize contact. The authors study this problem in C. elegans, which is amenable to genetic and cellular manipulations and allow the authors to define cellular and signaling mechanisms. This paper extends the original idea to now implicate signaling and transcriptional pathways within a particular neuron (ASJ) and the gut in mediating avoidance behaviour.

Strengths:<br /> The work is rigorous and elegant and the data are convincing. The authors make superb use of mutant strains in C. elegans, as well tissue specific gene inactivation and expression and genetic methods of cell ablation. to demonstrate how a gene, NPR15 controls behavioral changes in pathogen infection. The results suggest that ASJ neurons and the gut mediate such effects. I expect the paper will constitute an important contribution to our understanding of how the nervous system coordinates immune and behavioral responses to infection.

Reviewer #2 (Public Review):

This is a nice story about auricular chondrocyte maintenance, its molecular mechanism, and the role of the BMP 1 receptor in microtia disease conditions. A detailed analysis of different parts of the ear, their proliferation, and their differentiation condition with histological and immunofluorescence analysis strengthens the evidence. Further validation with patient sample RNA-Seq also helps the study end with an informative story.

From the public point of view, I want to say that the authors want to explain how auricular chondrocytes differ from growth plates or other chondrocytes. The authors show that Prxx1 is a good marker to differentiate auricular chondrocytes from different types of chondrocytes, which I doubt because other chondrocytes have an expression of Prxx1 at a lower level.

Another thing the authors mention is that microtia conditions develop through reduced size without affecting proliferation and apoptosis. The authors never provide any evidence about how the ablation of Bmpr1a affects the size, protein trafficking, and ECM organization.

Crosstalk between BMP-PKA in auricular chondrocytes and switching the chondrocytes' cell fate in osteoblast cells are not entirely stable by these studies for physiological functions.

Reviewer #3 (Public Review):

Summary:<br /> The authors set out to characterize the anatomical connectivity profile and the functional responses of chandelier cells (ChCs) in the mouse primary visual cortex. Using retrograde rabies tracing, optogenetics, and in vitro electrophysiology, they found that the primary source of input to ChCs are local layer 5 pyramidal cells, as well as long-range thalamic and cortical connections. ChCs provided input to local layer 2/3 pyramidal neurons, but did not receive reciprocal connections.

With two-photon calcium imaging recordings during passive viewing of drifting gratings, the authors showed that ChCs exhibit weakly selective visual responses, high correlations within their own population, and strong responses during periods of arousal (assessed by locomotion and pupil size). These results were replicated and extended in experiments with natural images and prediction of receptive field structure using a convolutional neural network.

Furthermore, the authors employed a learned visuomotor task in a virtual corridor to show that ChCs exhibit strong responses to mismatches between visual flow and locomotion, locomotion-related activation (similar to what was shown above), and visually-evoked suppression. They also showed the existence of two clusters of pyramidal neurons with functionally different responses - a cluster with "classically visual" responses and a cluster with locomotion- and mismatch-driven responses (the latter more correlated with ChCs). Comparing naive and trained mice, the authors found that visual responses of ChCs are suppressed following task learning, accompanied by a shortening of the axon initial segment (AIS) of pyramidal cells and an increase in the proportion of AIS contacted by ChCs. However, additional controls would be required to identify which component(s) of the experimental paradigm led to the functional and anatomical changes observed.

Strengths:<br /> The authors bring a comprehensive, state-of-the-art methodology to bear, including rabies tracing, in vivo two-photon calcium imaging, in vitro electrophysiology, optogenetics and chemogenetics, and deep neural networks. Their analyses and statistical tests are sound and for the most part, support their claims. Their results are in line with previous findings and extend them to the primary visual cortex.

Weaknesses:<br /> - Some of the results (e.g. arousal-related responses) are not entirely surprising given that similar results exist in other cortical areas.

Reviewer #2 (Public Review):

Summary:<br /> Nakai-Futatsugi et al. present a novel method to analyze the correlation between the degree of pigmentation and the gene expression profile of human-induced pluripotent stem cell-derived RPE (iPSC-RPE) cells at the single cell level. This was achieved with the use of ALPS (Automated Live imaging and cell Picking System), an invention developed by the same authors. Briefly, it allows one to choose and photograph a specific cell from a culture dish and proceed to single-cell digital RNA-seq. The authors identify clusters of cells that present differential gene expression, but this shows no association with the degree of pigmentation of the cells. Further data analysis allowed the authors to correlate the degree of pigmentation to some degree with the expression of complement and lysosome-related genes.

Strengths:<br /> An important amount of data related to gene expression and heterogeneity of the iPSC-RPE population has been generated in this work.

Weaknesses:<br /> However, the justification of the analysis, and the physiological relevance of the hypothesis and the findings could be strengthened.

Importantly, I fail to grasp from the introduction what is the previous evidence that leads to the hypothesis. Why would color intensity be related to the quality of cell transplantation? In fact, cell transplantation is not evaluated at all in this work. The authors mention "quality metrics for clinical use", but this concept is not further explained. Neither is the concept of "sufficient degree of pigmentation" explained.

On the other hand, the positive correlation of cluster formation with complement and lysosome-related genes is not discussed.

As a consequence, it is very difficult to evaluate the impact of these findings on the field.

Reviewer #2 (Public Review):

This paper introduces a new model that aims to explain the generators of temporal decoding matrices (TGMs) in terms of underlying signal properties. This is important because TGMs are regularly used to investigate neural mechanisms underlying cognitive processes, but their interpretation in terms of underlying signals often remains unclear. Furthermore, neural signals are often variant over different instances of stimulation despite behaviour being relatively stable. The author aims to tackle these concerns by developing a generative model of electrophysiological data and then showing how different parameterizations can explain different features of TGMs. The developed technique is able to capture empirical observations in terms of fundamental signal properties. Specifically, the model shows that complexity is necessary in terms of spatial configuration, frequencies and latencies to obtain a TGM that is comparable to empirical data.

The major strength of the paper is that the novel technique has the potential to further our understanding of the generators of electrophysiological signals which are an important way to understand brain function. The paper clearly outlines how the method can be used to capture empirical data. Furthermore, the used techniques are state-of-the-art and the developed model is publicly shared in open source code.

On the other hand, there is no unambiguous mapping between neurobiological mechanisms and different signal generators, making it hard to draw firm conclusions about neural underpinnings based on this analysis.

Reviewer #2 (Public Review):

This manuscript by Petersen and colleagues investigates the mechanistic underpinnings of activation of the ion channel TREK-1 by mechanical inputs (fluid shear or membrane stretch) applied to cells. Using a combination of super-resolution microscopy, pair correlation analysis and electrophysiology, the authors convincingly show that the application of shear to a cell can lead to changes in the distribution of TREK-1 and the enzyme PhospholipaseD2 (PLD2), relative to lipid domains defined by either GM1 or PIP2. The activation of TREK-1 by mechanical stimuli was shown to be sensitized by the presence of PLD2, but not a catalytically inactive xPLD2 mutant. In addition, the activity of PLD2 is increased when the molecule is more associated with PIP2, rather than GM1 defined lipid domains. The presented data do not exclude direct mechanical activation of TREK-1, rather suggest a modulation of TREK-1 activity, increasing sensitivity to mechanical inputs, through an inherent mechanosensitivity of PLD2 activity. The authors additionally demonstrate that cellular uptake of cholesterol inhibits TREK-1 activation and, in ex vivo studies, that depletion of cholesterol from astrocytes reduces correlation of TREK-1 and G1 lipids in mouse brain slices. In vivo studies, using Drosophila melanogaster behavioural assays, were used to demonstrate that disrupting PLD2 altered behavioural responses to mechanical and electrical inputs. These data demonstrate that manipulation of PLD2 analogue in the fly can alter sensory transduction, suggesting that PLD functions to regulate sensitivity to mechanical force. However, as the authors note, there is no TREK-1 homologue in this organism: thus the identity of the downstream effectors of PLD in D. melanogaster remain unknown. This work will be of interest to the growing community of scientists investigating the myriad mechanisms that can tune mechanical sensitivity of cells, providing valuable insight into the role of functional PLD2 in sensitizing TREK-1 activation in response to mechanical inputs, in some cellular systems.

The authors convincingly demonstrate that, post application of shear, an alteration in the distribution of TREK-1 and mPLD2 (in HEK293T cells) from being correlated with GM1 defined domains (no shear) to increased correlation with PIP2 defined membrane domains (post shear). The association of TREK-1 with PIP2 required functional mPLD2. These data were generated using super-resolution microscopy to visualise, at sub diffraction resolution, the localisation of labelled protein, compared to labelled lipids. The use of super-resolution imaging enabled the authors to visualise changes in cluster association that would not have been achievable with diffraction limited microscopy.

This work provides further evidence of the astounding flexibility of mechanical sensing in cells. By outlining how mechanical activation of TREK-1 can be sensitised by mechanical regulation of PLD2 activity, the authors highlight a mechanism by which TREK-1 sensitivity could be regulated under distinct physiological conditions.

Reviewer #2 (Public Review):

In their submitted manuscript, Blin et al. describe differences in the olfactory-driven behaviors of river-dwelling surface forms and cave-dwelling blind forms of the Mexican tetra, Astyanax mexicanus. They provide a dataset of unprecedented detail, that compares not only the behaviors of the two morphs but also that of a significant number of F2 hybrids, therefore also demonstrating that many of the differences observed between the two populations have a clear (and probably relatively simple) genetic underpinning.

To complete the monumental task of behaviorally testing 425 six-week-old Astyanax larvae, the authors created a setup that allows for the simultaneous behavioral monitoring of multiple larvae and the infusion of different odorants without introducing physical perturbations into the system, thus biasing the responses of cavefish that are particularly fine-tuned for this sensory modality. During the optimization of their protocol, the authors also found that for cave-dwelling forms one hour of habituation was insufficient and a full 24 hours were necessary to allow them to revert to their natural behavior. It is also noteworthy that this extremely large dataset can help us see that population averages of different morphs can mask quite significant variations in individual behaviors.

Testing with different amino-acids (applied as relevant food-related odorant cues) shows that cavefish are alanine- and histidine-specialists, while surface fish elicit the strongest behavioral responses to cysteine. It is interesting that the two forms also react differently after odor detection: while cave-dwelling fish decrease their locomotory activity, surface fish increase it. These differences are probably related to different foraging strategies used by the two populations, although, as the observations were made in the dark, it would be also interesting to see if surface fish elicit the same changes in light as well.

Further work will be needed to pinpoint the exact nature of the genetic changes that underlie the differences between the two forms. Such experimental work will also reveal how natural selection acted on existing behavioral variations already present in the SF population.

It will be equally interesting, however, to understand what lies behind the large individual variation of behaviors observed both in the case surface and cave populations. Are these differences purely genetic, or perhaps environmental cues also contribute to their development? Does stochasticity provided by the developmental process has also a role in this? Answering these questions will reveal if the evolvability of Astyanax behavior was an important factor in the repeated successful colonization of underground caves.

Reviewer #2 (Public Review):

The mechanisms that mediate female aggression remain poorly understood. Chiu, Schretter, and colleagues, employed circuit dissection techniques to tease apart the specific roles of particular doublesex and fruitless expressing neurons in the fly Drosophila in generating a persistent aggressive state. They find that activating the fruitless positive alPg neurons, generated an aggressive state that persisted for >10min after the stimulation ended. Similarly, activating the doublesex positive pC1de neurons also generated a peristent state. Activating pC1d or pC1e individually did not induce a persistent state. Interestingly, while neural activation of alPGs and pC1d+e neurons induced a persistent behavioural states it did not induce persistent activity in the neurons being activated.

The authors have revised the manuscript in accordance with comments of the reviewers. The conclusions of this paper are by and large well supported by the data. These data will be a useful addition to the literature on the circuit basis of female aggression, and open up intriguing avenues for further studies to explore.

Reviewer #2 (Public Review):

In this study, the authors describe a pipeline to sequence expressed var genes from RNA sequencing that improves on a previous one that they had developed. Importantly, they use this approach to determine how var gene expression changes with short-term culture. Their finding of shifts in the expression of particular var genes is compelling and casts some doubt on the comparability of gene expression in short-term culture versus var expression at the time of participant sampling.

Other studies have relied on short-term culture to understand var gene expression in clinical malaria studies. This study indicates the need for caution in over-interpreting findings from these studies.

We appreciate the careful attention of the authors to our comments and the edits that have been made. One additional suggestion that would be helpful to readers is to include in Table S1 the new approach described in the manuscript. This will provide the reader a direct means of comparing what the authors have done to past work.

Reviewer #2 (Public Review):

In this study, a minimalist setup was used to investigate the selectivity of the nuclear pore complex as a function of its diameter. To this end, a series of solid-state pores in a free-standing palladium membrane were designed and attached to a PDMS-based fluid cell that could be mounted on a confocal microscope. In this way, the frequency of translocation events could be measured in an unbiased manner. Furthermore, the pores were designed to exhibit the key properties of the nuclear pore complex: (i) the size of the pore, (ii) disordered FG Nups specifically located in the central channel; (ii) transport receptors that can shuttle through the central channel by binding to the FG-Nups. Additionally, such system offered the advantage of monitoring the translocation of multiple fluorescently labeled molecules (e.g. Kap95 and BSA) simultaneously and under well-controlled conditions.

The authors were able to demonstrate convincingly that the pore selectivity depends on the pore diameter, the FG Nup layer organization within the pore and the transport receptors concentration that can specifically interact with FG Nups. It was shown that the pores coated with FG Nups (e.g. Nsp1 in this case) and smaller than 50-60 nm are highly selective and such selectivity is increasing with the decrease of the pore diameter. Also, it was shown that the pore selectivity moderately enhances at the high Kap95 concentration (1 µM). Importantly, it was also shown that the selectivity is becoming negligible for the pores, which are larger than 60-75 nm.

The experimental data are well supported by coarse-grained modelling of Nsp1-coated pores, and the theoretical prediction correlates qualitatively with the experimentally obtained data.

Reviewer #2 (Public Review):

This study explores the breadth of effects of one important metabolite, azelaic acid, on marine microbes, and reveals in-depth its pathway of uptake and catabolism in one model bacterial strain. This compound is known to be widely produced by phytoplankton and plants, and to have complex effects on associated microbiomes.

This work uses transcriptomics to assay the response of two strains that show contrasting responses to the metabolite: one catabolizes the compound and assimilates the carbon, while the other shows growth inhibition and stress response. A highly induced TRAP transporter, adjacent to a previously identified regulator, is inferred to be the specific uptake system for azelaic acid, though this function was not directly tested via genetic or biochemical methods. Nevertheless, this is a significant finding that will be useful for exploring the distribution of azelaic acid uptake capability across metagenomes and other bacteria.

The authors use pulse-chase style metabolomics experiments to beautifully demonstrate the fate of azelaic acid through catabolic pathways. They also measure an assimilation rate per cell, though it remains unclear how this measured rate relates to natural systems. The metabolomics approach is an elegant way to show carbon flux through cells, and could serve as a model for future studies.

The study seeks to extend the results from two model strains to complex communities, using seawater mesocosm experiments and soil/Arabidopsis experiments. The seawater experiments show a community shift in mesocosms with added azelaic acid. The mechanisms for the shift were not determined in this study; further work is necessary to demonstrate which community members are directly assimilating the compound, benefitting indirectly, or experiencing inhibition. The authors also took the unusual and creative step of performing similar experiments in a soil - Arabidopsis system. I admire the authors' desire to identify unifying themes across ecosystems. The parallels are intriguing, and future experiments could determine the different modes of action in aquatic vs terrestrial microbial communities.

This work is a nice illustration of how we can begin to tease apart the effects of chemical currencies on marine ecosystems. A key strength of this work is the combination of transcriptomics and metabolomics methods, along with assaying the impacts of the metabolite on model strains of bacteria and whole communities. Given the sheer number of compounds that probably play critical roles in community interactions, a key challenge for the field will be navigating the tradeoffs between breadth and depth in future studies of metabolite impacts. This study offers a good compromise and will be a useful model for future studies.

Reviewer #2 (Public Review):

In this study, the authors utilize a compendium of public genomic data to identify transcription factors (TF) that can identify their DNA binding motifs in the presence of nuclosome-wrapped chromatin and convert the chromatin to open chromatin. This class of TFs are termed Pioneer TFs (PTFs). A major strength of the study is the concept, whose premise is that motifs bound by PTFs (assessed by ChIP-seq for the respective TFs) should be present in both "closed" nucleosome wrapped DNA regions (measured by MNase-seq) as well as open regions (measured by DNAseI-seq) because the PTFs are able to open the chromatin. Use of multiple ENCODE cell lines, including the H1 stem cell line, enabled the authors to assess if binding at motifs changes from closed to open. Typical, non-PTF TFs are expected to only bind motifs in open chromatin regions (measured by DNaseI-seq) and not in regions closed in any cell type. This study contributes to the field a validation of PTFs that are already known to have pioneering activity and presents an interesting approach to quantify PTF activity.

For this reviewer, there were a few notable limitations. One was the uncertainty regarding whether expression of the respective TFs across cell types was taken into account. This would help inform if a TF would be able to open chromatin. Another limitation was the cell types used. While understandable that these cell types were used, because of their deep epigenetic phenotyping and public availability, they are mostly transformed and do not bear close similarity to lineages in a healthy organism. Next, the methods used to identify PTFs were not made available in an easy-to-use tool for other researchers who may seek to identify PTFs in their cell type(s) of interest. Lastly, some terms used were not defined explicitly (e.g., meaning of dyads) and the language in the manuscript was often difficult to follow and contained improper English grammar.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Yu and colleagues sought to identify new susceptibility genes for adolescent idiopathic scoliosis (AIS). The significance of this work is high, especially given the still large knowledge gap of the mechanistic underpinnings for AIS. In this multidisciplinary body of work, the authors first performed a genetic association study of AIS case-control cohorts (combined 9,161 cases and 80,731 controls) which leveraged common SNPs in 1027 previously defined matrisome genes. Two nonsynonymous variants were found to be significantly associated with AIS: MMP14 p.Asp273Asn and COL11A1 p.Pro1153Leu, the latter of which had the more robust association and remained significant when females were tested independent of males. Next, the authors followed a series of functional validation experiments to support biological involvement of COL11A1 p.Pro1153Leu in AIS through expression, biochemical, and histological studies in physiologically relevant cell and mouse models. Together, the authors propose a hitherto unreported model for AIS that involves the interplay of the COL11A1 susceptibility locus with estrogen signaling to alter a Pax1-Col11a1-Mmp3 signaling axis at the growth plate.

Strengths:

The manuscript is clearly written and follows a series of logical steps toward connecting multiple matrisome genes and putative AIS effectors in a new framework of pathomechanism. The multidisciplinary nature of the work makes it a strong body of work wherein multiple models offer multiple lines of supportive data. Thus, this manuscript remains an important multidisciplinary study of the genetic and functional basis of adolescent idiopathic scoliosis (AIS). To the benefit of the overall manuscript quality, the reviewers have addressed most concerns to satisfaction. Please include the list of three rare missense variants mentioned in the response to reviewers as a supplementary table. Please also include methods for the SKATO rare variant burden analysis.

Reviewer #2 (Public Review):

Summary:<br /> In Ferrareti et al. they identify adaptively introgressed genes using VolcanoFinder and then identify pathways enriched for adaptively introgressed genes. They also use a signet to identify pathways that are enriched for Denisovan alleles. The authors find that angiogenesis and nitric oxide induction are enriched for archaic introgression.

Strengths:<br /> Most papers that have studied the genetic basis of high altitude (HA) adaptation in Tibet have highly emphasized the role of a few genes (e.g. EPAS1, EGLN1), and in this paper, the authors look for more subtle signals in other genes (e.g EP300, NOS 2) to investigate how archaic introgression may be enriched at the pathway level.

Looking into the biological functions enriched for Denisovan introgression in Tibetans is important for characterizing the impact of Denisovan introgression.

Weaknesses:<br /> The manuscript lacks details or justification about how/why some of the analyses were performed. Below are some examples where the authors could provide additional details.

The authors made specific choices in their window analysis. These choices are not justified or there is no comment as to how results might change if these choices were perturbed. For example, in the methods, the authors write "Then, the genome was divided into 200 kb windows with an overlap of 50 kb and for each of them we calculated the ratio between the number of significant SNVs and the total number of variants."

Additional information is needed for clarity. For example, "we considered only protein-protein interactions showing confidence scores {greater than or equal to} 0.7 and the obtained protein frameworks were integrated using information available in the literature regarding the functional role of the related genes and their possible involvement in high-altitude adaptation." What do the confidence scores mean? Why 0.7?

In the method section (Identifying gene networks enriched for Denisovan-like derived alleles), the authors write "To validate VolcanoFinder results by using an independent approach". Does this mean that for signet the authors do not use the regions identified as adaptively introgressed using volcanofinder? I thought in the original signet paper, the authors used a summary describing the amount of introgression of a given region.

Later, the authors write "To do so, we first compared the Tibetan and Denisovan genomes to assess which SNVs were present in both modern and archaic sequences. These loci were further compared with the ancestral reconstructed reference human genome sequence (1000 Genomes Project Consortium et al., 2015) to discard those presenting an ancestral state (i.e., that we have in common with several primate species)." It is not clear why the authors are citing the 1000 genomes project. Are they comparing with the reference human genome reference or with all populations in the 1000 genomes project? Also, are the authors allowing derived alleles that are shared with Africans? Typically, populations from Africa are used as controls since the Denisovan introgression occurred in Eurasia.

The methods section for Figures 4B, 4C, and 4D is a little hard to understand. What is the x-axis on these plots? Is it the number of pairwise differences to Denisovan? The caption is not clear here. The authors mention that "Conversely, for non-introgressed loci (e.g., EGLN1), we might expect a remarkably different pattern of haplotypes distribution, with almost all haplotype classes presenting a larger proportion of non-Tibetan haplotypes rather than Tibetan ones." There is clearly structure in EGLN1. There is a group of non-Tibetan haplotypes that are closer to Denisovan and a group of Tibetan haplotypes that are distant from Denisovan...How do the authors interpret this?

In the original signet paper (Guoy and Excoffier 2017), they apply signet to data from Tibetans. Zhang et al. PNAS (2021) also applied it to Tibetans. It would be helpful to highlight how the approach here is different.

Reviewer #2 (Public Review):

In the manuscript by Rajan et al., the authors have highlighted the direct interaction between Dbp5 and tRNA, wherein Dbp5 serves as a mediator for tRNA export. This export process is subject to spatial regulation, as Dbp5 ATPase activation occurs specifically at nuclear pore complexes. Notably, this regulation is independent of the Los1-mediated pre-tRNA export route and instead relies on Gle1. The manuscript is well constructed and nicely written.

Reviewer #2 (Public Review):

In their study, Stajich and co-authors present a new 1.03 Gb genome assembly for an isolate of the fungal insect parasite Entomophthora muscae (Entomophthoromycota phylum, isolated from Drosophila hydei). Many species of the Entomophthoromycota phylum are specialised insect pathogens with relatively large genomes for fungi, with interesting yet largely unexplored biology. The authors compare their new E. muscae assembly to those of other species in the Entomophthorales order and also more generally to other fungi. For that, they first focus on repetitive DNA (transposons) and show that Ty3 LTRs are highly abundant in the E. muscae genome and contribute to ~40% of the species' genome, a feature that is shared by closely related species in the Entomophthorales. Next, the authors describe the major differences in protein content between species in the genus, focusing on functional domains, namely protein families (pfam), carbohydrate-active enzymes, and peptidases. They highlight several protein families that are overrepresented/underrepresented in the E. muscae genome and other Entomophthorales genomes. The authors also highlight differences in components of the circadian rhythm, which might be relevant to the biology of these insect-infecting fungi. To gain further insights into E. muscae specificities, the authors identify orthologous proteins among four Entomophthorales species. Consistently with a larger genome and protein set in E. muscae, they find that 21% of the 17,111 orthogroups are specific to the species. To finish, the authors examine the consistency between methods for species delineation in the genus using molecular (ITS + 28S) or morphological data (# of nuclei per conidia + conidia size) and highlight major incongruences between the two.

Although most of the methods applied in the frame of this study are appropriate with the scripts made available, I believe there are some major discrepancies in the datasets that are compared which could undermine most of the results/conclusions. More precisely, most of the results are based on the comparison of protein family content between four Entomophthorales species. As the authors mention on page 5, genome (transcriptome) assembly and further annotation procedures can strongly influence gene discovery. Here, the authors re-annotated two assemblies using their own methods and recovered between 30 and 60% more genes than in the original dataset, but if I understand it correctly, they perform all downstream comparative analyses using the original annotations. Given the focus on E. muscae and the small sample size (four genomes compared), I believe performing the comparisons on the newly annotated assemblies would be more rigorous for making any claim on gene family variation.

The authors also investigate the putative impact of repeat-induced point mutation on the architecture of the large Entomophthorales genomes (for three of the eight species in Figure 1) and report low RIP-like dinucleotide signatures despite the presence of RID1 (a gene involved in the RIP process in Neurospora crassa) and RNAi machinery. They base their analysis on the presence of specific PFAM domains across the proteome of the three Entomophthorales species. In the case of RID1, the authors searched for a DNA methyltransferase domain (PF00145), however other proteins than RID1 bear such functional domain (DNMT family) so that in the current analysis it is impossible to say if the authors are actually looking at RID1 homologs (probably not, RID1 is monophyletic to the Ascomycota I believe). Similar comments apply to the analysis of components of the RNAi machinery. A more reliable alternative to the PFAM analysis would be to work with full protein sequences in addition to the functional domains.

Reviewer #2 (Public Review):

Summary:<br /> The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in N-fixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.

Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.

Strengths:<br /> • This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic nitrogen fixation.<br /> • The findings suggest that GmRIN4a/b could mediate appropriate responses during infection, whether it is by friendly or hostile organisms.

Weaknesses:<br /> • The study did not explore the immune response in the rin4 mutant. Therefore, it remains unknown how GmRIN4a/b distinguishes between friend and foe.

Reviewer #2 (Public Review):

This manuscript proposed a new link between the formation of chloroplast budding vesicles (Rubisco-containing bodies [RCBs]) and the development of chloroplast-associated autophagosomes. The authors' previous work demonstrated two types of autophagy pathways involved in chloroplast degradation, including piecemeal degradation of partial chloroplast and whole chloroplast degradation. However, the mechanisms underlying piecemeal degradation are largely unknown, particularly regarding the initiation and release of the budding structures. Here, the authors investigated the progression of piecemeal-type chloroplast trafficking by visualizing it with a high-resolution time-lapse microscope. They provide evidence that autophagosome formation is required for the initiation of chloroplast budding, and that stromule formation is not correlated with this process. In addition, the authors also demonstrated that the release of chloroplast-associated autophagosome is independent of a chloroplast division factor, DRP5b.

Overall, the findings are interesting, and in general, the experiments are very well executed. Although the mechanism of how Rubisco-containing bodies are processed is still unclear, this study suggests that a novel chloroplast division machinery exists to facilitate chloroplast autophagy, which will be valuable to investigate in the future.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Knights et al set out to identify the specific immune cells and their contribution to the development of osteoarthritis. They performed a comprehensive analysis of scRNA-seq and flow cytometry using different stages of the PTOA model and sought to identify specific synovial macrophages in OA. Computational analysis revealed that M-CSF signaling in synovium plays an important role in stromal-immune crosstalk in OA. They also found that four transcription factors including Pu.1, Cebp-alpha, Cebp-beta, and Jun regulate the differentiation of monocytes into pro-inflammatory synovial macrophages in OA.

Strengths:<br /> The main strength of this study is the profiling of immune cells which will be a valuable resource for better understanding the pathogenesis of OA. The work is technically sound, and the level of analysis of gene expression, clustering, cell-cell communication, and dynamic changes in gene modules over time is state-of-the-art.

The reviewer appreciates that the authors uncovered the transcriptional network that regulates the differentiation of synovial macrophages in OA. In addition, the identification of M-CSF signaling as a major crosstalk axis in OA development is also intriguing.

Weaknesses:<br /> Although the scRNA-seq analysis of immune cells in OA is quite convincing, the data has been rather descriptive and superficial at this stage. The authors did not show the in vivo significance of their findings in OA development.

Reviewer #2 (Public Review):

Summary:

The study introduces BRAID, a novel approach for targeting drugs to specific cell types, addressing the challenges of pleiotropic drug actions. Unlike existing methods, this one involves breaking a protein drug molecule into inactive parts that are then put back together using a bridging receptor on the target cell. The individual components of this assembly are not required to be together, thereby affording it a degree of flexibility. The authors applied this idea to the WNT/-catenin signaling pathway by splitting a WNT mimic into two parts with FZD and LRP binding domains and bridging receptors. This combined method, which is called SWIFT, showed that WNT signaling was turned on in target cells, showing that cell-specific targeting is. The technique shows promise for the development of therapeutics, as it provides a way to more precisely target signaling pathways.

The authors have effectively elucidated their strategy through visually appealing diagrams, providing clear and thorough visual aids that facilitate comprehension of the concept. In addition, the authors have provided convincing evidence that the C-terminal region of FGF21 is essential for the binding process. Their meticulous and thorough presentation of experimental results emphasizes the significance of this specific binding domain and validates their findings.

Strengths:

BRAID, a novel cell targeting method, divides an active drug molecule into inactive components formed by a bridging receptor. This novel approach to cell-specific drug action may reduce systemic toxicity.

The SWIFT approach successfully targets cells in the WNT/β-catenin signaling pathway. The approach activates WNT signaling only in target cells (hepatocytes), proving its specificity.

The study indicates that the BRAID approach can target various signaling systems beyond WNT/β-catenin, indicating its versatility. Therapeutic development may benefit from this adaptability.

Weaknesses:

The study shows the SWIFT approach works in vitro using cell lines, primary human hepatocytes, and human intestinal organoids, but it lacks in vivo animal model or clinical validation. I believe future studies will determine this aspect.

The success of SWIFT depends on the presence and expression of the bridging receptor (βKlotho) on target cells. The approach may fail if the target receptor is not expressed.

Reviewer #2 (Public Review):

Zirin, Jusiak, and Lopes et al presented an efficient pipeline for making LexA-GAD and QF2 drivers. The tools can be combined with a large collection of existing GAL4 drivers for a dual genetic control of two cell populations. This is essential when studying inter-organ communications since most of the current genetic drivers are biased toward the expression of the central nervous system. In this manuscript, the authors described the methodology for efficiently generating T2A-LexA-GAD and T2A-QF2 knock-ins by CRISPR, targeting a number of genes with known tissue-specific expression patterns. The authors then validated and compared the expression of double as well as single drivers and found the tissue-specific expression results were largely consistent as expected. Finally, a collection of plasmids for LexA-GAD and QF,2 as well as the corresponding LexAop and QUAS plasmids were generated to facilitate the expansion of these tool kits. In general, this study will be of considerable interest to the fly community and the resources can be readily generalized to make drivers for other genes. I believe this toolkit will have a significant, immediate impact on the fly community.

Reviewer #2 (Public Review):

In this manuscript, Chen et al. determined the structural basis for pre-RNA processing by Las1-Grc3 endoribonuclease and polynucleotide kinase complexes from S. cerevisiae (Sc) and C. jadinii (Cj). Using a robust set of biochemical assays, the authors identify that the sc- and CjLas1-Grc3 complexes can cleave the ITS2 sequence in two specific locations, including a novel C2' location. The authors then determined X-ray crystallography and cryo-EM structures of the ScLas1-Grc3 and CjLas1-Grc3 complexes, providing structural insight that is complimentary to previously reported Las1-Grc3 structures from C. thermophilum (Pillon et al., 2019, NSMB). The authors further explore the importance of multiple Las1 and Grc3 domains and interaction interfaces for RNA binding, RNA cleavage activity, and Las1-Grc3 complex formation. Finally, evidence is presented that indicates Las1 undergoes a conformational change upon Grc3 binding that stabilizes the Las1 HEPN active site, providing a possible rationale for the stimulation of Las1 cleavage by Grc3.

In the revised manuscript, the authors have made significant efforts towards addressing initial reviewer comments. This includes further clarification for key biochemical experiments, significant improvement in structural model quality, and additional structural analysis that further strengthens major conclusions in the manuscript. Overall, the authors conclusions are now well supported by the biochemical and structural data provided.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript shows evidence from a dataset with awake movie-watching in infants, that the infant brain contains areas with distinct functions, consistent with previous studies using resting state and awake task-based infant fMRI. However, substantial new analyses would be required to support the novel claim that movie-watching data in infants can be used to identify retinotopic areas or to capture within-area functional organization.

Strengths:<br /> The authors have collected a unique dataset: the same individual infants both watched naturalistic animations and a specific retinotopy task. These data position the authors to test their novel claim, that movie-watching data in infants can be used to identify retinotopic areas.

Weaknesses:<br /> To claim that movie-watching data can identify retinotopic regions, the authors should provide evidence for two claims:

- Retinotopic areas defined based only on movie-watching data, predict retinotopic responses in independent retinotopy-task-driven data.

- Defining retinotopic areas based on the infant's own movie-watching response is more accurate than alternative approaches that don't require any movie-watching data, like anatomical parcellations or shared response activation from independent groups of participants.

Both of these analyses are possible, using the (valuable!) data that these authors have collected, but these are not the analyses that the authors have done so far. Instead, the authors report the inverse of (1): regions identified by the retinotopy task can be used to predict responses in the movies. The authors report one part of (2), shared responses from other participants can be used to predict individual infants' responses in the movies, but they do not test whether movie data from the same individual infant can be used to make better predictions of the retinotopy task data, than the shared response maps.

So to be clear, to support the claims of this paper, I recommend that the authors use the retinotopic task responses in each individual infant as the independent "Test" data, and compare the accuracy in predicting those responses, based on:

- The same infant's movie-watching data, analysed with MELODIC, when blind experimenters select components for the SF and meridian boundaries with no access to the ground-truth retinotopy data.<br /> - Anatomical parcellations in the same infant.<br /> - Shared response maps from groups of other infants or adults.<br /> - (If possible, ICA of resting state data, in the same infant, or from independent groups of infants).

Or, possibly, combinations of these techniques.

If the infant's own movie-watching data leads to improved predictions of the infant's retinotopic task-driven response, relative to these existing alternatives that don't require movie-watching data from the same infant, then the authors' main claim will be supported.

The proposed analysis above solves a critical problem with the analyses presented in the current manuscript: the data used to generate maps is identical to the data used to validate those maps. For the task-evoked maps, the same data are used to draw the lines along gradients and then test for gradient organization. For the component maps, the maps are manually selected to show the clearest gradients among many noisy options, and then the same data are tested for gradient organization. This is a double-dipping error. To fix this problem, the data must be split into independent train and test subsets.

Reviewer #2 (Public Review):

In this study, Mao and co-workers deliver a substantial suite of genetic tools in support of the senior author's recent proposal to create a "chemoconnectomic" tool kit for the expression mapping and conditional disruption of specific neurotransmitter systems with fly neurons of interest. Specifically, they describe the creation of two toolsets for recombination-based and CRISPR/Cas9-based conditional knockouts of genes supporting neurotransmitter and neuromodulator function and Flp-Out and Split-LexA toolkit for the examination of gene expression within defined subsets of neurons. The authors report the creation of conditional genetic tools for the disruption/mapping of approximately 200 chemoconnectomic gene products, an examination of the general effectiveness of these tools in the fly brain, and apply them to the circadian clock network in an attempt to reveal new information regarding the transmitter/modulator systems involved in daily behavioral timing. The authors provide clear evidence of the effectiveness of the new methods along with a transparent assessment of the variability of the tools. In addition, they present evidence that the neuro peptide CNMa influences the morning peak of daily activity in the fly by regulating the timing of activity increases in anticipation of dawn.

A major strength of the study is the transparent assessment of the effectiveness and variability of the conditional genetic approaches developed by the authors. The authors have largely achieved their aims and the study therefore represents a major delivery on the promise of chemoconnectomics made by the senior author in 2019 (Neuron, Vol. 101, p. 876). Though there are some concerns about the variability of knockout effectiveness, off-target effects of the knockout strategies, and (especially) the accuracy of the gene expression approach, the tools created for this study will almost certainly be useful for the field and support a great deal of future work.

Reviewer #2 (Public Review):

In "Characterization of caffeine response regulatory variants in vascular endothelial cells", Boye et. al. employ a massively parallel reporter assay, bi-allelic targeted STARR-seq (BiT-STARR-seq), to characterize how non-coding variants affect gene expression in HUVECs after treatment with caffeine. After measuring the differential activity of the individual MPRA constructs in their cells, they test for both allele-specific effects (ASE) in each condition. They likewise test for conditional allele-specific effects (cASE). The authors identify an enrichment cASE variants with stronger allelic effects in caffeine vs control conditions and use a combination of transcription factor motif identification, open chromatin enrichment, caffeine response factor binding site identification, and eQTL fine-mapping to identify 25 SNPs that meet their selection criteria. The authors finally highlight one example SNP from this set, rs22871, as a potential candidate for further analysis.

Reviewer #2 (Public Review):

In this manuscript, Shaku and colleagues investigated if the deletion of the enzymatic pair MurT-GatD from Mycobacterium bovis BCG leads to more effective immune activation and protection against tuberculosis disease. MurT-GatD are enzymes implicated in the amidation of peptidoglycan sidechains, an immune evasion mechanism used by virulent mycobacteria to avoid recognition by the pathogen recognition receptor NOD-1.<br /> Using CRISPRi, the authors show that D-glutamate diaminopimelate (iE-DAP) gets unmasked in BCG when MurT-GatD are deleted. They call the resulting recombinant BCG strain in which the induction of the CRISPRi construct is achieved via anhydrotetracycline, BCG::iE-DAP.<br /> Subsequently, the authors characterize the growth kinetics of the strain and show that MurT-GatD deletion results in cell wall defects (as expected) and increased susceptibility to antibiotics. They use in vitro assays with bone marrow-derived macrophages to show that rBCG::iE-DAP leads to an enhanced 'training effect' of the macrophages and increased killing of subsequent Mtb infection. They go on to show that the growth of the rBCG strain can be inhibited both in vitro and in vivo via the addition of doxycycline. Finally, the authors vaccinate Balb/c mice with wildtype BCG or their rBCG strain, deliver doxycycline via oral gavage, and challenge mice with Mycobacterium tuberculosis 6 weeks later. At 4 and 8 weeks after M. tuberculosis infection the mice get assessed for bacterial burden and histopathology. They show that rBCG::iE-DAP leads to reduced bacterial burden, but increased pathology in the lung compared to parental BCG.

The conclusions of this paper are mostly supported by data, but the in vivo protection results against TB need to be clarified and extended.

Strength:<br /> The authors demonstrate an important new pathway by which to improve immunogenicity of BCG - the unmasking of DAP. This is an exciting finding and could lead to the improvement of multiple existing rBCG strains.<br /> The authors also show a rigorous characterization of the rBCG strain and robust in vitro data, demonstrating the effect of MuRT-GatD deletion on cell wall morphology, antibiotic susceptibility and immune training of macrophages.

Weaknesses:<br /> The in vivo part of the manuscript is much weaker than the in vitro findings, and the in vivo experiments are only performed with 5 mice per group and time-point in one single experiment. Scientific standards require that each experiment is repeated at least once to show reproducibility and robustness. The low number of mice for the in vivo experiments also don't allow for strong statistical power.

Reviewer #2 (Public Review):

This paper demonstrates that model-free reinforcement learning, with relatively small networks, is sufficient to observe collaborative hunting in predator prey environments. The paper then studies the conditions under which collaborative hunting emerges (namely, difficulty of hunting and sharing of the spoils) which is an interesting question to study and the paper contains a fascinating study in which a human is tasked with controlling the prey. However, the simplicity of the environment, a 2-d particle world with simple dynamics, makes it unclear how generalizable the results are and the results rely heavily on visual interpretation of t-SNE plots rather than more direct metrics.

Strengths:<br /> - The distinct behaviors uncovered between the predators in shared vs. not-shared reward are quite interesting!<br /> - The realization that the ability of deep RL models to solve predator-prey problems has implication for models of what is needed for collaborative hunting is clever.

Weaknesses:<br /> - The paper seems to make a claim that since this problem is solvable with model-free learning or a model-free decision tree, complicated cognition is not needed for collaborative hunting. However, the settings under which this hunting is done is exceedingly simple and it is possible that in more complex settings such as more partially observable settings or settings where the capabilities of the partners are unknown then more complicated forms of cognition might still be needed.<br /> - The problem is fully observed (I think), so there may be one uniquely good strategy that the predators can use that will work successfully against all prey. If this is the case, the human studies are of limited value, they are just confirming that the problem has a near-deterministic solution on the part of the predators.

Reviewer #2 (Public Review):

In this interesting work on the neuropharmacological effects of ketamine, the authors conducted a pharmacological functional magnetic resonance imaging (fMRI) study in 40 healthy participants receiving bolus and constant infusion of ketamine during resting-state fMRI. Data were preprocessed with the human connectome-based standard pipeline previously successfully used by the lab (FS parcellation and application of an atlas published by the group, HCP pipeline, FSL, global brain connectivity with and without global-signal regression). Briefly, GBC and principle component maps of the positive and negative syndrome scale (PANSS) were related to somatostatin and parvalbumin cortical gene expression patterns. In addition, the authors compared the effective dimensionality, i.e. eigenvalues of covariance matrices of drug vs. placebo, and found higher complexity of responses in ketamine vs. LSD and psilocybin, which is very interesting. Also, there was substantial inter-individual variation in behavioral and neurobehavioral results, which was captured by PC and GBC maps. In supplementary results, the authors also showed that the principle component PS1 highly correlated with the fMRI global signal.

Although a complex set of analyses is presented, the paper is written very clearly and understandable. The authors did a good job of outlining the steps of their analyses in supplemental diagrams and the source code is provided. As a general remark, I consider the main strength of this work, to acknowledge the very diverse inter-individual variation of ketamine's effects and to use advanced methodological approaches to disentangle these.

Since the drug also exhibits strong variation in clinical antidepressant responses, the methodology applied here will very likely yield interesting results applied in clinical datasets of patients with major depressive disorder.

Reviewer #2 (Public Review):

The authors set out to study the potent HIV capsid inhibitor lenacapavir (LEN) and how it alters capsid stability. They use a previously developed single-molecule fluorescence imaging assay to take two measurements of individual viral particles over time: 1) they track the release of GFP from GFP-loaded particles to determine whether the capsid is intact or open, and 2) they track the disassembly of the capsid lattice by measuring the signal intensity of a capsid binding fluorophore (AF568-CypA), which diminishes as the capsid lattice subunits disassociate.

As in their previous work, the authors report that most of their capsids are "leaky" and rapidly lose GFP after the viral membrane is permeabilized, followed by disassembly of the capsid lattice. A subset of capsids maintain GFP signal for various periods of time until they spontaneously "open," and a smaller subset remains closed for the entire length of the imaging experiment (typically 30 min). Interestingly, the authors find that LEN has two effects in this assay: it not only promotes a more rapid release of GFP (interpreted to mean loss of capsid integrity), but it also prevents the capsid lattice from disassembling after opening. As expected, the cellular cofactor IP6 (which stabilizes capsids in cells and in vitro) was found to protect against capsid rupture and counteracted the effects of LEN (although high concentrations of LEN could override any protective effects of IP6).

Their single-molecule experiments are nicely buttressed by in vitro assembly reactions of purified CA protein, with IP6 promoting cone formation and LEN promoting aberrant assembly into tubes. The authors go further to test the kinetics of LEN's effects on HIV infection and reverse transcription, and they perform experiments in comparison to other factors that target the FG binding pocket (BI-2, PF-74, and a peptide from the host factor CPSF6). They find that LEN works differently than these other capsid binders, and stabilizes the lattice structure much more effectively, which the authors suggest is due to how well LEN bridges between CA-CA monomers and rigidifies CA hexamers.

It's particularly interesting that the results of their kinetic studies indicate that LEN's effects on capsid strain (which may ultimately promote rupture) may not happen immediately, but instead, take time to build as the drug occupies more and more binding sites. The authors estimate that roughly 30% of binding sites need to be occupied by LEN to reach half-maximal inhibition of infection, and based on their binding curves, it may take ~20h to reach this level of occupancy in the presence of sub nM concentrations of LEN. Although other mechanisms in addition to catastrophic rupture of capsids are likely at play during inhibition of infection (such as inhibition of host factor binding), these kinetics support previous reports that the most potent functions of capsid inhibition occur at or between the steps of nuclear entry and integration.

It is important to note that although in vitro uncoating assays can help us understand the physical nature of HIV capsid and capsid inhibitor interactions, the assays in this paper might not accurately model the capsid dynamics that are experienced in a cell during infection. The authors report that more than half of their capsids are "leaky" at the start of their assay, but this could be an artifact of the experimental system. Several groups have now demonstrated that capsids remain intact or largely intact for several hours after infection. Thus, while their method is valuable to the research community and can provide insight into capsid stability (and how it can be influenced by capsid binding factors), the authors should be cautious about using pore-forming proteins to permeabilize the virion and interpreting the release of GFP in their single-molecule fluorescence system as an accurate reflection of HIV dynamics in vivo.

In this regard, it would be helpful to establish whether the pore-forming proteins used in vitro to permeabilize the virus membrane have an impact on capsid integrity. It's possible that the concentration of pore-forming proteins used in this paper (200nM) actually promotes "leaky" capsids and rapid opening of capsids in vitro, whereas capsids in their native state in the cytoplasm could remain mostly intact until disrupted by host factors and/or small molecules. Determining whether lower concentrations of DLY/SLO (or PFO as used in Marquez et al., 2018) change the ratio of leaky to closed capsids, or delay the time to capsid opening (either in the presence of IP6 or in the presence of LEN) would be informative. It may be possible to optimize the concentration of pore-forming proteins (and other buffer constituents) to achieve permeabilization of the membrane with minimal disruption to capsid integrity, which could approximate conditions within the cell.

Experiments with capsid mutations that stabilize or destabilize the lattice structure (and exhibit different sensitivities to IP6) could help support the authors' conclusions, as would testing mutations that confer resistance to LEN (e.g. Q67H+N74D, M66I, etc...). It would be of great interest to find if CA mutations affect either GFP release or the CypA paint signal, and whether resistance mutations mitigate the effects of LEN in single-molecule experiments.

The discussion section of this paper is expertly written and places the work into the larger context of HIV research. The authors have thoughtfully analyzed their experiments with capsid inhibitors in relation to kinetics, occupancy, the potential for rigidification, and cofactor binding. They offer reasonable explanations for how LEN exhibits opposing effects on the HIV capsid at high occupancy through inducing capsid rupture while simultaneously preventing the dissociation of CA subunits. Many lines of evidence are now converging on the concept that the capsid evolved to be stable enough to protect its contents, yet flexible enough to navigate the steps of reverse transcription, nuclear entry, and uncoating. With this paper, the authors make a strong case that LEN functions as an antiviral, at least in part, through engaging "lethal hyperstabilization" of the capsid, promoting rigid lattice formations that are incompatible with closed cone structures.

Reviewer #2 (Public Review):

The core planar cell polarity (PCP) pathways are known to control tissue morphogenesis in vertebrates and also in a number of developing tissues in the fruitfly Drosophila. However, it has long been observed that beyond effects on hair polarity, core PCP activity does not have dramatic effects on Drosophila wing morphogenesis. Here the authors carry out detailed quantitative studies of cell behaviors in flies mutant for core PCP genes during pupal wing morphogenesis between about 16 to 32 hours of pupal life to further try to determine if core PCP activity affects cell behaviors in the wing.

Their overall conclusion is that there is no effect on tissue morphogenesis. However, the number of wings looked at for each genotype is low due to the enormous amount of work required to analyze the cell behaviors on an entire wing surface over 16 hours of development. Thus, rigorous statistics cannot be applied to support the statement that there is no change in morphogenesis. Moreover, by eye, the average cell behaviors do appear different and the authors themselves say there are subtle differences. They also note that adult wings have a change in size. Also, a previous publication suggested a change in cell arrangements at the late stages of the period studied (Sugimura & Ishihara 2013).

Interestingly, the authors do report a change in local mechanical properties of the tissue in flies with altered core PCP pathway activity, by using laser ablation to study tissue rheology. This seems to support the view that there could be a subtle change in tissue morphogenesis.

Ultimately, this is a valuable set of results that help to clarify core PCP pathway function in Drosophila tissues. It clearly demonstrates effects on tissue mechanics, but also indicates that this does not result in gross changes in tissue morphogenesis - the latter being consistent with previous observations.

DOI: 10.1016/j.neuron.2023.11.008

Resource: ZFIN_ZDB-ALT-200915-2

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-200915-2

What is this?

Reviewer #2 (Public Review):

In the present study, Liu et al present an analysis of benign and HCC liver samples which were subjected to a new technology (LOOP-Seq) and paired WES. By integrating these data, the authors find isoforms, fusions and mutations which uniquely cluster within HCC samples, such as in the HLA locus, which serve as candidate leads for further investigation. The main appeal of the study is in the potential of LOOP-Seq as a method to present isoform-resolved data without actually performing long-read sequencing.

Comments on revised version:

I made several comments on the previous version which have been adequately addressed.

Reviewer #2 (Public Review):

This is a very interesting paper with several important findings related to the working mechanism of the cartwheel cells (CWC) in the dorsal cochlear nucleus (DCN). These cells generate spontaneous firing that is inhibited by the activation of α2-adrenergic receptors, which also enhances the synaptic strength in the cells, but the mechanisms underlying the spontaneous firing and the dual regulation by α2-adrenergic receptor activation have remained elusive. By recording these cells with the NALCN sodium-leak channel conditionally knocked, the authors discovered that both the spontaneous firing and the regulation by noradrenaline (NA) require NALCN. Mechanistically, the authors found that activation of the adrenergic receptor or GABAB receptor inhibits NALCN. Interestingly, these receptor activations also suppress the low [Ca2+] "activation" of NALCN currents, suggesting crosstalk between the pathways. The finding of such dominant contribution of the NALCN conductance to the regulation of firing by NA is somewhat surprising considering that NA is known to regulate K+ conductances in many other neurons.

The studies reveal the molecular mechanisms underlying well known regulations of the neuronal processes in the auditory pathway. The results will be important to the understanding of auditory information processing in particular, and, more generally, to the understanding of the regulation of inhibitory neurons and ion channels. The results are convincing and are clearly presented.

In this revision, the authors have satisfactorily addressed all my previous comments.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript explores infants' attention patterns in real-world settings and their relationship with autonomic arousal and EEG oscillations in the theta frequency band. The study included 5- and 10-month-old infants during free play. The results showed that the 5-month-old group exhibited a decline in HR forward-predicted attentional behaviors, while the 10-month-old group exhibited increased theta power following shifts in gaze, indicating the start of a new attention episode. Additionally, this increase in theta power predicted the duration of infants' looking behavior.

Strengths:<br /> The study's strengths lie in its utilization of advanced protocols and cutting-edge techniques to assess infants' neural activity and autonomic arousal associated with their attention patterns, as well as the extensive data coding and processing. Overall, the findings have important theoretical implications for the development of infant attention.

Weaknesses:<br /> Certain methodological procedures require further clarification, e.g., details on EEG data processing. Additionally, it would be beneficial to eliminate possible confounding factors and consider alternative interpretations, e,g., whether the differences observed between the two age groups were partly due to varying levels of general arousal and engagement during the free play.

Reviewer #2 (Public Review):

In this paper, Portillo-Ledesma et al. study chromatin organization in the length scale of a gene, simulating the polymer at nucleosome resolution. The authors have presented an extensive simulation study with an excellent model of chromatin. The model has linker DNA and nucleosomes with all relevant interactions (electrostatics, tails, etc). Authors simulate 10 to 26 kb chromatin with varying linker lengths, linker histones (LH), and acetylated tails. The authors then study the effect of a transcription factor (TF) Myc: Max binding. The critical physical feature of the TF in the model is that it binds to the linker region and bends the DNA to make loops/intra-chromatin contacts. Authors systematically investigate the interplay between different variables such as linker DNA length, LH density, and the TF concentration in determining chromatin compaction and 3D organization.

The manuscript is well-written and is a relevant study with many useful results. The biggest strength of the work is the fact that the authors start with a relevant model that incorporates well-known biophysical properties of DNA, nucleosomes, linker histones, and the transcription factor Myc:Max. One of the novel results is the demonstration of how linker lengths play an important role in chromatin compaction (measured by computing packing ratio) in the presence of DNA-bending TFs. As the TF concentration increases, chromatin with short linker lengths does not compact much (only a small change in packing ratio). If the linker lengths are long, a higher percentage of TFs leads to an increase in packing ratio (higher compaction). Authors further show that TFs are able to compact Life-like chromatin fiber with linker length taken from a realistic distribution. The authors compute inter-nucleosomal contact maps from their simulated configurations and show that the map has features similar to what is observed in Hi-C/Micro-C experiments. Authors study the compaction of the Eed gene locus and show that TF binding leads to the formation of small domains known as micro-domains. Authors have predicted many relevant and testable quantities. Many of the results agree with known experiments like the formation of the micro-domains. Hence, the conclusions made in this study are justified - they follow from the simulation results.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, the authors explore the role of the Homeodomain Transcription Factor Bsh in the specification of Lamina neuronal types in the optic lobe of Drosophila. Using the framework of terminal selector genes and compelling data, they investigate whether the same factor that establishes early cell identity is responsible for the acquisition of terminal features of the neuron (i.e., cell connectivity and synaptogenesis).

The authors convincingly describe the sequential expression and activity of Bsh, termed here as 'primary HDTF', and of Ap in L4 or Pdm3 in L5 as 'secondary HDTFs' during the specification of these two neurons. The study demonstrates the requirement of Bsh to activate either Ap and Pdm3, and therefore to generate the L4 and L5 fates. Moreover, the authors show that in the absence of Bsh, L4 and L5 fates are transformed into a L1 or L3-like fates.

Finally, the authors used DamID and Bsh:DamID to profile the open chromatin signature and the Bsh binding sites in L4 neurons at the synaptogenesis stage. This allows the identification of putative Bsh target genes in L4, many of which were also found to be upregulated in L4 in a previous single-cell transcriptomic analysis. Among these genes, the paper focuses on Dip-β, a known regulator of L4 connectivity. They demonstrate that both Bsh and Ap are required for Dip-β, forming a feed-forward loop. Indeed, the loss of Bsh causes abnormal L4 synaptogenesis and therefore defects in several visual behaviors.

The authors also propose the intriguing hypothesis that the expression of Bsh expanded the diversity of Lamina neurons from a 3 cell-type state to the current 5 cell-type state in the optic lobe.

Strengths:<br /> Overall, this work presents a beautiful practical example of the framework of terminal selectors: Bsh acts hierarchically with Ap or Pdm3 to establish the L4 or L5 cell fates and, at least in L4, participates in the expression of terminal features of the neuron (i.e., synaptogenesis through Dip-β regulation).

The hierarchical interactions among Bsh and the activation of Ap and Pdm3 expression in L4 and L5, respectively, are well established experimentally. Using different genetic drivers, the authors show a window of competence during L4 neuron specification during which Bsh activates Ap expression. Later, as the neuron matures, Ap becomes independent of Bsh. This allows the authors to propose a coherent and well-supported model in which Bsh acts as a 'primary' selector that activates the expression of L4-specific (Ap) and L5-specific (Pdm3) 'secondary' selector genes, that together establish neuronal fate.

Importantly, the authors describe a striking cell fate change when Bsh is knocked down from L4/L5 progenitor cells. In such case, L1 and L3 neurons are generated at the expense of L4 and L5. The paper demonstrates that Bsh in L4/L5 represses Zfh1, which in turn acts as the primary selector for L1/L3 fates. These results point to a model where the acquisition of Bsh during evolution might have provided the grounds for the generation of new cell types, L4 and L5, expanding lamina neuronal diversity for a more refined visual behaviors in flies. This is an intriguing and novel hypothesis that should be tested from an evo-devo standpoint, for instance by identifying a species when L4 and L5 do not exist and/or Bsh is not expressed in L neurons.

To gain insight into how Bsh regulates neuronal fate and terminal features, the authors have profiled the open chromatin landscape and Bsh binding sites in L4 neurons at mid-pupation using the DamID technique. The paper describes a number of genes that have Bsh binding peaks in their regulatory regions and that are differentially expressed in L4 neurons, based on available scRNAseq data. Although the manuscript does not explore this candidate list in depth, many of these genes belong to classes that might explain terminal features of L4 neurons, such as neurotransmitter identity, neuropeptides or cytoskeletal regulators. Interestingly, one of these upregulated genes with a Bsh peak is Dip-β, an immunoglobulin superfamily protein that has been described by previous work from the author's lab to be relevant to establish L4 proper connectivity. This work proves that Bsh and Ap work in a feed-forward loop to regulate Dip-β expression, and therefore to establish normal L4 synapses. Furthermore, Bsh loss of function in L4 causes impairs visual behaviors.

Weaknesses:<br /> ● The last paragraph of the introduction is written using rhetorical questions and does not read well. I suggest rewriting it in a more conventional direct style to improve readability.

● A significant concern is the way in which information is conveyed in the Figures. Throughout the paper, understanding of the experimental results is hindered by the lack of information in the Figure headers. Specifically, the genetic driver used for each panel should be adequately noted, together with the age of the brain and the experimental condition. For example, R27G05-Gal4 drives early expression in LPCs and L4/L5, while the 31C06-AD, 34G07-DBD Split-Gal4 combination drives expression in older L4 neurons, and the use of one or the other to drive Bsh-KD has dramatic differences in Ap expression. The indication of the driver used in each panel will facilitate the reader's grasp of the experimental results.

● Bsh role in L4/L5 cell fate:<br /> o It is not clear whether Tll+/Bsh+ LPCs are the precursors of L4/L5. Morphologically, these cells sit very close to L5, but are much more distant from L4.<br /> o Somatic CRISPR knockout of Bsh seems to have a weaker phenotype than the knockdown using RNAi. However, in several experiments down the line, the authors use CRISPR-KO rather than RNAi to knock down Bsh activity: it should be explained why the authors made this decision. Alternatively, a null mutant could be used to consolidate the loss of function phenotype, although this is not strictly necessary given that the RNAi is highly efficient and almost completely abolishes Bsh protein.<br /> o Line 102: Rephrase "R27G05-Gal4 is expressed in all LPCs and turned off in lamina neurons" to "is turned off as lamina neurons mature", as it is kept on for a significant amount of time after the neurons have already been specified.<br /> o Line 121: "(a) that all known lamina neuron markers become independent of Bsh regulation in neurons" is not an accurate statement, as the markers tested were not shown to be dependent on Bsh in the first place.<br /> o Lines 129-134: Make explicit that the LPC-Gal4 was used in this experiment. This is especially important here, as these results are opposite to the Bsh Loss of Function in L4 neurons described in the previous section. This will help clarify the window of competence in which Bsh establishes L4/L5 neuronal identities through ap/pdm3 expression.

● DamID and Bsh binding profile:<br /> ○ Figure 5 - figure supplement 1C-E: The genotype of the Control in (C) has to be described within the panel. As it is, it can be confused with a wild type brain, when it is in fact a Bsh-KO mutant.<br /> ○ It Is not clear how L4-specific Differentially Expressed Genes were found. Are these genes DEG between Lamina neurons types, or are they upregulated genes with respect to all neuronal clusters? If the latter is the case, it could explain the discrepancy between scRNAseq DEGs and Bsh peaks in L4 neurons.

● Dip-β regulation:<br /> ○ Line 234: It is not clear why CRISPR KO is used in this case, when Bsh-RNAi presents a stronger phenotype.<br /> ○ Figure 6N-R shows results using LPC-Gal4. It is not clear why this driver was used, as it makes a less accurate comparison with the other panels in the figure, which use L4-Split-Gal4. This discrepancy should be acknowledged and explained, or the experiment repeated with L4-Split-Gal4>Ap-RNAi.<br /> ○ Line 271: It is also possible that L4 activity is dispensable for motion detection and only L5 is required.

● Discussion: It is necessary to de-emphasize the relevance of HDTFs, or at least acknowledge that other, non-homeodomain TFs, can act as selector genes to determine neuronal identity. By restricting the discussion to HDTFs, it is not mentioned that other classes of TFs could follow the same Primary-Secondary selector activation logic.

Reviewer #2 (Public Review):

Summary:

In this work, the authors explore how Notch activity acts together with Bsh homeodomain transcription factors to establish L4 and L5 fates in the lamina of the visual system of Drosophila. They propose a model in which differential Notch activity generates different chromatin landscapes in presumptive L4 and L5, allowing the differential binding of the primary homeodomain TF Bsh (as described in the co-submitted paper), which in turn activate downstream genes specific to either neuronal type. The requirement of Notch for L4 vs. L5 fate is well supported, and complete transformation from one cell type into the other is observed when altering Notch activity. However, the role of Notch in creating differential chromatin landscapes is not directly demonstrated. It is only based on correlation, but it remains a plausible and intriguing hypothesis.

Strengths:<br /> The authors are successful in characterizing the role of Notch to distinguish between L4 and L5 cell fates. They show that the Notch pathway is active in L4 but not in L5. They identify L1, the neuron adjacent to L4 as expressing the Delta ligand, therefore being the potential source for Notch activation in L4. Moreover, the manuscript shows molecular and morphological/connectivity transformations from one cell type into the other when Notch activity is manipulated.

Using DamID, the authors characterize the chromatin landscape of L4 and L5 neurons. They show that Bsh occupies distinct loci in each cell type. This support their model that Bsh acts as a primary selector gene in L4/L5 that activates different target genes in L4 vs L5 based on the differential availability of open chromatin loci.

Overall, the manuscript presents an interesting example of how Notch activity cooperates with TF expression to generate diverging cell fates. Together with the accompanying paper, it helps thoroughly describe how lamina cell types L4 and L5 are specified and provides an interesting hypothesis for the role of Notch and Bsh in increasing neuronal diversity in the lamina during evolution.

Weaknesses:<br /> Differential Notch activity in L4 and L5:<br /> ● The manuscript focuses its attention on describing Notch activity in L4 vs L5 neurons. However, from the data presented, it is very likely that the pool of progenitors (LPCs) is already subdivided into at least two types of progenitors that will rise to L4 and L5, respectively. Evidence to support this is the activity of E(spl)-mɣ-GFP and the Dl puncta observed in the LPC region. Discussion should naturally follow that Notch-induced differences in L4/L5 might preexist L1-expressed Dl that affect newborn L4/L5. Therefore, the differences between L4 and L5 fates might be established earlier than discussed in the paper. The authors should acknowledge this possibility and discuss it in their model.<br /> ● The authors claim that Notch activation is caused by L1-expressed Delta. However, they use an LPC driver to knock down Dl. Dl-KD should be performed exclusively in L1, and the fate of L4 should be assessed.<br /> ● To test whether L4 neurons are derived from NotchON LPCs, I suggest performing MARCM clones in early pupa with an E(spl)-mɣ-GFP reporter.<br /> ● The expression of different Notch targets in LPCs and L4 neurons may be further explored. I suggest using different Notch-activity reporters (i.e., E(spl)-GFP reporters) to further characterize these differences. What cause the switch in Notch target expression from LPCs to L4 neurons should be a topic of discussion.

Notch role in establishing L4 vs L5 fates:<br /> ● The authors describe that 27G05-Gal4 causes a partial Notch Gain of Function caused by its genomic location between Notch target genes. However, this is not further elaborated. The use of this driver is especially problematic when performing Notch KD, as many of the resulting neurons express Ap, and therefore have some features of L4 neurons. Therefore, Pdm3+/Ap+ cells should always be counted as intermediate L4/L5 fate (i.e., Fig3 E-J, Fig3-Sup2), irrespective of what the mechanistic explanation for Ap activation might be. It's not accurate to assume their L5 identity. In Fig4 intermediate-fate cells are correctly counted as such.<br /> ● Lines 170-173: The temporal requirement for Notch activity in L5-to-L4 transformation is not clearly delineated. In Fig4-figure supplement 1D-E, it is not stated if the shift to 29{degree sign}C is performed as in Fig4-figure supplement 1A-C.<br /> ● Additionally, using the same approach, it would be interesting to explore the window of competence for Notch-induced L5-to-L4 transformation: at which point in L5 maturation can fate no longer be changed by Notch GoF?

L4-to-L3 conversion in the absence of Bsh<br /> ● Although interesting, the L4-to-L3 conversion in the absence of Bsh is never shown to be dependent on Notch activity. Importantly, L3 NotchON status is assumed based on their position next to Dl-expressing L1, but it is not empirically tested. Perhaps screening Notch target reporter expression in the lamina, as suggested above, could inform this issue.<br /> ● Otherwise, the analysis of Bsh Loss of Function in L4 might be better suited to be included in the accompanying manuscript that specifically deals with the role of Bsh as a selector gene for L4 and L5.

Different chromatin landscape in L4 and L5 neurons<br /> ● A major concern is that, although L4 and L5 neurons are shown to present different chromatin landscapes (as expected for different neuronal types), it is not demonstrated that this is caused by Notch activity. The paper proves unambiguously that Notch activity, in concert with Bsh, causes the fate choice between L4 and L5. However, that this is caused by Notch creating a differential chromatin landscape is based only in correlation (NotchON cells having a different profile than NotchOFF). Although the authors are careful not to claim that differential chromatin opening is caused directly by Notch, this is heavily suggested throughout the text and must be toned down.<br /> e.g.: Line 294: "With Notch signaling, L4 neurons generate distinct open chromatin landscape" and Line 298: "Our findings propose a model that the unique combination of HDTF and open chromatin landscape (e.g. by Notch signaling)" . These claims are not supported well enough, and alternative hypotheses should be provided in the discussion. An alternative hypothesis could be that LPCs are already specified towards L4 and L5 fates. In this context, different early Bsh targets in each cell type could play a pioneer role generating a differential chromatin landscape.

● The correlation between open chromatin and Bsh loci with Differentially Expressed genes is much higher for L4 than L5. It is not clear why this is the case, and should be discussed further by the authors.

Reviewer #2 (Public Review):

This study developed a setup for laminar fMRI at 3T that aimed to get the best from all worlds in terms of brain coverage, temporal resolution, sensitivity to detect functional responses, and spatial specificity. They used a gradient-echo EPI readout to facilitate sensitivity, brain coverage and temporal resolution. The former was additionally boosted by NORDIC denoising and the latter two were further supported by parallel-imaging acceleration both in-plane and across slices. The authors evaluated whether the implementation of velocity-nulling (VN) gradients could mitigate macrovascular bias, known to hamper the laminar specificity of gradient-echo BOLD.

The setup allows for 0.9 mm isotropic acquisitions with large coverage at a reasonable TR (at least for block designs) and the fMRI results presented here were acquired within practical scan-times of 12-18 minutes. Also, in terms of the availability of the method, it is favorable that it benefits from lower field strength (additional time for VN-gradient implementation, afforded by longer gray matter T2*).

The well-known double peak feature in M1 during finger tapping was used as a test-bed to evaluate the spatial specificity. They were indeed able to demonstrate two distinct peaks in group-level laminar profiles extracted from M1 during finger tapping, which was largely free from superficial bias. This is rather intriguing as, even at 7T, clear peaks are usually only seen with spatially specific non-BOLD sequences. This is in line with their simple simulations, which nicely illustrated that, in theory, intravascular macrovascular signals should be suppressible with only minimal suppression of microvasculature when small b-values of the VN gradients are employed. However, the authors do not state how ROIs were defined making the validity of this finding unclear; were they defined from independent criteria or were they selected based on the region mostly expressing the double peak, which would clearly be circular? In any case, results are based on a very small sub-region of M1 in a single slice - it would be useful to see the generalizability of superficial-bias-free BOLD responses across a larger portion of M1.

As repeatedly mentioned by the authors, a laminar fMRI setup must demonstrate adequate functional sensitivity to detect (in this case) BOLD responses. The sensitivity evaluation is unfortunately quite weak. It is mainly based on the argument that significant activation was found in a challenging sub-cortical region (LGN). However, it was a single participant, the activation map was not very convincing, and the demonstration of significant activation after considerable voxel-averaging is inadequate evidence to claim sufficient BOLD sensitivity. How well sensitivity is retained in the presence of VN gradients, high acceleration factors, etc., is therefore unclear. The ability of the setup to obtain meaningful functional connectivity results is reassuring, yet, more elaborate comparison with e.g., the conventional BOLD setup (no VN gradients) is warranted, for example by comparison of tSNR, quantification and comparison of CNR, illustration of unmasked-full-slice activation maps to compare noise-levels, comparison of the across-trial variance in each subject, etc. Furthermore, as NORDIC appears to be a cornerstone to enable submillimeter resolution in this setup at 3T, it is critical to evaluate its impact on the data through comparison with non-denoised data, which is currently lacking.

The proposed setup might potentially be valuable to the field, which is continuously searching for techniques to achieve laminar specificity in gradient echo EPI acquisitions. Nonetheless, the above considerations need to be tackled to make a convincing case.

Reviewer #2 (Public Review):

Summary<br /> Song et al investigate the role of the frontal eye field (FEF) and the intraparietal sulcus (IPS) in mediating the shift in ocular dominance (OD) observed after a period of dichoptic stimulation during which attention is selectively directed to one eye. This manipulation has been previously found to transiently shift OD in favor of the unattended eye, similar to the effect of short-term monocular deprivation. To this aim, the authors combine psychophysics, fMRI, and transcranial magnetic stimulation (TMS). In the first experiment, the authors determine the regions of interest (ROIs) based on the responses recorded by fMRI during either dichoptic or binocular stimulation, showing selective recruitment of the right FEF and IPS during the dichoptic condition, in line with the involvement of eye-based attention. In a second experiment, the authors investigate the causal role of these two ROIs in mediating the OD shift observed after a period of dichoptic stimulation by selectively inhibiting with TMS (using continuous theta burst stimulation, cTBS), before the adaptation period (50 min exposure to dichoptic stimulation). They show that, when cTBS is delivered on the FEF, but not the IPS or the vertex, the shift in OD induced by dichoptic stimulation is reduced, indicating a causal involvement of the FEF in mediating this form of short-term plasticity. A third control experiment rules out the possibility that TMS interferes with the OD task (binocular rivalry), rather than with the plasticity mechanisms. From this evidence, the authors conclude that the FEF is one of the areas mediating the OD shift induced by eye-selective attention.

Strengths<br /> 1. The experimental paradigm is sound and the authors have thoroughly investigated the neural correlates of an interesting form of short-term visual plasticity combining different techniques in an intelligent way.

2. The results are solid and the appropriate controls have been performed to exclude potential confounds.

3. The results are very interesting, providing new evidence both about the neural correlates of eye-based attention and the involvement of extra-striate areas in mediating short-term OD plasticity in humans, with potential relevance for clinical applications (especially in the field of amblyopia).

Weaknesses<br /> 1. Ethics: more details about the ethics need to be included in the manuscript. It is only mentioned for experiment 1 that participants "provided informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of the Institute of Psychology, Chinese Academy of Sciences". (Which version of the Declaration of Helsinki? The latest version requires the pre-registration of the study. The code of the approved protocol together with the code and date of the approval should be provided.) There is no mention of informed consent procedures or ethics approval for the TMS experiments. This is a huge concern, especially for brain stimulation experiments!

2. Statistics: the methods section should include a sub-section describing in detail all the statistical analyses performed for the study. Moreover, in the results section, statistical details should be added to support the fMRI results. In the current version of the manuscript, the claims are not supported by statistical evidence.

3. Interpretation of the results: the TMS results are very interesting and convincing regarding the involvement of the FEF in the build-up of the OD shift induced by dichoptic stimulation, however, I am not sure that the authors can claim that this effect is related to eye-based attention, as cTBS has no effect on the blob detection task during dichoptic stimulation. If the FEF were causally involved in eye-based attention, one would expect a change in performance in this task during dichoptic stimulation, perhaps a similar performance for the unattended and attended eye. The authors speculate that the sound could have an additional role in driving eye-based attention, which might explain the lack of effect for the blob discrimination task, however, this hypothesis has not been tested.

4. Writing: in general, the manuscript is well written, but clarity should be improved in certain sections.

a. fMRI results: the first sentence is difficult to understand at first read, but it is crucial to understand the results, please reformulate and clarify.

b. Experiment 3: the rationale for experiment one should be straightforward, without a long premise explaining why it would not be necessary.

c. Discussion: the language is a bit familiar here and there, a more straightforward style should be preferred (one example: p.19 second paragraph).

5. Minor: the authors might consider using the term "participant" or "observer" instead of "subject" when referring to the volunteers who participated in the study.

Reviewer #2 (Public Review):

Summary:<br /> This study by Tünte et al. investigated the development of interoceptive sensitivity in the first year of life, focusing specifically on cardiac and respiratory sensitivity in infants aged 3, 9, and 18 months. The research employed a previously developed experimental paradigm in the cardiac domain and adapted it for a novel paradigm in the respiratory domain. This approach assessed infants' cardiac and respiratory sensitivity based on their preferential-looking behavior toward visuo-auditory stimuli displayed on a monitor, which moved either in sync or out of sync with the infants' own heartbeats or breathing. The results for the cardiac domain showed that infants, across all age groups, preferred stimuli moving synchronously rather than asynchronously with their heartbeat, suggesting the presence of cardiac sensitivity as early as 3 months of age. However, it is noteworthy that the direction of this preference contradicts a previous study, which found that 5-month-old infants looked longer at stimuli moving asynchronously, rather than synchronously, with their heartbeat (Maister et al., 2017). In the respiratory domain, only the younger age group(s) of infants showed a preference for stimuli presented synchronously with their breathing, unlike the 18-month-olds. The authors conducted various statistical analyses to thoroughly examine the obtained data, an effort that provides deeper insights and is valuable for future research in this field.

Strengths:<br /> Few studies have explored the early development of interoception, making the replication of the original study by Maister et al. (2017) particularly valuable. Beyond replication, this study expands the investigation into the respiratory domain, significantly enhancing our understanding of interoceptive development. The provision of longitudinal and cross-sectional data from infants at 3, 9, and 18 months of age is instrumental in understanding their developmental trajectory.

Weaknesses:<br /> (1) My primary concern is that this study did not counterbalance the conditions of the first trial in both iBEAT and iBREATH tests for the 9-month and 18-month age groups. In these tests, the first trial invariably involved a synchronous stimulus. I believe that the order of trials can significantly influence an infant's looking duration, and this oversight could potentially impact the results, especially where a marked preference for synchronous stimuli was observed among infants.<br /> (2) The analysis indicated that the study's sample size was too small to effectively assess the effects within each age group. This limitation fundamentally undermines the reliability of the findings.<br /> (3) The authors attribute the infants' preferential-looking behavior solely to the effects of familiarity and novelty. However, the meaning of "familiarity" in relation to external stimuli moving in sync with an infant's heartbeat or breathing is not clearly defined. A deeper exploration of the underlying mechanisms driving this behavior, such as from the perspectives of attention and perception, is necessary.

Reviewer #2 (Public Review):

In this manuscript, Wang and colleagues analyze the shapes of cerebral cortices from several primate species, including subgroups of young and old humans, to characterize commonalities in patterns of gyrification, cortical thickness, and cortical surface area. The work builds on the scaling law introduced previously by co-author Mota, and Herculano-Houzel. The authors state that the observed scaling law shares properties with fractals, where shape properties are similar across several spatial scales. One way the authors assess this is to perform a "cortical melting" operation that they have devised on surface models obtained from several primate species. The authors also explore differences in shape properties between the brains of young (~20 year old) and old (~80) humans. My main criticism of this manuscript is that the findings are presented in too abstract a manner for the scientific contribution to be recognized.

1. The series of operations to coarse-grain the cortex illustrated in Figure 1, constitute a novel procedure, but it is not strongly motivated, and it produces image segmentations that do not resemble real brains. The process to assign voxels in downsampled images to cortex and white matter is biased towards the former, as only 4 corners of a given voxel are needed to intersect the original pial surface, but all 8 corners are needed to be assigned a white matter voxel (section S2). This causes the cortical segmentation, such as the bottom row of Figure 1B, to increase in thickness with successive melting steps, to unrealistic values. For the rightmost figure panel, the cortex consists of several 4.9-sided voxels and thus a >2 cm thick cortex. A structure with these morphological properties is not consistent with the anatomical organization of a typical mammalian neocortex.

2. For the comparison between 20-year-old and 80-year-old brains, a well-documented difference is that the older age group possesses more cerebral spinal fluid due to tissue atrophy, and the distances between the walls of gyri becomes greater. This difference is born out in the left column of Figure 4c. It seems this additional spacing between gyri in 80-year-olds requires more extensive down-sampling (larger scale values in Figure 4a) to achieve a similar shape parameter K as for the 20-year-olds. A case could be made that the familiar way of describing brain tissue - cortical volume, white matter volume, thickness, etc. - is a more direct and intuitive way to describe differences between young and old adult brains than the obscure shape metric described in this manuscript. At a minimum, a demonstration of an advantage of the Figure 4a and 4b analyses over current methods for interpreting age-related differences would be valuable.

3. In Discussion lines 199-203, it is stated that self-similarity, operating on all length scales, should be used as a test for existing and future models of gyrification mechanisms. First, the authors do not show, (and it would be surprising if it were true) that self-similarity is observed for length scales smaller than the acquired MRI data for any of the datasets analyzed. The analysis is restricted to coarse (but not fine)-graining. Therefore, self-similarity on all length scales would seem to be too strong a constraint. Second, it is hard to imagine how this test could be used in practice. Specific examples of how gyrification mechanisms support or fail to support the generation of self-similarity across any length scale, would strengthen the authors' argument.

Some additional, specific comments are as follows:

4. The definition of the term A_e as the "exposed surface" was difficult to follow at first. It might be helpful to state that this parameter is operationally defined as the convex hull surface area. Also, for the pial surface, A_t, there are several who advocate instead for the analysis of a cortical mid-thickness surface area, as the pial surface area is subject to bias depending on the gyrification index and the shape of the gyri. It would be helpful to understand if the same results are obtained from mid-thickness surfaces.

5. In Figure 2c, the surfaces get smaller as the coarse-graining increases, making it impossible to visually assess the effects of coarse-graining on the shapes. Why aren't all cortical models shown at the same scale?

6. Text in Section 3.2 emphasizes that K is invariant with scale (horizontal lines in Figure 3), and asserts this is important for the formation of all cortices. However, I might be mistaken, but it appears that K varies with scale in Figure 4a, and the text indicates that differences in the S dependence are of importance for distinguishing young vs. old brains. Is this an inconsistency?

Reviewer #2 (Public Review):

Summary:<br /> Yan, Hu, and colleagues introduce BADGERS, a new method for biobank-wide scanning to find associations between a phenotype of interest, and the genetic component of a battery of candidate phenotypes. Briefly, BADGERS capitalizes on publicly available weights of genetic variants for a myriad of traits to estimate polygenic risk scores for each trait, and then identify associations with the trait of interest. Of note, the method works using summary statistics for the trait of interest, which is especially beneficial for running in population-based cohorts that are not enriched for any particular phenotype (ie. with few actual cases of the phenotype of interest).

Here, they apply BADGERS on Alzheimer's disease (AD) as the trait of interest, and a battery of circa 2,000 phenotypes with publicly available precalculated genome-wide summary statistics from the UK Biobank. They run it on two AD cohorts, to discover at least 14 significant associations between AD and traits. These include expected associations with dementia, cognition (educational attainment), and socioeconomic status-related phenotypes. Through multivariate modelling, they distinguish between (1) clearly independent components associated with AD, from (2) by-product associations that are inflated in the original bivariate analysis. Analyses stratified according to APOE inclusion show that this region does not seem to play a role in the association of some of the identified phenotypes. Of note, they observe overlap but significant differences in the associations identified with BADGERS and other Mendelian randomization (MR), hinting at BADGERS being more powerful than classical top variant-based MR approaches. They then extend BADGERS to other AD-related phenotypes, which serves to refine the hypotheses about the underlying mechanisms accounting for the genetic correlation patterns originally identified for AD. Finally, they run BADGERS on a pre-clinical cohort with mild cognitive impairment. They observe important differences in the association patterns, suggesting that this preclinical phenotype (at least in this cohort) has a different genetic architecture than general AD.

Strengths:<br /> BADGERS is an interesting new addition to a stream of attempts to "squeeze" biobank data beyond pure association studies for diagnosis. Increasingly available biobank cohorts do not usually focus on specific diseases. However, they tend to be data-rich, opening for deep explorations that can be useful to refine our knowledge of the latent factors that lead to diagnosis. Indeed, the possibility of running genetic correlation studies in specific sub-settings of interest (e.g. preclinical cohorts) is arguably the most interesting aspect of BADGERS. Classical methods like LDSC or two-sample MR capitalize on publicly available summary statistics from large cohorts, or having access to individual genotype data of large cohorts to ensure statistical power. Seemingly, BADGERS provides a balanced opportunity to dissect the correlation between traits of interest in settings with small sample size in which other methods do not work well.

Weaknesses:<br /> However, the increased statistical power is just hinted, and for instance, they do not explore if LDSC would have identified these associations. Although I suspect that is the case, this evidence is important to ensure that the abovementioned balance is right. Finally, as discussed by the authors, the reliance on polygenic risk scoring necessarily undermines the causality evidence gained through BADGERS. In this sense, BADGERS provides an alternative to strict instrumental-variable based analysis, which can be particularly useful to generate new mechanistic hypotheses.

In summary, after 15 years of focus on diagnosis that would require having individual access to large patient cohorts, BADGERS can become an excellent tool to dig into trait heterogeneity, especially if it turns out to be more powerful than other available methodologies.

Reviewer #2 (Public Review):

Summary:<br /> This is an exciting paper that explores the in vitro assembly of recombinant alpha-synuclein into amyloid filaments. The authors changed the pH and the composition of the assembly buffers, as well as the presence of different types of seeds, and analysed the resulting structures by cryo-EM.

Strengths:<br /> By doing experiments at different pHs, the authors found that so-called type-2 and type-3 polymorphs form in a pH-dependent manner. In addition, they find that type-1 filaments form in the presence of phosphate ions. One of their in vitro assembled type-1 polymorphs is similar to the alpha-synuclein filaments that were extracted from the brain of an individual with juvenile-onset synucleinopathy (JOS). They hypothesize that additional densities in a similar place as additional densities in the JOS fold correspond to phosphate ions.

Weaknesses:<br /> The paper contains multiple instances of non-scientific language, as indicated below. It would also benefit from additional details on the cryo-EM structure determination in the Methods and inclusion of commonly accepted requirements for cryo-EM structures, like examples of 2D class averages, raw micrographs, and FSC curves (between half-maps as well as between rigid-body fitted (or refined) atomic models of the different polymorphs and their corresponding maps). In addition, cryo-EM maps for the control experiments F1 and F2 should be presented in Figure 9.

Reviewer #2 (Public Review):

In this study, Ninagawa et al., shed light on UGGT's role in ER quality control of glycoproteins. By utilizing UGGT1/UGGT2 DKO cells, they demonstrate that several model misfolded glycoproteins undergo early degradation. One such substrate is ATF6alpha where its premature degradation hampers the cell's ability to mount an ER stress response.

While this study convincingly demonstrates early degradation of misfolded glycoproteins in the absence of UGGTs, my major concern is the need for additional experiments to support the "tug of war" model involving UGGTs and EDEMs in influencing the substrate's fate - whether misfolded glycoproteins are pulled into the folding or degradation route. Specifically, it would be valuable to investigate how overexpression of UGGTs and EDEMs in WT cells affects the choice between folding and degradation for misfolded glycoproteins. Considering previous studies indicating that monoglucosylation influences glycoprotein solubility and stability, an essential question is: what is the nature of glycoproteins in UGGTKO/EDEMKO and potentially UGGT/EDEM overexpression cells? Understanding whether these substrates become more soluble/stable when GM9 versus mannose-only translation modification accumulates would provide valuable insights.

The study delves into the physiological role of UGGT, but is limited in scope, focusing solely on the effect of ATF6alpha in UGGT KO cells' stress response. It is crucial for the authors to investigate the broader impact of UGGT KO, including the assessment of basal ER proteotoxicity levels, examination of the general efflux of glycoproteins from ER, and the exploration of the physiological consequences due to UGGT KO. This broader perspective would be valuable for the wider audience. Additionally, the marked increase in ATF4 activity in UGGTKO requires discussion, which the authors currently omit.

The discussion section is brief and could benefit from being a separate section. It is advisable for the authors to explore and suggest other model systems or disease contexts to test UGGT's role in the future. This expansion would help the broader scientific community appreciate the potential applications and implications of this work beyond its current scope.