3,978 Matching Annotations
  1. Sep 2023
    1. Reviewer #2 (Public Review):

      Summary: The authors seek to elucidate the early evolution of cnidarians through computer modeling of fluid flow in the oral region of very small, putative medusozoan polyps. They propose that the evolutionary advent of the free-swimming medusoid life stage was preceded by a sessile benthic life stage equipped with circular muscles that originally functioned to facilitate feeding and that later became co-opted for locomotion through jet propulsion.

      Strengths: Assumptions of the modeling exercise laid out clearly; interpretations of the results of the model runs in terms of functional morphology plausible. An intriguing investigation that should stimulate further discussion and research.

      Weaknesses: Speculation on the origin of the medusoid life stage in cnidarians heavily dependent on prior assumptions concerning the soft part anatomy and material properties of the skeleton of the modeled fossil organism that may be open to alternative interpretations.

    1. 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.<br /> 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 - a question I have here is how to actually release eggs, and could variability in the efficiency of this aspect be modelled in the sensitivity analysis? It seems to me like this could be a challenge and inherently very variable.

    1. Reviewer #2 (Public Review):

      The authors were trying to survey reservoir viral sequences in different anatomical sites in the body, with the brain being of special interest. This is a study that is technically demanding and here is well done, providing insights that prompt new and more sophisticated questions.

      The authors use end-point dilution PCR to identify individual proviruses that can then be sequenced with high accuracy. These are high quality data sets but given the technical requirements of this approach the number of sequenced proviruses is limiting given the scope of questions this study addresses. Nonetheless, there is a lot of data here to draw many useful conclusions.

      It will be important to realize how clones of infected T cells can move around the body, including into the CNS compartment. It will also be important to remember that there are limits in sampling depth of proviruses in any one tissue meaning the failure to detect something has a limit in sensitivity of detection when trying to interpret a negative result.

      As noted in the next section, it is important to emphasize that there is another entry phenotype beyond X4 that will ultimately be important in interpreting these results. Macrophage-tropic viruses are often found in the CNS compartment and it will be important to understand whether these CNS-derived sequences are macrophage-tropic viruses there infecting macrophages and microglia or if they are all T-tropic viruses brought in by wandering infected T cells (or both).

    1. Reviewer #2 (Public Review):

      Summary:<br /> In the current study, the authors tested the hypothesis that Aβ42 toxicity arises from its proven affinity for γ-secretases. Specifically, the increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. They showed that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including (CTFs of APP, p75 and pan-cadherin. Moreover, Aβ42 dysregulated cellular homeostasis by inducing p75-dependent neuronal death. Because γ-secretases process many other membrane proteins, including NOTCH, ERB-B2<br /> receptor tyrosine kinase 4 (ERBB4), N-cadherin (NCAD) and p75 neurotrophin receptor (p75-NTR), revealing a broad range of downstream signaling pathways, including those critical for neuronal structure and function. Hence, they propose to identification of a selective role for the Aβ42 peptide, and raise the intriguing possibility that compromised γ-secretase activity against the CTFs of APP and/or other neuronal substrates contributes to the pathogenesis of AD. Overall, the data are not very convincing to support the main claim.

      Strengths.

      Different in vitro and cellular approaches are employed to test the hypothesis.

      Weaknesses.

      The experimental concentrations for Aβ42 peptide in the assay are too high, which are far beyond the physiological concentrations or pathological levels. The artificial observations are not supported by any in vivo experimental evidence.

    1. Reviewer #2 (Public Review):

      The authors describe the synthesis and testing of the anti-cancer activity of a new molecule CK21 against pancreatic cancer mouse models. This part of the study is very strong showing regression of pancreatic tumors at non-toxic concentrations, which is very hard to achieve for practically uncurable pancreatic cancer. Authors synthesized CK21 as an analog of a known inhibitor of RNA synthesis which is very toxic. The authors did very little attempt to understand whether the mechanism of anti-cancer efficacy of CK2 is similar to this known inhibitor of transcription or not. One cannot compare gene expression profiles between untreated and CK21-treated cells, taking into account that CK2 may inhibit the expression of all genes. The effect of CK2 on general transcription needs to be tested first, and then based on this data absolute changes in the expression of genes may be considered for the revealing of the mechanism of activity of CK21.

    1. Reviewer #2 (Public Review):

      In this manuscript, Ruesseler and colleagues use a continuous task to examine how neural correlates of decision-making change when subjects face conditions with different durations and frequencies of occurrence of signals embedded in noise. The authors develop a novel task where subjects must report the direction of relatively sustained (3 or 5 s) signal changes in average coherence of a random dot kinetogram that are intermittent among relatively transient noise fluctuations (<1 s) of motion coherence that is continuous. Subjects adjust their behavior to changes in the duration of signal events and the frequency of their occurrence. The authors estimate a decay time constant of leaky integration of evidence based on the average coherence leading up to decision responses. Interestingly, there is considerable inter-subject variability in decay time constants even under identical conditions. In addition, the average time constants are shorter when signal periods occur more frequently as opposed to when they are more rare. The authors use EEG to find that a component of the Centroparietal Positivity (CPP) regressed to the magnitude of changes in the noise coherence is larger in conditions when the signal periods occur less frequently. Using a control condition, the authors show that this component of the CPP is not simply based on surprise because it is smaller for changes in motion coherence in irrelevant directions with matched statistics as the changes in relevant directions. The authors also find that a different component of the CPP related to the magnitude of the motion coherence co-varies with the inter-subject variability in decay time constants estimated from behavior.

      Overall, the authors use a clever experimental design and approach to tackle an important set of questions in the field of decision-making. The manuscript is easy to follow with clear writing. The analyses are well thought-out and generally appropriate for the questions at hand. From these analyses, the authors have a number of intriguing results. So, there is considerable potential and merit in this work. That said, I have a number of important questions and concerns that largely revolve around putting all the pieces together. I describe these below.

      1) Quite sensibly, the authors hypothesize that "decay time constant" for past evidence and "decision threshold" would be altered between the different task conditions. They find clear and compelling evidence of behavioral alterations with the conditions. They also have a method to estimate the decay time constant. However, it is unclear to what extent the decision threshold is changing between subjects and conditions, how that might affect the empirical integration kernel, and how well these two factors can together explain the overall changes in behavior.

      To be more specific, the authors state that the lower false alarm rates and slower reaction times for the LONG condition are consistent with a more cautious response threshold for LONG. The empirical integration kernels lead to the suggestion that the decay time constant is not changing between SHORT and LONG, while it is changing between FREQUENT and RARE. Does the lack of change in false alarm rate between FREQUENT and RARE imply no change in the decision threshold? Is this consistent with the behavior shown in Figure 2? I would expect that less decay in RARE would have led to more false alarms, higher detection rates, and faster RTs unless the decision threshold also increased (or there was some other additional change to the decision process). The CPP for motor preparatory activity reported in Fig. 5 is also potentially consistent with a change in the decision threshold between RARE and FREQUENT. If the decision threshold is changing, how would that affect the empirical integration kernel? These are important questions on their own and also for interpreting the EEG changes.

      2) The authors find an interesting difference in the CPP for the FREQUENT vs RARE conditions where they also show differences in the decay time constant from the empirical integration kernel. As mentioned above, I'm wondering what else may be different between these conditions. Do the authors have any leverage in addressing whether the decision threshold differs? What about other factors that could be important for explaining the CPP difference between conditions? Big picture, the change in CPP becomes increasingly interesting the more tightly it can be tied to a particular change in the decision process.

      I'll note that I'm also somewhat skeptical of the statements by the authors that large shifts in evidence are less frequent in the RARE compared to FREQUENT conditions (despite the names) - a central part of their interpretation of the associated CPP change. The FREQUENT condition obviously has more frequent deviations from the baseline, but this is countered to some extent by the experimental design that has reduced the standard deviation of the coherence for these response periods. I think a calculation of overall across-time standard deviation of motion coherence between the RARE and FREQUENT conditions is needed to support these statements, and I couldn't find that calculation reported. The authors could easily do this, so I encourage them to check and report it.

      3) The wide range of decay time constants between subjects and the correlation of this with another component of the CPP is also interesting. However, in trying to interpret this change in CPP, I'm wondering what else might be changing in the inter-subject behavior. For instance, it looks like there could be up to 4 fold changes in false alarm rates. Are there other changes as well? Do these correlate with the CPP? Similar to my point above, the changes in CPP across subjects become increasingly interesting the more tightly it can be tied to a particular difference in subject behavior. So, I would encourage the authors to examine this in more depth.

    1. Reviewer #2 (Public Review):

      The study by Yang et al. examines the interactions between a model host, the nematode C. elegans, and its gut bacteria during aging, focusing on how the host responds to progressing bacterial colonization. In a sense, this work follows up on a previous report describing the activation of DAF-16 in middle-aged worms. Here they test the importance of DAF-16 for aging-dependent accumulation of E. coli in the worm gut, as a model for responses to, and mitigation of, dysbiosis, which in humans is associated with pathology.

      The mechanism unraveled in this study includes the sensing of increasing concentrations of indole, a tryptophan metabolite that is secreted by the accumulating gut bacteria, which dependent on the neuronal cation channel TRPA-1 (and NOT through the known indole receptor AHR-1), activates intestinal DAF-16, driving its nuclear translocation and leading to subsequent induction of downstream targets, of which LYS-7 and LYS-8 are essential for diminishing bacterial colonization and mitigating the associated damage.

      The authors provide very clean and very strong evidence to support the described mechanism, clean identification of indole as the metabolite responsible for DAF-16 nuclear localization, and good indole supplementation experiments and measurements of indole levels inside of worms to support its function. At the same time, some of the methods are not completely clear - for example, how did the authors obtain pure bioactive fraction to run their NMR analysis and identify indole as the activating molecule (this should be clarified in, or added to the method section); or how were indole supplementation experiments carried out? On solid media, i.e. NGM plates, or in solution; with live bacteria, or heat-killed ones? (this is important for figuring out if indole sensing is from the outside or from the gut); and in a few cases the results appear too clear-cut, like the contribution of lys-7 and lys-8 to controlling gut bacteria - these two lysozymes seem to be sufficient to account for the entire contribution of DAF-16, which is surprising considering the large number of downstream targets this transcription factor has, as well as the very redundant nature of innate immune protection, which would have suggested the partial ability to protect at best; this should be considered and discussed.

      Overall, though, the study is strong, and the conclusions are well supported. Given this, its potential impact is high, to inform our understanding of how animals respond to dysbiosis and the mechanisms aimed at mitigating potential detrimental effects of dysbiosis. Here, dysbiosis is manifested as increased colonization of aging worms by bacteria that cannot colonize young adults. In humans, dysbiosis manifests as imbalances in microbiome composition, which may include the proliferation of some gut bacteria at the expense of others. Thus, the mechanisms characterized here, which are conserved in humans, may play similar roles in human pathology and may offer handles to try and mitigate the detrimental effects of dysbiosis.

    1. Reviewer #2 (Public Review):

      Hybridization events between species are known to result in substantial genomic upheaval, requiring subsequent coordination between gene copies to ensure proper control of gene expression and embryonic viability. An example of such an event happened over 18 million years ago between two frog species that resulted in Xenopus laevis-an allotetraploid that has largely retained copies of both genes from this event, known as L-alleles and S-alleles. Often, the presence of both copies presents an experimental and bioinformatic hurdle for researchers and is a feature of the biology of X. laevis that renders cross-species comparisons difficult. Phelps et al, however, take advantage of this feature of Xenopus biology and use it to their advantage to ask how the hybridization event in this species altered gene regulatory architecture. They find that a handful of pluripotency genes are largely responsible for activating gene expression in the early embryo, but that L and S alleles are differentially activated in many cases. Moreover, they find extensive differences in cis-regulatory architecture between L/S alleles. Despite these differences in alleles, however, they find that their combined gene expression output is largely conserved, possibly reflecting strong selection pressures acting to maintain gene expression output at specific levels. This work represents a significant advance in how hybridization events are something greatly understudied in developmental biology-influence gene regulatory programs and how evolutionary pressures have shaped these programs in response to such events.

    1. Reviewer #2 (Public Review):

      Zou et al. presented a comprehensive study where they generated single-cell RNA profiling of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC, and their matched normal tissues, covering comprehensive clinical courses of cSCC. Using bioinformatics analysis, they identified keratinocytes, CAFs, immune cells, and their subpopulations. The authors further compared signatures within subpopulations of keratinocytes along with the clinical progression, especially basal cells, and identified many interesting genes. They also further validate some of the markers in an independent cohort using IHC, followed by some knockdown experiments using cSCC cell lines.

      The strength of this study is the unique data set they have created, providing the community with invaluable resources to study and validate their findings. However, a lot of analyses were not robust enough to support the claims and conclusions in the paper. More clarification and cross-comparison with polished data are needed to further strengthen the study and claims.

      1) Stemness markers were used. The authors used COL17A1, TP63, ITGB1, and ITGA3 to represent stemness markers. However, these were not common classic stemness markers used in cSCC. What is the source claiming these genes were stemness markers in cSCC? TP63 is a master regulator and early driver event in SCC, while COL17A1, ITGB1, and ITGA3 are all ECM genes. The authors need to use commonly well-known stem cell markers in cSCC, e.g., LGR5, to mark stem-like cells.

      2) Cell proportion analysis. The authors used the mean proportions to compare different clinical groups for subpopulations of keratinocytes, e.g., Figure 2B, and Figure 5B. This is not robust, as no statistics can be derived from this. For example, from Fig 2A, it is clearly shown there is a high level of heterogeneity of cellular compositions for normal samples. One cannot say which group is higher or lower simply based on mean not variance as well.

      3) Basal tumour cells in SCCIS and SCC. To make the findings valid, authors need to compare these cells/populations with the keratinocyte cell populations defined by Ji et al. Cell 2020. Do basal-SCCIS-tumours cells, also in SCC samples, resemble any of the population defined in Ji et al. Ji et al. also had 10 match normal, thus the authors need to validate their findings of SCC vs normal analysis using the Ji et al. dataset.

      4) Copy number analysis. Authors used inferCNV to perform copy number analysis using scRNA-seq data and identified CNVs in subpopulations of keratinocytes in SCCIS and SCC. To ensure these CNVs were not artefacts, were some of the CNVs identified by inferCNV well-known copy number changes previously reported in cSCC?

      5) Pseudotime analysis lines 308-313. Not sure the pseudotime analysis added much as, as it is unclear two distinct subgroups were identified from this analysis. Suggest removing this to keep it neater

      6) Selection of candidate genes for validation using IHC and cell line work. For example, lines 205-206, lines 352-356 and lines 437-441, authors selected several genes associated with AK and SCC to further validate using IHC and cell line knockdown work. What are the criteria for selecting those genes for validation? It is unclear to readers how these were selected. It reads like a fishing experiment, then followed by a knockdown. Clear rationale/criteria need to be elaborated.

      7) TME. Compared to keratinocytes populations, the investigation of TME cells was weak. (a) can authors produce UMAP files just for T cells, DC cells, and fibroblasts separately? Figure 7B is not easy to see those subclusters. (b) similar to what was done for keratinocytes, can authors find differentially expressed clusters and genes among the different clinical groups, associated with disease progression? (c) where are the myeloid cell populations, also B cells?

      8) Heat shock protein genes line 327-329. HSP signature was well-known to be induced via tissue dissociation and library prep during the scRNA experiment. How could the authors be sure these were not artefacts induced by the experiment? If authors regress their gene expression against HSP gene signatures, would this cluster still be identified?

      9) Cell-cell communication analysis. The authors claimed that that cell-to-cell interaction was significantly enhanced in poorly-differentiated cSCC, and multiple interaction pathways were significantly active. How was this kind of analysis carried out? How did the authors define significance? what statistical method was used? these were all unclear. Furthermore, it is difficult to judge the robustness of the cell-cell communication analysis. Were these findings also supported by another method, such as celltalker, and cellphoneDB?

      10) Statistics and significance. In general, the detail of statistics and significance was lacking throughout the paper. Authors need to specify what statistical tests were used, and the p-values. It is difficult to judge the correctness of the test, and robustness without seeing the stats.

      11) Overall, this manuscript needs a lot of re-writing. A lot of discussion was also included in the results, making it really difficult to read overall. The authors should simplify the results sections, remove the discussion bits, and further highlight and streamline with the key results of this paper.

    1. Reviewer #2 (Public Review):

      The manuscript employs multiple approaches, including molecular docking, molecular dynamic simulations, and functional experiments to uncover a distinct uridine diphosphate-sugar-binding site on P2Y14 - a key drug target for inflammation and immune responses. Overall, the manuscript is clearly written and the experimental techniques are well-documented. However, it may benefit from further analysis, particularly in terms of validating the binding pose.

    1. Reviewer #2 (Public Review):

      There currently are several hundreds of kinase inhibitors described and available for purchase. However, most of the target the ATP binding site of the protein kinase domain and, since it is pretty well conserved across the whole protein family, it means that the inhibitors are rarely selective, and most are able to simultaneously inhibit several kinases with, sometimes, different binding affinities. In this m/s, the authors present a strategy to combine kinase inhibitors with the aim of reducing off-target effects while preserving the inhibition potency in the intended target. To develop the methodology, the authors have used a set of publicly available data (protein kinase inhibitor set-2, or PKIS-2) containing affinity data on 406 kinases and 645 inhibitors. The authors run a series of simulations suggesting that, in a few cases, the identified combination of inhibitors is superior to the most specific single kinase inhibitor (i.e. show fewer off-target effects while maintaining the inhibition of the on-target). Finally, they test one of these examples in cells using nanoBRET.

      The manuscript tackles an interesting problem (i.e. poor selectivity of kinase inhibitors) that, in some cases, has important clinical bearings. The approach is novel, interesting, and well-executed. However, unfortunately, I am not convinced that the strategy presents a real advantage over the most selective inhibitor.

    1. Reviewer #2 (Public Review):

      Respiratory chain complexes assemble in higher-ordered structures termed supercomplexes or respirasomes. The functional significance of these assemblies is currently investigated, there are two main hypothesis tested, namely that supercomplexes provide kinetic advantages or structural stability. Here, the authors use the fruitfly to reveal that, while the respiratory chain in the organism normally does not form higher-order assemblies, it does so under conditions when their assembly is impaired. Because the rather moderate increase in supercomplex formation does not change oxygen consumption stimulated by CI or CII substrate, the authors conclude that supercomplex formation has more a structural than a functional role. The main strength of this work is that the technical quality of the experiments is high and that the authors induced defects in respiratory chain assembly through sets of well-controlled genetic models. The obtained data are mostly descriptive using standard approaches and are very well executed. The authors claim that their experiments allow to conclude that the role of supercomplex formation is restricted to a structural role and, hence, exclude a function directly related to electron transport efficiency. However, while the authors can show convincingly that supercomplexes form in the mutants, but not in the wild type, the main questions still remain, namely what is the structural mechanism of supercomplex formation and what is the significance of their formation. Given that the fly system does not show supercomplex formation under normal conditions, it is likely that it evolved functionally to work different than systems having supercomplexes. Because these differences are yet unknown, it remains questionable whether the fly system can be used to inform about the general significance of supercomplexes found in the other systems.

    1. Reviewer #2 (Public Review):

      This manuscript by Martin-Flores et al. has examined the role of DKK3 in Alzheimer's disease, focusing on the regulation of synaptic numbers. By using human AD brain databases and tissue samples, the authors showed that DKK3 protein and mRNA levels are increased in the brains of AD patients. DKK3 is expressed in the excitatory neurons in WT mouse brains and accumulates at atrophic neurites around amyloid plaques in AD mouse brains. Interestingly, secretion of DKK3 appears to be regulated by NMDAR antagonist as well as chemical LTD. Through gain and loss of function studies, the authors showed that DKK3 regulates the number of excitatory as well as inhibitory synapses with distinct downstream pathways. Finally, the authors investigated the contribution of DKK3 to synaptic changes in AD and found that DKK3 loss of function rescues both the excitatory and inhibitory synaptic defects, resulting in the improvement of memory function in J20 mice.

      Overall, the data is clearly presented and deals with 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.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Zung et al. use a comparative approach to examine the volatile headspace of diverse mammals and host species to understand the differences in chemical profiles that may provide mosquitoes with signatures of appropriate hosts. The authors collect the volatiles from hair samples and conduct qualitative analyses of the headspace composition. The authors' results suggest that mammals share overlapping volatile signatures, although the sampling method and statistical approaches reduce the veracity of the authors' findings. Additional comparisons between mammalian and floral odours were conducted, although the datasets were limited.

      The inter-species comparisons will be helpful in the field, although the data pipeline and approaches may underestimate the headspace chemical diversity, and sampling artifacts and contaminants occur in the datasets, which further weakens the study's findings.

      Strengths:<br /> The comparative approach is a strength of the manuscript. The authors identify an important gap in mosquito natural history by attempting to characterize the odours from various mammalian, bird, and reptile species that mosquitoes may use as blood hosts. Although others have compared the skin volatiles of humans, apes, and ungulates (Verhulst et al., 2018, not cited in the current manuscript), Zung and coworkers expand this sampling by using hair samples from collections and zoos. Unfortunately, the sampling approach leads to potential artifacts associated with the collected volatiles and statistical analyses.

      Weaknesses:<br /> There are three major points of weakness associated with the manuscript: (1) sampling approach and analysis pipeline; (2) statistical analyses; and (3) premise and prior work.

      1. Sampling approach and pipeline<br /> A. The authors have described their sampling and analysis as quantitative, but they use a qualitative approach by not quantifying their samples and using a low-res MS. I outline several approaches that would allow the authors to quantitate their samples. The authors must run synthetic standards for peak verification (the mass spectra alone are insufficient for compound identification). The authors are also encouraged to run the standards in a concentration curve to allow quantification of the compounds. The authors have only tentatively identified 120 compounds. Using an autosampler and standard analyses in the software, the authors could easily quantify their samples which would take less than a week's time (this is not impossible, as the authors state in the methods). Based on the volatile fragmentation and the MS detector, the compounds will differ in their relative abundances - running calibration curves, co-injection of authentic standards, and using multiple column types are necessary for the resulting statistical analyses to prevent mischaracterization of the abundances in the hair samples. Using an internal standard, by spiking the Tenax before collection, would also allow determination if column conditions change over the course of the experiment. These measurements would provide some quantitative measures to explore the differences in host odors. Details on these approaches can be found in Methods in Chemical Ecology, Techniques in Pheromone Research, and article reviews that describe more recent approaches and analyses (Tholl and Rose, 2006; Stashenko and Martínez, 2008; Spicer et al., 2017; Tholl et al., 2020; Eisen et al., 2021; Schulz and Mollerke, 2022).

      B. Abundant contaminants in the samples. In the supplemental table of partially identified compounds, many contaminants are associated with the headspace collection method and environmental contaminants. Under thermal deadsorption, Tenax degradation produces many compounds, including quinolones and benzenoid compounds. Phenyl-substituted carbonyl compounds (benzaldehyde, acetophenone, benzene acetaldehyde) are formed as artifacts from the oxidation of Tenax with environmental contaminants. Other compounds, like phenol or -ethyl and methylated benzene compounds, are known to be released from the Tenax traps. The authors' pipeline and blank subtraction should have identified these compounds.

      C. Hair and live headspace volatiles. I appreciate the authors' experiments comparing the composition and abundance of volatiles from live collections and hair samples. However, the results demonstrate that the hair does not always match the volatiles from the live animal. Humans 1, 3, and 4 differ significantly in their aldehyde abundances, especially nonanal. The hamster and mice samples also differ significantly. The matrix of the hair will adsorb and modify the emissions and ratios of compounds, which makes the inter-species comparisons difficult if not impossible if the headspace collection approaches differ. The authors need to change their phrasing of the host odours to "hair odours", and soften their statements associated with the complete host odour profile, and use hair samples as a standard matrix for the headspace collections. The comparison of human odour collections relative to hair samples is like the comparison of apples and oranges.

      D. The authors need to use another column type to characterize their peaks further. Some of the compounds are enantiomers or closely elute from the column. Although the authors suggest their methods may separate these compounds, they may be misidentified without a different GC temperature ramp or column.

      E. The authors should replace their retention indices with KRI values to further identify their compounds. The methods section does not describe whether the alkane standards were run parallel to the hair samples, and the manuscript's retention indices do not match published KRI values.

      F. The number of compounds across species (including flower compounds) is very low (approximately 120 compounds) and surprising. This suggests that the analysis pipeline and thresholding may miss many compounds in the headspace. I would encourage the authors to lower their threshold to 10^-5 AU, or to perform a sensitivity analysis on their ability to identify the peaks. Running authentic standards would also allow the identification of compounds missed in the analysis.

      G. I understand the difficulty in obtaining these samples across the different species. However, additional information is needed for those species that are limited in the number of replicates (individuals). Sampling the individual multiple times may indicate the variability in the hair volatiles. Although the authors and many others have shown the reproducibility of human skin volatiles through time, additional sampling would indicate this also occurs for other mammals while strengthening the authors' approach.

      H. An important measure of natural odour statistics is the odor emission rates, and normalizing across samples by the sample mass. More information on the methods would have clarified these aspects. It needs to be clarified why the samples were collected for different time periods (5 to 80 minutes). The sample mass for each specimen should also be included as this would allow normalization by time and mass, and should be described in the methods. This would allow quantitative measurements of the samples.

      I. A critical missing component in the headspace is the acids. Tenax does not perform well at collecting these compounds. However, Gerstel Twisters and other collection matrices can capture those compounds. The authors must use these other collection methods to sample the hair specimens and identify those compounds to include in their table and analyses. Without this information, the manuscript lacks a critical dimension in the human odour landscape that is critical for mosquito attraction.

      2. Statistical Analyses<br /> A. Sampling effort and the replicate numbers used in the analyses is an important consideration that the authors do not address, but should be discussed in more detail. In many subfields of chemical ecology, a minimum of ten replicates per species has been suggested to accurately identify the composition of compounds, and even with ten samples, this may not be enough to characterize the volatile profile (Raguso and Pellmyr, 1998; Campbell et al 2019). The authors could perform a power analysis, or an accumulation curve to represent the needed sample number to identify the number of compounds in the hair headspace accurately.

      B. It would be worthwhile for the authors to provide more detail on their supervised and unsupervised approaches, and how their data fits the assumptions of the analyses. The PCA parametric method may require log or square root transformation of the data to make residuals fit the normality assumption, but it's unclear if this was the case with the authors' datasets.

      C. PCA is also not appropriate when many samples have zero values in the data matrix, which occurs in the authors' data. In such a case, the approaches of NMDS or canonical analysis of principal coordinates would be more appropriate, and allow distance measures (the Bray-Curtis distance) to define dissimilarity of different groups. An analysis of similarity (ANOSIM) could be used to determine if the data clustered significantly by species or by mosquito host.

      D. The authors are encouraged to use alternate approaches, such as random forest (ML) approach, to determine if the odor classification is based on host or non-host. This method has been used for the last fifteen years in chemical ecology and human odor analysis (Cutler et al, 2007, Kwak et al 2008).

      E. The authors use a phylogenetic framework for their analyses. Multivariate methods are now available to test evolutionary hypotheses about scent composition in a phylogenetic framework (Goolsby, 2017), and the authors are encouraged to use these approaches.

      F. Comparison to floral odour space section. I would encourage the authors to examine other datasets of plant headspace samples, including plants used by mosquitoes. There are many datasets out there that the authors could use (El-Sayed 2021, Farré-Armengol et al 2020). Expanding the authors' dataset would provide more statistical power, and provide control of differences in plant visitor and plant phylogenetic relatedness.

      G. Adding context related to mosquito olfaction. The authors describe how their work could provide insight into the coding of olfactory information by the mosquito. I would encourage the authors to analyze their data further by collapsing the host volatiles into groups based on biochemical pathways, or knowledge of the detection of the volatiles by the mosquitoes (such as using electroantennogram responses) to filter and identify only those responsive volatiles to keep in their dataset.

      Premise and Background Knowledge<br /> A. Analyses of odour headspace have been known for the last three decades, e.g. (Methods in Chemical Ecology, Techniques in Pheromone Research, George Petri's work, Tholl and Rose, 2006; Stashenko and Martínez, 2008; Spicer et al., 2017; Tholl et al., 2020; Eisen et al., 2021; Schulz and Mollerke, 2022). But in many places, the paper conveys the impression that these are new discoveries and analyses. For example,<br /> -"Yet we remain remarkably ignorant of the composition of the chemical world."<br /> -"Our work provides one of the first quantitative descriptions of a natural odour space"<br /> -"Progress in understanding natural odours has also been hindered by the technical challenges of capturing and analyzing odour, especially the complex blends that constitute most natural odours"<br /> The Introduction and Discussion are rife with these overblown statements. I found this frustrating as the authors were not giving due credit to prior work on that topic while (maybe unintentionally) giving an impression that this specific idea was a new contribution. More care is needed to delineate which aspects of the study are 1) based on prior understanding, or 2) totally new). The authors are adding to an already extensive field of chemical ecology and olfactory processing of mixtures, and are contributing to this knowledge by adding datasets related to mammalian odor. I plead that the authors clearly describe these gaps, and place their results into proper context.

      B. Similarly to the above statements relating to chemical ecology, the authors have numerous statements about gaps in odour processing. Mixture processing has been an important topic of study for the last forty years (Shorey, 1973, Caprio, 1988, Riffell et al 2009, Su et al 2009, Rokni et al 2014, Mathis et al 2016), which is based on encoding the temporal and concentration-dependent statistics of the odour.<br /> -"Yet compared to visual and auditory scenes, we know very little about the statistics of natural olfactory scenes"<br /> As described above, this is surprising and frustrating because of the rich literature on these topics (searching for "odour mixtures" provides 32,000 articles). In their manuscript, the authors are providing a strawman argument for their analyses by focusing on single odorant signatures, when the literature has repeatedly demonstrated the importance of odour mixtures for behavior and combinatorial processing.

      C. There are increasing studies examining the mosquito behavioral and electrophysiological responses to hosts and other odours. However, this literature is not cited or included in the authors' analyses. The chemical ecology of mosquito attractants and natural odours has been studied in the Carde, Leal, Ignell, Carlson, Kline, Riffell, Takken, Torto, Verlhurst, Vosshall labs, and many others. The authors could use this information in their analyses and cite the literature.

    1. Reviewer #2 (Public Review):

      It is well known that DMRT proteins and more specifically, DMRT1 plays a key role in the sex determination processes of many species. While DMRT1 has been shown to be critical for the sex determination of fish, birds, and reptiles, it seems less crucial at the sex determination stages of the mice. It is important though for adult sex maintenance in mice.

      Unlike its minor role in mouse sex determination, it seems that variants in DMRT1 in humans cause 46, XY DSD and sex reversal.

      The paper by Dujardin et al. is a beautiful study that provides an answer to this long-lasting discrepancy of the difference between the two common mammal species: human and mouse. It is a really nice example of how working with other mammal species, like the rabbit, could serve as a nice model for understanding mammalian sex determination.

      In this study the researchers first described the expression patterns of DMRT1 in the rabbit XY and XX gonads throughout the window of sex determination.

      They then used CRISPR/Cas9 to generate DMRT1 KO rabbits and analysed the phenotype in XY and XX rabbits. They show that XY rabbits present with complete XY male-to-female sex reversal, very similar to what observed in human 46, XY DSD patients (but not the mice model). They further show that in the XY sex reversed gonads, germ cells fail to enter meiosis. They next analysed XX gonads and while there is no major effect on sex determination (as expected), the germ cells in these ovaries fail to enter meiosis, highlighting the critical role that DMRT1 has in germ cells.

      I think it is really important that we start to embrace other mammal models that are not the mouse as we find many instances that the mouse is not the optimal system for understanding human sex determination.

      The study is well explained and presented. The data is clear, and the paper is fluent to read.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors used next-generation sequencing approaches combined with ribosome trapping to investigate gene expression in neurons and glia in the heads of adult fruit flies. Ribosome footprinting was further used to investigate the translational efficiency (TE) of particular RNAs in these two tissues. The evidence convincingly demonstrated that translation of specific messages is repressed in glia while others are repressed in neurons. Further evidence suggests that cis-acting elements within the 5'UTR of neuronal transcripts cause the repression of translation in glia. For instance, a fluorescent reporter using the 5'UTR of Rhodopsin-1 is highly translated in neurons but fluorescence from this reporter is nearly undetectable in glia. Furthermore, pausing of ribosomes on start codons of upstream Open Reading Frames (uORFs) is seen on the 5'UTR of this and other messages in glia but not in neurons.

      Strengths:<br /> The main strength of the manuscript is its use of cutting-edge next-generation sequencing and bioinformatic approaches to investigate the tissue-specific translatome of Drosophila.

      Weaknesses:<br /> A minor weakness is that little insight is provided into the mechanism that leads to ribosome stalling on uORFs in glia but not in neurons. The manuscript could be improved by some discussion on potential pathways that might control the differential TE through uORF pausing.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript by Xu et al., is an interesting study aiming to identify novel features of macaque cortical development. This study serves as a valuable atlas of single cell data during macaque neurogenesis, which extends the developmental stages previously explored. Overall, the authors have achieved their aim of collecting a comprehensive dataset of macaque cortical neurogenesis and have identified a few unknown features of macaque development.

      Strengths:<br /> The authors have accumulated a robust dataset of developmental time points and have applied a variety of informatic approaches to interrogate this dataset. One interesting finding in this study is the expression of previously unknown receptors on macaque oRG cells. Another novel aspect of this paper is the temporal dissection of neocortical development across species. The identification that the regulome looks quite different, despite similar expression of transcription factors in discrete cell types, is intriguing.

      Weaknesses:<br /> Due to the focus on demonstrating the robustness of the dataset, the novel findings in this manuscript are underdeveloped. There is also a lack of experimental validation. This is a particular weakness for newly identified features (like receptors in oRG cells). It's important to show expression in relevant cell types and, if possible, perform functional perturbations on these cell types. The presentation of the data highlighting novel findings could also be clarified at higher resolution, and dissected through additional informatic analyses. Additionally, the presentation of ideas and goals of this manuscript should be further clarified. A major gap in the study rationale and results is that the data was collected exclusively in the parietal lobe, yet the rationale and interpretation of what this data indicates about this specific cortical area was not discussed. Last, a few textual errors about neural development are also present and need to be corrected.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors examined several defensive responses elicited during Pavlovian conditioning using a serial compound stimulus (SCS) as the conditioned stimulus (CS) and a shock unconditioned stimulus (US) in male and female mice. The SCS consisted of tone pips followed by white noise. Their design included 3 treatment groups that were either exposed to the CS and US in a paired fashion, in an unpaired fashion, or only exposed to the shock US. They compared freezing, jumping, darting, and tail rattling across all groups during conditioning and extinction. During conditioning, strong freezing responses to the tone pips followed by strong jumping and darting responses to the white noise were present in the paired group but less robust or not present in the unpaired or shock only groups. During extinction, tone-induced freezing diminished while the jumping was replaced by freezing and darting in the paired group. Together, these findings support the idea that associative pairings are necessary for conditioned defensive responses.

      Strengths:<br /> The study has strong control groups including a group that receives the same stimuli in an unpaired fashion and another control group that only receives the shock US and no CS to test the associative value of the SCS to the US. The authors examine a wide variety of defensive behaviors that emerge during conditioning and shift throughout extinction: in addition to the standard freezing response, jumping, darting, and tail rattling were also measured.

      Weaknesses:<br /> This study could have greater impact and significance if additional conditions were added (e.g., using other stimuli of differing salience during the SCS), and determining the neural correlates or brain regions that are differentially recruited during different phases of the task across the different groups.

    1. Reviewer #2 (Public Review):

      The manuscript investigates the function of basal forebrain cholinergic axons in mouse primary visual cortex (V1) during locomotion using two-photon calcium imaging in head-fixed mice. Cholinergic modulation has previously been proposed to mediate the effects of locomotion on V1 responses. The manuscript concludes that the activity of basal forebrain cholinergic axons in visual cortex provides a signal which is more correlated with binary locomotion state than locomotion velocity of the animal. Cholinergic axons did not seem to respond to grating stimuli or visuomotor prediction error. Optogenetic stimulation of these axons increased the amplitude of responses to visual stimuli and decreased the response latency of layer 5 excitatory neurons, but not layer 2/3 neurons. Moreover, optogenetic or chemogenetic stimulation of cholinergic inputs reduced pairwise correlation of neuronal responses. These results provide insight into the role of cholinergic modulation to visual cortex and demonstrate that it affects different layers of visual cortex in a distinct manner. The experiments are well executed and the data appear to be of high quality. However, further analyses are required to fully support several of the study's conclusions.

      1) In experiments analysing the activity of V1 neurons, GCaMP6f was expressed using a ubiquitous Ef1a promoter, which is active in all neuronal cell types as well as potentially non-neuronal cells. The manuscript specifically refers to responses of excitatory neurons but it is unclear how excitatory neuron somata were identified and distinguished from that of inhibitory neurons or other cell types.

      2) The manuscript concludes that cholinergic axons convey a binary locomotion signal and are not tuned to running speed. The average running velocity of mice in this study is very slow - slower than 15 cm/s in the example trace in Figure 1D and speeds <6 cm/s were quantified in Figure 2E. However, mice can run at much faster speeds both under head-fixed and freely moving conditions (see e.g. Jordan and Keller, 2020, where example running speeds are ~35 cm/s). Given that the data in the present manuscript cover such a narrow range of running speeds, it is not possible to determine whether cholinergic axons are tuned to running speed or convey a binary locomotion signal.

      3) The analyses in Figure 4 only consider the average response to all grating orientations and directions. Without further analysing responses to individual grating directions it is unclear how stimulation of cholinergic inputs affects visual responses. Previous work (e.g. Datarlat and Stryker, 2017) has shown that locomotion can have both additive and multiplicative effects and it would be valuable to determine the type of modulation provided by cholinergic stimulation.

      4) The difference between the effects of locomotion and optogenetic stimulation of cholinergic axons in Figure 5 may be confounded by differences in the visual stimulus. These experiments are carried out under open-loop conditions, where mice may adapt their locomotion based on the speed of the visual stimulus. Consequently, locomotion onsets are likely to occur during periods of higher visual flow. Since optogenetic stimulation is presented randomly, it is likely to occur during periods of lower visual flow speed. Consequently, the difference between the effect of locomotion and optogenetic stimulation may be explained by differences in visual flow speed and it is important to exclude this possibility.

      5) It is unclear why chemogenetic manipulations of cholinergic inputs had no effect on pairwise correlations of L2/3 neuronal responses while optogenetic stimulation did.

      6) The effects of locomotion and optogenetic stimulation on the latency of L5 responses in Figure 7 are very large - ~100 ms. Indeed, typical latencies in mouse V1 measured using electrophysiology are themselves shorter than 100 ms (see e.g. Durand et al., 2016). Visual response latencies in stationary conditions or without optogenetic stimulation appear surprisingly long - much longer than reported in previous studies even under anaesthesia. Such large and surprising results require careful analysis to ensure they are not confounded by artefacts. However, as in Figure 4, this analysis is based only on average responses across all gratings and no individual examples are shown.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors develop a computational approach-avoidance-conflict (AAC) task, designed to overcome the limitations of existing offer based AAC tasks. The task incorporated likelihoods of receiving rewards/ punishments that would be learned by the participants to ensure computational validity and estimated model parameters related to reward/punishment and task induced anxiety. Two independent samples of online participants were tested. In both samples participants who experienced greater task induced anxiety avoided choices associated with greater probability of punishment. Computational modelling revealed that this effect was explained by greater individual sensitivities to punishment relative to rewards.

      Strengths:

      Large internet-based samples, with discovery sample (n = 369), pre-registered replication sample (n = 629) and test-retest sub group (n = 57). Extensive compliance measures (e.g. audio checks) seek to improve adherence.

      There is a great need for RL tasks that model threatening outcomes rather than simply loss of reward. The main model parameters show strong effects and the additional indices with task based anxiety are a useful extension. Associations were broadly replicated across samples. Fair to excellent reliability of model parameters is encouraging and badly needed for behavioral tasks of threat sensitivity.

      The task seems to have lower approach bias than some other AAC tasks in the literature.

      Appraisal and impact:<br /> Overall this is a very strong paper, describing a novel task that could help move the field of RL forward to take account of threat processing more fully. The large sample size with discovery, replication and test-retest gives confidence in the findings. The task has good ecological validity and associations with task-based anxiety and clinical self-report demonstrate clinical relevance. Test-retest of the punishment learning parameter is the only real concern. Overall this task provides an exciting new probe of reward/threat that could be used in mechanistic disease models.

      Additional context:

      The sex differences between the samples are interesting as effects of sex are commonly found in AAC tasks. It would be interesting to look at the main model comparison with sex included as a covariate.

    1. Reviewer #2 (Public Review):

      This manuscript links the distinctive stinging behavior of sea anemones in different ecological niches to varying inactivation properties of voltage-gated calcium channels that are conferred by the identity of auxiliary Cavbeta subunits. Previous work from the Bellono lab established that the burrowing anemone, Nematostella vectensis, expresses a CaV channel that is strongly inactivated at rest which requires a simultaneous delivery of prey extract and touch to elicit a stinging response, reflecting a precise stinging control adapted for predation. They show here that by contrast, the anemone Exaiptasia diaphana which inhabits exposed environments, indiscriminately stings for defense even in the absence of prey chemicals, and that this is enabled by the expression of a CaVbeta splice variant that confers weak inactivation. They further use the heterologous expression of CaV channels with wild type and chimeric anemone Cavbeta subunits to infer that the variable N-termini are important determinants of Cav channel inactivation properties.

    1. Reviewer #2 (Public Review):

      Summary:

      C. difficile infection (CDI) is clinically important as a hospital-acquired infection and a frequent cause of antibiotic-associated diarrhea, which is associated with high morbidity and mortality and increases in prevalence. It is also the prime example of a disease that is associated with gut microbiome dysbiosis and successfully treated with fecal microbiota transfer, highlighting the important but unclear functional or structural role of this bacterial pathogen and the condition of CDI for the gut microbiome, which is the focus of this study.

      Ferretti et al. assembled an impressive gut metagenome dataset from previous and ongoing microbiome studies, which involves a large number of samples from patients with CDI or other diarrheal and non-diarrheal diseases and from healthy individuals, as well as from infants, adolescents, and adults. The authors analyze the prevalence and relative abundance of C. difficile in this dataset in relation to CDI diagnosis, host age and disease background, and the composition of the remaining microbiota. They detect C. difficile only in a minority of samples labelled as originating from CDI patients but frequently identify other pathogens and their toxin genes in the same samples. In infants, they detect C. difficile at high frequency and relative abundance in samples without clinical symptoms. They associate C. difficile presence in infant samples with "multiple indicators of healthy gut microbiome maturation' and suggest 'distinct biotic and physiological contexts in infants and adults' for C. difficile.

      Strengths:

      The manuscript provides an important overview of the complex relationship of C. difficile with the gut microbiome of healthy and diseased infants and adults, mostly due to the large studied dataset and convincing applied analysis that underlies the presented findings. This includes a number of interesting findings including, for example, that CDI can be reliably predicted based on taxonomic microbiota compositions, without including C. difficile itself or that C. difficile in infants appears not to originate from maternal sources.

      Weaknesses:

      Inconsistent associations of C. difficile with what is clinically labeled CDI, as well as the frequent detection of C. difficile in healthy infants, have been reported before and the manuscript does not reveal to what extent this bacterium reflects or even directly influences the gut microbiome of infants and adults. Whether the increased microbiota diversity, richness, and compositional similarity of C. difficile-positive infants to their mothers is sufficient to associate this bacterium with "healthy gut microbiome maturation" seems questionable, since C. difficile was also found to be more prevalent in preterm infants, formula-fed or antibiotically treated infants, and infants born by C-section, all of which are typically considered detrimental influences on microbiota development. The conclusion that "C. difficile may be a transient hallmark of healthy gut microbiome maturation" therefore appears too strong.

      In addition, the statement that "its asymptomatic carriage in adults depends on microbial context" is not sufficiently supported by the presented data. Apparently, the authors are unable to define or measure "asymptomatic carriage", as they convincingly show that many patients diagnosed with "CDI" appear not to carry C. difficile, suggesting that neither asymptomatic nor symptomatic "CDI" conditions are necessarily linked to C. difficile.

      The manuscript includes a large number of samples from poorly defined, but diverse patient backgrounds. It might be helpful to better define these samples (e.g. fecal samples vs. other gut samples) and to specify subcategories for samples from "diseased control subjects without CDI". Maybe this information could help validate the interesting suggestion from the manuscript that C. difficile may be (one of several) dysbiosis marker rather than the cause of (CDI) dysbiosis.

      The phylogenetic analysis of C. difficile from metagenomic sequence data seems to suggest that there is a large mostly toxin gene-free cluster that is only identified in infants (Supplementary Figure 13). Could this indicate that there are, in fact, less pathogenic C. difficile lineages that are more prevalent in infants?

      The authors argue in the Discussion that "Differential diagnosis against multiple enteropathogens may therefore stratify patients with CDI-like symptoms, towards adapted therapeutic interventions." It might be helpful to expand this discussion of different clinical options that could be adapted to highlight the clinical applicability of the presented findings.

    1. Reviewer #2 (Public Review):

      In this work, the authors reported cryo-EM structures of four types of zinc-binding site mutants of a bacterial Zn2+/H+ antiporter YiiP, and proposed distinct structural/functional roles of each of the binding sites in the intramolecular Zn2+ relay and the integrity of the homodimeric structure of YiiP. MST analysis using the mutants with a single Zn2+-binding site at different pH further clarified the pH dependence of Zn2+ binding affinity of each site. Moreover, the inverse Multibind approach refined the CpHMD pKa values of the key Zn2+-binding residues so that they agreed with the MST data. Consequently, energetic coupling of Zn2+ export to the proton-motive force has been suggested. These findings definitely provide new mechanistic insight into this Zn2+/H+ antiporter.

    1. Reviewer #2 (Public Review):

      This paper makes important and novel advances that significantly enhance our understanding of the ClC-2 channel. The EM data are of high quality, and the most important argument, concerning the role of the N-terminus of the protein as an occluding inactivation gate, is very well supported by structural, computational, and functional data (some of which is previously published). The proposal that the "run up" observed in patch clamp experiments represents relief of inactivation is interesting and compelling. The model predicts that mutations at the hairpin binding site should influence this "run up", which should be tested in the near future. Finally, the confirmation of the AK-42 binding site further solidifies evidence that this is a pore-blocking compound; the authors' argument about determinants of specificity is convincing.

    1. Reviewer #2 (Public Review):

      Using standard and widely used tools, the author revealed the factors (cultural, phenotypic, phylogenetic, etc.) shaping societal and scientific interest in natural species around the globe. The strength of this ms (and the authors) lies in its command of the available literature, database and variable management and analysis, and its solid discussion. The authors thus achieved a manuscript that was pleasant to read.

      While I agree that doing a global study requires losing details of local patterns, maybe this is exactly the biggest shortcoming of the manuscript, oblivious to how different cultures (compare USA to PNG, for example) are reflected in these global patterns.

    1. Reviewer #2 (Public Review):

      The paper presents new mitochondrial sequence data from baboons from a museum collection and from one ancient Egyptian mummified baboon. By comparing the mitochondrial sequence of the mummified baboon with the new and existing data, they conclude that it originated from present-day Eritrea, specifically the ancient city of Adulis.

      The paper is well-written and an interesting read. The background and details of the study are well-described and logical. Not knowing much about the history of the region I learned a lot. The data also seem sound and the analysis robust, with the exception of one check that should be added (in particular, to assess contamination by looking at mismatching reads).

      The main limitation of the paper is just down to the N=1 sample and the limits of mitochondrial phylogeography. Based on the present-day distribution of hamadryas, the baboon must either come from the area of Africa around present-day Eritrea/Ethiopia/Sudan, or from Arabia. All the authors can really reasonably establish here is that this particular baboon did not come from Arabia. But beyond that, there is not much more they can say. Fig 2b makes it clear that the G3Y clade extends over a large range. Given the limited sampling, this is a minimum bound for the range, which probably includes most of the non-Arabian hamadryas range. The link to Adulis is speculative. There may be historical or archaeological evidence to support this but the genetic data really do not come close to establishing this. The authors do acknowledge this in the text, though the abstract makes a much stronger claim. And of course, it also remains possible that other baboons in the assemblage came from other places.

    1. Reviewer #2 (Public Review):

      The study by Ciabatti et al examined the mutation issue for self-inactivating rabies (SiR), which was found by other labs. The authors identified the mutations in the rabies genome and showed that this mutation occurred more frequently after multiple passage of production cell lines with suboptimal TEVp expressions. The authors further showed that such mutation did not accumulate in vivo and that SiR-labeled cells remained alive across longitudinal imaging in vivo.

      In this study, the rabies genome is rigorously examined by sequencing many viral particles from independent preparations. The rabies with point mutation in the PEST domain is directly engineered for sequencing and infection test. Overall, the mutation issue is well addressed by the authors and the conclusions are well supported, but some more aspects of discussion and data analysis need to be extended for an easier production of SiR in a condition not that optimal.

      1) The authors stated that one should produce SiR from cDNA in order to avoid the potential mutation in SiR. From a practical point of view, it would be much better to amplify the rabies from a stock virus directly in the production cell lines. Any discussion or exploration on this direction would be appreciated in the field.

      2) 6 passages of production cell lines are not that extensive. In Fig.2C, there was already some level of TEVp activity reduction at 2nd passage. It is not clear to me that how the TEVp activity reduction naturally happens. Is there some room to play around puromycin concentration to maintain high TEVp activity?

    1. Reviewer #2 (Public Review):

      Wang et al. investigate the LGN in the tree shrew as a potential target for artificial vision. They report that (a) animals pre-trained on a visual detection task can generalize from visual to optogenetic detection and (b) optogenetic activation of the LGN results in reliable field potential activity in V1.

      In this revised version of the manuscript, the authors have done a commendable job of addressing the critiques from the previous round of reviews.

      Among the new results, the analysis of V1 LFP entrainment with optogenetic stimulation in the LGN is quite interesting and convincing. However, I found the spiking results in V1 to be underwhelming (which the authors also acknowledge). I find this a little surprising, given the robustness of the LFP results. Was this a matter of finding a better alignment of LGN and V1 sites? Might the authors have found more convincing spiking activity results if they use laminar electrodes in V1 to find monosynaptic connectivity between the LGN injection sites and their targets in V1?

    1. Reviewer #2 (Public Review):

      While the hypothesis that MEMO1 plays a key role in cell iron homeostasis remains to be directly tested, the data presented herein clearly support further delineation of the underlying mechanisms. The key findings in this regard are the facts, as established herein, that: 1) MEMO1 binds ferrous iron (the appropriate valence state for cell iron) along with glutathione (Fig. 5A); 2) the structure of MEMO1 in complex with Fe(II)-GSH reveals the coordination site within the protein for this complex (Fig. 5B/c); 3) oxidative stress and sensitivity to ferroptosis correlate with MEMO1 protein abundance in a consistent fashion (Fig. 4); and 4) while the effect is limited, there are data that indicate a relation between cell iron content and MEMO1 abundance (Fig. 4A/B).

      Experimentally, it is thorough and well-documented and offers a new look at a protein that has been at the edges of iron metabolism (and copper, but I agree with the authors that this is not likely to be the case). This work and its subject will stimulate much further research.

    1. Reviewer #2 (Public Review):

      In this study, the authors validated a positive feedback loop between ZEB2 and ACSL4 in breast cancer, which regulates lipid metabolism to promote metastasis.

      Overall, the study is original, well structured, and easy to read.

    1. Reviewer #2 (Public Review):

      Summary:

      Molecular dynamics (MD) data is deposited in public, non-specialist repositories. This work starts from the premise that these data are a valuable resource as they could be used by other researchers to extract additional insights from these simulations; it could also potentially be used as training data for ML/AI approaches. The problem is that mining these data is difficult because they are not easy to find and work with. The primary goal of the authors was to discover and index these difficult-to-find MD datasets, which they call the "dark matter of the MD universe" (in contrast to data sets held in specialist databases).

      The authors developed a search strategy that avoided the use of ill-defined metadata but instead relied on the knowledge of the restricted set of file formats used in MD simulations as a true marker for the data they were looking for. Detection of MD data marked a data set as relevant with a follow-up indexing strategy of all associated content. This "explore-and-expand" strategy allowed the authors for the first time to provide a realistic census of the MD data in non-specialist repositories.

      As a proof of principle, they analyzed a subset of the data (primarily related to simulations with the popular Gromacs MD package) to summarize the types of simulated systems (primarily biomolecular systems) and commonly used simulation settings.

      Based on their experience they propose best practices for metadata provision to make MD data FAIR (findable, accessible, interoperable, reusable).

      A prototype search engine that works on the indexed datasets is made publicly available. All data and code are made freely available as open source/open data.

      Strengths:

      - The novel search strategy is based on relevant data to identify full datasets instead of relying on metadata and thus is likely to have many true positives and few false positives.

      - The paper provides a first glimpse at the potential hidden treasures of MD simulations and force field parametrizations of molecules.

      - Analysis of parameter settings of MD simulations from how researchers *actually* run simulations can provide valuable feedback to MD code developers for how to document/educate users. This approach is much better than analyzing what authors write in the Methods sections.

      - The authors make a prototype search engine available.

      - The guidelines for FAIR MD data are based on experience gained from trying to make sense of the data.

      Weaknesses:

      - So far the work is a proof-of-concept that focuses on MD data produced by Gromacs (which was prevalent under all indexed and identified packages).

      As discussed in the manuscript, some types of biomolecules are likely underrepresented because different communities have different preferences for force fields/MD codes (for example: carbohydrates with AMBER/GLYCAM using AMBER MD instead of Gromacs).

      - Materials sciences seem to be severely under-represented --- commonly used codes in this area such as LAMMPS are not even detected, and only very few examples could be identified. As it is, the paper primarily provides an insight into the *biomolecular* MD simulation world.

      The authors succeed in providing a first realistic view on what MD data is available in public repositories. In particular, their explore-expand approach has the potential to be customized for all kinds of specialist simulation data, whereby specific artifacts are<br /> used as fiducial markers instead of metadata. The more detailed analysis is limited to Gromacs simulations and primarily biomolecular simulations (even though MD is also widely used in other fields such as the materials sciences). This restricted view may simply be correlated with the user community of Gromacs and hopefully, follow-up studies from this work will shed more light on this shortcoming.

      The study quantified the number of trajectories currently held in structured databases as ~10k vs ~30k in generalist repositories. To go beyond the proof-of-principle analysis it would be interesting to analyze the data in specialist repositories in the same way as the one in the generalist ones, especially as there are now efforts underway to create a database for MD simulations (Grant 'Molecular dynamics simulation for biology and chemistry research' to establish MDDB' DOI 10.3030/101094651). One should note that structured databases do not invalidate the approach pioneered in this work; if anything they are orthogonal to each other and both will likely play an important role in growing the usefulness of MD simulations in the future.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This work investigates how increased temperature affects pollen production and fertility of Arabidopsis thaliana plants grown at selected temperature conditions ranging from 16C to 30C. They report that pollen production and fertility decline with increasing temperature. To identify the cause of reduced pollen and fertility, they resort to living cell imaging of male meiotic cells to identify that the duration of meiosis increases with an increase in temperature. They also show that pollen sterility is associated with the increased presence of micronuclei likely originating from heat stress-induced impaired meiotic chromosome segregation. They correlate abnormal meiosis to weakened centromere caused by meiosis-specific defective loading of the centromere-specific histone H3 variant (CenH3) to the meiotic centromeres. Similar is the case with kinetochore-associated spindle assembly checkpoint(SAC) protein BMF1. Intriguingly, they observe a reverse trend of strong CENH3 presence in the somatic cells of the tapetum in contrast to reduced loading of CENH3 in male meiocytes with increasing temperature. In contrast to CENH3 and BMF1, the SAC protein BMF3 persists for longer periods than the WT control, based on which authors conclude that the heat stress prolongs the duration of SAC at metaphase I, which in turn extends the time of chromosome biorientation during meiosis I. The study provides preliminary insights into the processes that affect plant reproduction with increasing temperatures which may be relevant to develop climate-resilient cultivars.

      Strengths:<br /> The authors have mastered the live cell imaging of male meiocytes which is a technically demanding exercise, which they have successfully employed to examine the time course of meiosis in Arabidopsis thaliana plants exposed to different temperature conditions. In continuation, they also monitor the loading dynamics and resident time of fluorescently tagged centromere/kinetochore proteins and spindle assembly checkpoint proteins to precisely measure the time duration of respective proteins to study their precise dynamics and function in male meiosis.

      Weaknesses:<br /> Here the authors use only one representative centromere protein CENH3, one kinetochore-associated SAC protein BMF1, and the SAC protein BMF3 to conclude that heat stress impairs centromere function and prolongs SAC with increased temperatures. Centromere and its associated protein complex the kinetochores and the SAC contain a multitude of proteins, some of which are well characterized in Arabidopsis thaliana. Hence the authors could have used additional such tagged proteins to further strengthen their claim. Though the results presented here are interesting and solid, the study lacks a deeper mechanistic understanding of what causes the defective loading of CenH3 to the centromeres, and why the SAC protein BMF3 persists only at meiotic centromeres to prolong the spindle assembly checkpoint. Also, this observation should be interpreted in light of the fact that SAC is not that robust in plants as several null mutants of plant SAC components are known to grow as healthy as wild-type plants at normal growth conditions without any vegetative and reproductive defects. One of the immediate responses to heat stress is the production of heat shock proteins(Hsps), which act as molecular chaperones to safeguard the proteome. It will be interesting to see if the expression levels of known HsPs can be correlated with their role in stabilizing the structure of SAC proteins like BMF1 to prolong its presence at the meiotic kinetochores.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The manuscript highlights a mechanistic insight into meiotic initiation in budding yeast. In this study, the authors addressed a genetic link between mitotic cell cycle regulator SBF (the Swi4-Swi6 complex) and a meiosis inducing regulator Ime1 in the context of meiotic initiation. The authors' comprehensive analyses with cytology, imaging, RNA-seq using mutant strains lead the authors to conclude that Swi4 levels regulates Ime1-Ume6 interaction to activate expression of early meiosis genes for meiotic initiation. The major findings in this paper are that (1) the higher level of Swi4, a subunit of SBF transcription factor for mitotic cell cycle regulation, is the limiting factor for mitosis-to-meiosis transition; (2) G1 cyclins (Cln1, Cln2), that are expressed under SBF, inhibit Ime1-Ume6 interaction under overexpression of SWI4, which consequently leads to downregulation of early meiosis genes; (3) expression of SWI4 is regulated by LUTI-based transcription in the SWI4 locus that impedes expression of canonical SWI4 transcripts; (4) expression of SWI4 LUTI is likely negatively regulated by Ime1; (5) Action of Swi4 is negatively regulated by Whi5 (homologous to Rb)-mediated inhibition of SBF, which is required for meiotic initiation. Thus, the authors proposed that meiotic initiation is regulated under the balance of mitotic cell cycle regulator SBF and meiosis-specific transcription factor Ime1.

      Strengths:<br /> The most significant implication in their paper is that meiotic initiation is regulated under the balance of mitotic cell cycle regulator and meiosis-specific transcription factor. This finding will provide a mechanistic insight in initiation of meiosis not only into the budding yeast also into mammals. The manuscript is overall well written, logically presented and raises several insights into meiotic initiation in budding yeast. Therefore, the manuscript should be open for the field. I would like to raise the following concerns, though they are not mandatory to address. However, it would strengthen their claims if the authors could technically address and revise the manuscript by putting more comprehensive discussion.

      Weaknesses:<br /> The authors showed that increased expression of the SBF targets, and reciprocal decrease in expression of meiotic genes upon SWI4 overexpression at 2 h in SPO (Figure 2F). However, IME1 was not found as a DEG in Supplemental Table 1. Meanwhile, IME1 transcript level was decreased at 2 h SPO condition in pATG8-CLN2 cells in Fig S4C.

      Now this reviewer still wonders with confusion whether expression of IME1 transcripts per se is directly or in directly suppressed under SBF-activated gene expression program at 2 h SPO in pATG8-SWI4 and pATG8-CLN2 cells. This reviewer wonders how Fig S4C data reconciles with the model summarized in Fig 6F.

      One interpretation could be that persistent overexpression of G1 cyclin caused active mitotic cell cycle, and consequently delayed exit from mitotic cell cycle, which may have given rise to an apparent reduction of cell population that was expressing IME1. For readers to better understand, it would be better to explain comprehensively this issue in the main text.

      The % of cells with nuclear Ime1 was much reduced in pATG8-CLN2 cells (Fig 2B) than in pATG8-SWI4 cells (Fig 4C). Is the Ime1 protein level comparable or different between pATG8-CLN2 strain and pATG8-SWI4 strain? Since it is difficult to compare the quantifications of Ime1 levels in Fig S1D and Fig S4B, it would be better to comparably show the Ime1 protein levels in pATG8-CLN2 and pATG8-SWI4 strains.<br /> Further, it is uncertain how pATG8-CLN2 cells mimics the phenotype of pATG8-SWI4 cells in terms of meiotic entry. It would be nice if the authors could show RNA-seq of pATG8-CLN2/WT and/or quantification of the % of cells that enter meiosis in pATG8-CLN2.

      The authors stated that reduced Ime1-Ume6 interaction is a primary cause of meiotic entry defect by CLN2 overexpression (Line 320-322, Fig 4J-L). This data is convincing. However, the authors also showed that GFP-Ime1 protein level was decreased compared to WT in pATG8-CLN2 cells by WB (Fig S4A). Further, GFP-Ime1 signals were overall undetectable through nuclei and cytosol in pATG8-CLN2 cells (Fig 4B), and accordingly cells with nuclear Ime1 were reduced (Fig 4C). Although the authors raised a possibility that the meiotic entry defect in the pATG8-CLN2 mutant arises from downregulation of IME1 expression (Line 282-283), causal relationship between meiotic entry defect and CLN2 overexpression is still not clear. Is the Ime1 protein level reduced in the pATG8-CLN2;UME6-⍺GFP strain compared to WT? It would be better to comparably show the Ime1 protein levels in the pATG8-CLN2 strain and the pATG8-CLN2;UME6-⍺GFP strain by WB. Also, it would be nice if the authors could show quantification of the % of cells that enter meiosis in the pATG8-CLN2;UME6-⍺GFP strain to see how and whether artificial tethering of Ime1 to Ume6 rescued normal meiosis program rather than simply showing % sporulation in Fig4A.

      The authors showed Ume6 binding at the SWI4LUTI promoter (Figure 5K). However, since Ume6 forms a repressive form with Rpd3 and Sin3a and binds to target genes independently of Ime1, Ume6 binding at the SWI4LUTI promoter bind does not necessarily represent Ime1-Ume6 binding there. Instead, it would be better to show Ime1 ChIP-seq at the SWI4LUTI promoter.

      The authors showed ∆LUTI mutant and WHI5-AA mutant did not significantly change the expression of SBF targets nor early meiotic genes relative to wildtype (Figure 6A, C). Accordingly, they concluded that LUTI- or Whi5-based repression of SBF alone was not sufficient to cause a delay in meiotic entry (Line451-452), and perturbation of both pathways led to a significant delay in meiotic entry (Figure 6E). This reviewer wonders whether Ime1 expression level and nuclear localization of Ime1 was normal in ∆LUTI mutant and WHI5-AA mutant.

    1. Reviewer #2 (Public Review):

      Nagy et al investigated the role of volume increase and swelling in neutrophils in response to the chemoattractant. Authors show that following chemoattractant response cells lose their volume slightly owing to the cell spreading phase and then have a relatively rapid increase in the cell volume that is concomitant with cell migration. The authors performed an impressive genome-wide CRISPR screen and buoyant density assay to identify the regulators of neutrophil swelling. This assay showed that stimulating cells with chemoattractant fMLP led to an increase in the cell volume that was abrogated with the FPR1 receptor knockout. The screen revealed a cascade that could potentially be involved in cell swelling including NHE1 (sodium-proton antiporter) and PI3K. NHE1 and PI3K are required for chemoattractant-induced swelling in human primary neutrophils. Authors also suggest slightly different functions of NHE1 and PI3K activity where PI3K is also required to maintain chemoattractant-induced cell shape changes. The authors convincingly show that chemoattractant-induced cell swelling is linked to cell migration and NHE1 is required for swelling at the later stages of swelling since the cells at the early point work on low-volume and low-velocity regime. Interestingly, the authors also show that lack of swelling in NHE1-inhibited cells could be rescued by mild hypo-osmotic swelling strengthening the argument that water influx followed chemoattractant stimulation is important for potentiation for migration.

      The conclusions of this paper are mostly well supported by data and are pretty convincing, but some aspects of image acquisition and data analysis need to be clarified and extended.

      Weaknesses<br /> 1) It would really help if the authors could add the missing graph for the footprint area when cells are treated with Latranculin. Graph S1F for volume changes with Lat treatment should be compared with DMSO-treated controls.<br /> 2) The authors show inhibition of NHE1 blocked cell swelling using Coulter counter, a similar experiment should be done with PI3K inhibitions especially since they see PI3K inhibition impact chemoattractant-induced cell shape change.<br /> 3) It would be more convincing visually if the authors could also include the movie of cell spreading (footprint) and then mobility with PI3K inhibition.<br /> 4) It is not clear how cell spreading and later volume increase are linked to overall mobility of neutrophils. Are authors suggesting that cell spreading is not required for cell mobility in neutrophils?<br /> 5) Volume fluctuations associated with motility were impacted by NHE1 inhibition at the baselines, what about PI3K inhibitions? Does that impact the actual fluctuations?<br /> 6) It would really help if the authors compared similar analyses and drew conclusions from that, for example, it is unclear what the authors mean by they found no change in the angular persistence of WT and NHE1 inhibited cells which is in contrast to PI3K inhibition since they do not really have an analysis for angular persistence in PI3K inhibited cells. (S4A and S4B).

    1. Reviewer #2 (Public Review):

      The authors present a pipeline for generating strain-specific genome-scale metabolic models for bacteria using Klebsiella spp. as the demonstrative data. This paper claims to provide a high-throughput tool for generating strain-specific models for bacteria. However, in reality, the tool requires a reference pan-genome-based complete model to generate the strain-specific model of the species of interest, which in this study is Klebsiella pneumoniae. This requirement renders the tool redundant for high-throughput purposes since the process of building or generating the pan-genome reference model is performed separately. Additionally, the quality of the newly built strain-specific model will depend on the reference model used. Therefore, this tool, on its own, can only work specifically with the available pan-genome model of reference, which in this case is only applicable to Klebsiella pneumoniae. Its effectiveness with other bacteria has not been proven. I would suggest that the authors either reframe the performance and results to be applicable only to Klebsiella or consider adding more reference pan-genome models for the study.

    1. Reviewer #2 (Public Review):

      In this manuscript, Funabiki and colleagues investigated the co-evolution of DNA methylation and nucleosome remolding in eukaryotes. This study is motivated by several observations: (1) despite being ancestrally derived, many eukaryotes lost DNA methylation and/or DNA methyltransferases; (2) over many genomic loci, the establishment and maintenance of DNA methylation relies on a conserved nucleosome remodeling complex composed of CDCA7 and HELLS; (3) it remains unknown if/how this functional link influenced the evolution of DNA methylation. The authors hypothesize that if CDCA7-HELLS function was required for DNA methylation in the last eukaryote common ancestor, this should be accompanied by signatures of co-evolution during eukaryote radiation.

      To test this hypothesis, they first set out to investigate the presence/absence of putative functional orthologs of CDCA7, HELLS and DNMTs across major eukaryotic clades. They succeed in identifying homologs of these genes in all clades spanning 180 species. To annotate putative functional orthologs, they use similarity over key functional domains and residues - such as ICF related mutations for CDCA7 and SNF2 domains for HELLS - as well as maximum likelihood phylogenetic analyses. Using established eukaryote phylogenies, the authors conclude that the CDCA7-HELLS-DNMT axis arose in the last common ancestor to all eukaryotes. Importantly, they found recurrent loss events of CDCA7-HELLS-DNMT in at least 40 eukaryotic species, most of them lacking DNA methylation.

      Having identified these factors, they successfully identify signatures of co-evolution between DNMTs, CDCA7 and HELLS using CoPAP analysis - a probabilistic model inferring the likelihood of interactions between genes given a set of presence/absence patterns. As a control, such interactions are not detected with other remodelers or chromatin modifying pathways also found across eukaryotes. Expanding on this analysis, the authors found that CDCA7 was more likely to be lost in species without DNA methylation.

      In conclusion, the authors suggest that the CDCA7-HELLS-DNMT axis is ancestral in eukaryotes and raise the hypothesis that CDCA7 becomes quickly dispensable upon the loss of DNA methylation and/or that CDCA7 might be the first step toward the switch from DNA methylation-based genome regulation to other modes.

      The data and analyses reported are significant and solid. Overall, this work is a conceptual advance in our understanding of the evolutionary coupling between nucleosome remolding and DNA methylation. It also provides a useful resource to study the early origins of DNA methylation related molecular process. Finally, it brings forward the interesting hypothesis that since eukaryotes are faced with the challenge of performing DNA methylation in the context of nucleosome packed DNA, loosing factors such as CDCA7-HELLS likely led to recurrent innovations in chromatin-based genome regulation.

      Strengths:<br /> - The hypothesis linking nucleosome remodeling and the evolution of DNA methylation.<br /> - Deep mapping of DNA methylation related process in eukaryotes.<br /> - Identification and evolutionary trajectories of novel homologs/orthologs of CDCA7.<br /> - Identification of CDCA7-HELLS-DNMT co-evolution across eukaryotes.

    2. Reviewer #2 (Public Review):

      In this manuscript, Funabiki and colleagues investigated the co-evolution of DNA methylation and nucleosome remolding in eukaryotes. This study is motivated by several observations: (1) despite being ancestrally derived, many eukaryotes lost DNA methylation and/or DNA methyltransferases; (2) over many genomic loci, the establishment and maintenance of DNA methylation relies on a conserved nucleosome remodeling complex composed of CDCA7 and HELLS; (3) it remains unknown if/how this functional link influenced the evolution of DNA methylation. The authors hypothesize that if CDCA7-HELLS function was required for DNA methylation in the last eukaryote common ancestor, this should be accompanied by signatures of co-evolution during eukaryote radiation.

      To test this hypothesis, they first set out to investigate the presence/absence of putative functional orthologs of CDCA7, HELLS and DNMTs across major eukaryotic clades. They succeed in identifying homologs of these genes in all clades spanning 180 species. To annotate putative functional orthologs, they use similarity over key functional domains and residues - such as ICF related mutations for CDCA7 and SNF2 domains for HELLS - as well as maximum likelihood phylogenetic analyses. Using established eukaryote phylogenies, the authors conclude that the CDCA7-HELLS-DNMT axis arose in the last common ancestor to all eukaryotes. Importantly, they found recurrent loss events of CDCA7-HELLS-DNMT in at least 40 eukaryotic species, most of them lacking DNA methylation.

      Having identified these factors, they successfully identify signatures of co-evolution between DNMTs, CDCA7 and HELLS using CoPAP analysis - a probabilistic model inferring the likelihood of interactions between genes given a set of presence/absence patterns. As a control, such interactions are not detected with other remodelers or chromatin modifying pathways also found across eukaryotes. Expanding on this analysis, the authors found that CDCA7 was more likely to be lost in species without DNA methylation.

      In conclusion, the authors suggest that the CDCA7-HELLS-DNMT axis is ancestral in eukaryotes and raise the hypothesis that CDCA7 becomes quickly dispensable upon the loss of DNA methylation and/or that CDCA7 might be the first step toward the switch from DNA methylation-based genome regulation to other modes.

      The data and analyses reported are significant and solid. Overall, this work is a conceptual advance in our understanding of the evolutionary coupling between nucleosome remolding and DNA methylation. It also provides a useful resource to study the early origins of DNA methylation related molecular process. Finally, it brings forward the interesting hypothesis that since eukaryotes are faced with the challenge of performing DNA methylation in the context of nucleosome packed DNA, loosing factors such as CDCA7-HELLS likely led to recurrent innovations in chromatin-based genome regulation.

      Strengths:<br /> - The hypothesis linking nucleosome remodeling and the evolution of DNA methylation.<br /> - Deep mapping of DNA methylation related process in eukaryotes.<br /> - Identification and evolutionary trajectories of novel homologs/orthologs of CDCA7.<br /> - Identification of CDCA7-HELLS-DNMT co-evolution across eukaryotes.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This work follows previous work from the group where they have demonstrated the role of TASK1 in the regulation of glucose-stimulated insulin secretion. Moreover, a recent study links a mutation in KCNK16, the gene encoding TALK-1 channels to MODY. Here the authors have constructed a mouse model with the specific mutation (TALK-1 L114P mutation) and investigated the phenotype. They have to perform a couple of breeding tricks to find a model that is lethal in adult which might complicate the conclusions, however, the phenotype of the heterozygote model used has a MODY-like phenotype. The study is convincing and solid.

      Strengths:<br /> 1) The work is a natural follow-up from previous studies from the groups.

      2) The authors present convincing and solid data that in the long perspective will help patients with these mutations.

      3) Both in vivo and in vitro data are presented to give the full picture of the phenotype.

      4) Data from both female and male mice are presented.

      Weaknesses:<br /> 1) The authors perform an RNA-sequencing showing that the cAMP amplifying pathway is upregulated. A weakness is that this is not further followed up. The remaining questions include; Is this also true in humans with this mutation? Would treatment with incretins improve glucose-stimulated insulin secretion and and lower blood glucose?<br /> 2) The authors avoid further investigating what it means that the glucagon area and secretion are increased in the model.<br /> 3) The performance of measurements in both male and female mice is praiseworthy. However, despite differences in the response, the authors do not investigate the potential reason for this. Are hormonal differences of importance?

    1. Reviewer #2 (Public Review):

      Summary:

      The paper presents a novel approach to expand iPSC-derived pdx1+/nkx6.1+ pancreas progenitors, making them potentially suitable for GMP-compatible protocols. This advancement represents a significant breakthrough for diabetes cell replacement therapies, as one of the current bottlenecks is the inability to expand PP without compromising their differentiation potential. The study employs a robust dataset and state-of-the-art methodology, unveiling crucial signaling pathways (eg TGF, Notch...) responsible for sustaining pancreas progenitors while preserving their differentiation potential in vitro.

      Strengths:

      This paper has strong data, guided omics technology, clear aims, applicability to current protocols, and beneficial implications for diabetes research. The discussion on challenges adds depth to the study and encourages future research to build upon these important findings.

      Weaknesses:

      The paper does have some weaknesses that could be addressed to improve its overall clarity and impact. The writing style could benefit from simplification, as certain sections are explained in a convoluted manner and difficult to follow, in some instances, redundancy is evident. Furthermore, the legends accompanying figures should be self-explanatory, ensuring that readers can easily understand the presented data without the need to be checking along the paper for information.

      The culture conditions employed in the study might benefit from more systematic organization and documentation, making them easier to follow.

      Another important aspect is the functionality of the expanded cells after differentiation. While the study provides valuable insights into the expansion of pancreas progenitors in vitro and does the basic tests to measure their functionality after differentiation the paper could be strengthened by exploring the behavior and efficacy of these cells deeper, and in an in vivo setting.

      Quantifications for immunofluorescence (IF) data should be displayed.

      Some claims made in the paper may come across as somewhat speculative.

      Additionally, while the paper discusses the potential adaptability of the method to GMP-compatible protocols, there is limited elaboration on how this transition would occur practically or any discussion of the challenges it might entail.

    1. Reviewer #2 (Public Review):

      Summary: Walker et al have proposed that the tumor suppressor TMEM127 converges with RET activation to drive adrenal phenochromocytoma. RET is a common oncogene both in familial and sporadic forms of this cancer, and TMEM127 has also been observed as a loss of function mutation in sporadic disease. The authors hypothesize that loss of the TMEM127 might signal stabilization of RET on the cell surface, mimicking an activating mutation. Through a nice set of experiments, they show that TMEM127 loss impairs endosome function and promotes RET surface accumulation. This expression was resistant to GDNF, suggesting that recycling via endosome recirculation was impaired such that the half-life of RET on the cell surface was extended. RET interaction with clathrin-coated pits was also disrupted, as the CCPs themselves were significantly smaller, and plasma membrane organization was affected by the impaired endosome recycling. Notably, a number of proteins were found to be accumulating on the cell surface via the purported mechanism, EGFR, TFR1, N cadherin, integrin beta 3. The authors applied a RET inhibitor to cells, showing decreased cellular proliferation.

      Strengths: In summary, this is an interesting finding, that is preliminary in nature and is incompletely validated currently. It is certainly worth further investigation as a central feature linking TMEM127 mutations and pheochromocytoma through a common pathway of RET activation by fixing this factor in an active state on the cell surface.

      Weaknesses: Although this is a provocative finding, and the authors test the interaction in a number of ways, there are several factors that limit the enthusiasm for this work as currently presented. The work is limited to one isogenic cell line with limited validation.

    1. Reviewer #2 (Public Review):

      The authors tried to diagnose cancers and pinpoint tissues of origin using cfDNA. To achieve this goal, they developed a framework to assess methylation, CNA, and other genomic features. They established discovery and validation cohorts for systematic assessment and successfully achieved robust prediction power.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study is quite thorough, tackling this NO-dependent UV avoidance circuit with both breadth and depth. There are several novel discoveries throughout, but the whole package represents perhaps even more than the sum of these parts.

      Strengths:<br /> The presentation of the work is compelling. The introduction sets up the question and the state of the field very nicely. The discovery of the non-canonical NO receptor pathway in the ciliary photoreceptors is fascinating and will likely open up new avenues for future research into NO-pathways in different species. The use of genetic and pharmacological manipulations of circuit components was well thought-out. The authors applied different experimental techniques expertly throughout the study so that they could develop a comprehensive view from the molecular to the behavioral levels.

      Weaknesses:<br /> While I appreciate the intent of bringing together a large set of measurements from connectomics and calcium imaging in the framework of a model, the model seemed rather poorly constrained. How many parameters are in the model shown in Figure 6A? How many of them are well constrained by experimental measurements? The authors also don't perform sensitivity analysis on the parameters of the model. And ultimately, the conclusion over the model in Figure 7 is somewhat trivial within the unitless construction: larger amplitude and longer duration stimuli lead to increased activation of the downstream neuron thought to lead to the downward swim behavior. I could imagine that a large family of models would arrive at this same result, and without units, there is no way to really test it with new behavioral experiments.

    1. Reviewer #2 (Public Review):

      Kleinman and colleagues conducted an analysis of two datasets, one recorded from DLPFC in one monkey and the other from PMD in two monkeys. They also performed similar analyses on trained RNNs with various architectures.

      The study revealed four main findings. (1) All task variables (color coherence, target configuration, and choice direction) were found to be encoded in DLPFC. (2) PMD, an area downstream of PFC, only encoded choice direction. (3) These empirical findings align with the celebrated 'information bottleneck principle,' which suggests that FF networks progressively filter out task-irrelevant information. (4) Moreover, similar results were observed in RNNs with three modules.

      While the analyses supporting results 1 and 2 were convincing and robust, I have some concerns and recommendations regarding findings 3 and 4, which I will elaborate on below. It is important to note that findings 2 and 4 had already been reported in a previous publication by the same authors (ref. 43).

      Major recommendation/comments:<br /> The interpretation of the empirical findings regarding the communication subspace in relation to the information bottleneck theory is very interesting and novel. However, it may be a stretch to apply this interpretation directly to PFC-PMd, as was done with early vs. late areas of a FF neural network.

      In the RNN simulations, the main finding indicates that a network with three or more modules lacks information about the stimulus in the third or subsequent modules. The authors draw a direct analogy between monkey PFC and PMd and Modules 1 and 3 of the RNNs, respectively. However, considering the model's architecture, it seems more appropriate to map Area 1 to regions upstream of PFC, such as the visual cortex, since Area 1 receives visual stimuli. Moreover, both PFC and PMd are deep within the brain hierarchy, suggesting a more natural mapping to later areas. This contradicts the CCA analysis in Figure 3e. It is recommended to either remap the areas or provide further support for the current mapping choice.

    1. Reviewer #2 (Public Review):

      Summary:<br /> One often wishes to combine activation of a neural population via red light with simultaneous modulation of a different population via blue light, or simultaneous imaging of a blue-excited fluorescent reporter. The problem is that all red-shifted opsins have an action spectrum with a long blue tail, leading to spurious opsin activation by blue light.

      This valuable paper presents a clever solution to this problem, by pairing an engineered blue-shifted inhibitory chloride-conducting opsin with a red-shifted excitatory opsin. The combined effect is excitation by red light and shunting inhibition by blue light. The paper is very thorough, with convincing spectroscopic and patch clamp characterization of the tools, and tests in brain slices and in vivo. This tool is likely to be useful in the neuroscience community.

      Strengths:<br /> The methods are solid, including the complete characterization of each tool separately, as well as the combination in vivo. The array of testing gives a strong degree of confidence that this tool will work as expected.

      Weaknesses:<br /> There are two discussion points and one experimental point which would make the paper stronger.

      1) In the Introduction or Discussion, the authors could better motivate the need for a red-shifted actuator that lacks blue crosstalk, by giving some specific examples of how the tool could be productively used, e.g. pairing with another blue-shifted excitatory opsin in a different population, or pairing with a GFP-based fluorescent indicator, e.g. GCaMP. The motivation for the current tool is not obvious to non-experts.

      2) Simultaneous excitation and inhibition are not the same as non-excitation. The authors mentioned shunting briefly. Another possible issue is changes in osmotic balance. Activation of a Na+ channel and a Cl- channel will lead to net import of NaCl into the cell, possibly changing osmotic pressure. Please discuss.

      3) The authors showed that in ZipT-IvfChr, orange light drives excitation and blue light does not. But what about simultaneous blue and orange light? Can the blue light overwhelm the effect of the orange light? Since the stated goal is to open the blue part of the spectrum for other applications, one is now worried about "negative" crosstalk. Please discuss and, ideally, characterize this phenomenon.<br /> 3.1) Does the use of the new tool require careful balancing of the expression levels of the ZipT and the IvfChr? Does it require careful balancing of blue and orange light intensities?<br /> 3.2) Also, many opsins show complex and nonlinear responses to dual-wavelength illumination, so each component should be characterized individually under simultaneous blue + orange light.<br /> 3.3) I was expecting to see photocurrents at different holding potentials as a function of illumination wavelength for the co-expressed construct (i.e. to see at what wavelength it switches from being excitatory to inhibitory); and also to see I-V curves of the photocurrent at blue and orange wavelengths for the co-expressed constructs (i.e. to see the reversal potential under blue excitation). Overall, the patch clamp and spectroscopic characterization of the individual constructs was stronger than that of the combined constructs.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Silva et al. describe an experimental study conducted on cerebellar parallel fiber-to-molecular interneuron synapses to investigate the size of the readily releasable pool (RRP) of synaptic vesicles (SVs) per docking site in response to trains of action potentials. The study aims to determine whether there are multiple binding sites for SVs at each docking site, which could lead to a higher RRP size than previously thought.

      The researchers used this glutamatergic synapse to conduct their experiments. They employed various techniques and manipulations to enhance release probability, docking site occupancy, and synaptic depression. By counting the number of released SVs in response to action potential trains and normalizing the results based on the number of docking sites, they estimated the RRP size per docking site.

      The key findings and observations in the manuscript are as follows:

      Docking Site Occupancy and Release Probability Enhancement: The researchers used 4-amidopyridine (4-AP) and post-tetanic potentiation (PTP) protocols to enhance the release probability of docked SVs and the occupancy of docking sites, respectively.

      Synchronous and Asynchronous Release: Synchronous release refers to SVs released in response to individual action potentials, while asynchronous release involves SVs released after the initial release response due to calcium elevation. The study observed changes in the balance between synchronous and asynchronous release under different conditions, revealing the degree of filling of the RRP.

      Modeling of Release Dynamics: The researchers employed a modeling approach based on the "replacement site/docking site" (RS/DS) model, where SVs bind to a replacement site before moving to a docking site and eventually undergoing release. The model was adjusted to experimental conditions to estimate parameters like docking site occupancy and release probabilities.

      Comparison of Different Models: The study compared the RS/DS model with an alternative model known as the "loosely docked/tightly docked" (LS/TS) model. The LS/TS model assumes that a docking site can only accommodate one SV at a time, while the RS/DS model considers the possibility of accommodating multiple SVs.

      Maximum RRP Size: Through a combination of experimental results and model simulations, the study revealed that the maximum RRP size per docking site reached close to two SVs under certain conditions, supporting the idea that each docking site can accommodate multiple SVs.

      Strengths:<br /> The study is rigorously conducted and takes into consideration the previous work on RRP size and SV docking site estimation. The study addresses a long-standing question in synaptic physiology.

      Weaknesses:<br /> It remains unclear how generalizable the findings are to other types of synapses.

    1. Reviewer #2 (Public Review):

      Pak et al. report on a study using a computational method to assess differences in the relative proportion of six canonical brain cell types, across eleven neurodegenerative classes (defined as both clinical syndromes (e.g. FTD, PD), groups of neurogenerative diseases (e.g. 4-repeat tauopathies) or distinct neuropathological entities (e.g. FTLD-TDP type C), as they relate to a standard map of class-dependent volume loss. The study uses innovative methods and is commendable in its goal to highlight the contribution of non-neuronal cell types to the pathobiology of neurodegeneration. The findings of the study are in part contradicting expected results based on extensive literature on the biology of these diseases. The authors based their methodology on the use of a deconvolutional cell classifier; however, do not extensively recognize that their data on gene expression are based on normal brain levels rather than on diseased ones. Also, while predicted levels are uniquely based on patterns of brain atrophy, it is not possible to know whether this strategy is generalizable to all diseases (for instance, it is known that pure DLB, PD and ALS are not associated with extensive brain atrophy), or even adequately comparable between subtypes of diseases within the same class (e.g., different forms of FTLD). The authors do not acknowledge that only data based on true neuropathological assessment may prove whether their findings are true. Subject characteristics, numbers, and diagnostic criteria are hard to assess and only described in the methods section. This format prevents the reader from assessing data robustness while going through the results, especially when fundamental biological bases of nomenclature and differences between clinical syndromes and pathological entities are omitted or uncharacteristically provided.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study links rare human loss of function mutations in the zinc transporter family member SLC39A5 to increased circulating and hepatic concentrations of this trace element. Beneficial metabolic changes were observed in a corresponding convincing mouse model relevant to the development of NASH.

      Strengths:<br /> Authors combine human exome sequencing data, meta-analysis of four large European cohorts, and a patient recall approach to link the rare loss of function variants of SLC39A5 to the phenotype and protection from T2DM.

      Using a SLC39A5-null mouse model challenged either by cross-breeding with Lepr-/- mice or diet-induced obesity they unravel the metabolic impact of elevated circulating and hepatic zinc concentration with respect to T2DM, glucose homeostasis, hepatic steatosis, and NASH development. Some mechanistic aspects and a remarkable sex difference in the outcome are identified from mouse ex vivo readouts and supported by in vitro hepatocyte cellular studies. Authors present evidence that increased hepatic zinc concentrations inhibit zinc-regulated phosphatases resulting in activation of AMPK and AKT signalling with consequences for lipid and glucose metabolism and insulin sensitivity.

      Weaknesses:<br /> The reasons for the observed sex differences in the metabolic consequences of SLC39A5 inactivation in the mouse models remain unclear. While heterozygous rare SLC39A5 variants show distinct phenotypes only SLC39A5-null mice and no heterozygous mice are studied. The role of SLC39A5 in pancreatic islets and on insulin secretion remains unclear because authors do not address data published recently that claim a relevant role of SLC39A5 in b-cell function and glucose tolerance.

    1. Reviewer #2 (Public Review):

      Medwig-Kinney et al. explore the role of the transcription factor NHR-67 in distinguishing between AC and VU cell identity in the C. elegans gonad. NHR-67 is expressed at high levels in AC cells where it induces G1 arrest, a requirement for the AC fate invasion program (Matus et al., 2015). NHR-67 is also present at low levels in the non-invasive VU cells and, in this new study, the authors suggest a role for this residual NHR-67 in maintaining VU cell fate. What this new role entails, however, is not clear.

      The authors present two models: 1) That NHR-67 switches from a transcriptional activator in ACs to a transcriptional repressor in VUs by virtue of recruiting translational repressors, or 2) that these interactions sequester NHR-67 away from its transcription targets in VU cells. Neither model is fully supported by the data, leaving a paper with extensive data but no single compelling conclusions, and leaving open the question of what is the function, if any, of NHR-67 condensates in VU cells?

      While the authors report on interesting observations, in particular the co-localization of NHR-67 with UNC-37/Groucho and POP-1 in nuclear puncta, the functional significance of these observations remains unclear. The authors have not demonstrated that the "repressive condensates" are functional and play a role in the suppression of AC fate in VU cells as claimed. The colocalization data suggest that NHR-67 interacts with repressors, but additional experiments are needed to demonstrate that these interactions are specific to VUs, impact VU fate, and sequester NHR-67 from its targets or transform NHR-67 into a transcriptional repressor.

      [Editor's note: we feel that the current state of the data with respect to this question is best captured in the response by the authors to the original concerns expressed by reviewer 2, which we include in abbreviated form here]

      1) The authors report that NHR-67 forms "repressive condensates" (aka. puncta) in the nuclei of VU cells and imply that these condensates prevent VU cells from becoming ACs. However, there are also examples of AC cells presented that have NHR-67 puncta (these are less obvious simply due to the higher levels of NHR-67 in ACs). Similarly, there also are UNC-37 and LSY-22 also puncta in ACs. The presence of NHR-67 puncta in the AC seems to directly contradict the author's assumption that the puncta repress the AC fate.

      RESPONSE: The puncta formed by NHR-67 in the AC are different in appearance than those observed in the VU cells and furthermore do not exhibit strong colocalization with that of UNC-37 or LSY-22. The Manders' overlap coefficient between NHR-67 and UNC-37 is 0.181 in the AC, whereas it is 0.686 in the VU cells. Likewise, the Manders' overlap coefficient between NHR-67 and LSY-22 is 0.189 in the AC compared to 0.741 in the VU cells. We speculate that the areas of NHR-67 subnuclear enrichment in the AC may represent concentration around transcriptional targets, but testing this would require knowledge of direct targets of NHR-67.

      2) While a pool of NHR-67 localizes to "repressive condensates", it appears that a substantial portion of NHR-67 also exists diffusively in the nucleoplasm. This would appear to contradict a "sequestration model" since, for such a model to work, a majority of NHR-67 should be in puncta? What proportion of NHR-67 is in puncta? Is the concentration of NHR-67 in the nucleoplasm lower in VUs compared to ACs and does this depend on the puncta?

      RESPONSE: The proportion of NHR-67 localizing to puncta versus the nucleoplasm is dynamic, as these puncta form and dissolve over the course of the cell cycle. However, we estimate that approximately 25-40% of NHR-67 protein resides in puncta based on segmentation and quantification of fluorescent intensity. We also measured NHR-67 concentration in the nucleoplasm of VU cells and found that it is only 28% of what is observed in ACs (n = 10). We also disagree with the notion that the majority of NHR-67 protein should be located in puncta to support the sequestration model. As one example, previously published work examining phase separation of endogenous YAP shows that it is present in the nucleoplasm in addition to puncta (Cai et al., 2019, doi: 10.1038/s41556-019-0433-z). In our system, it is possible that the combination of transcriptional downregulation and partial sequestration away from DNA is sufficient to disrupt the normal activity of NHR-67.

      3) The authors do not report whether NHR-67, UNC-37, LSY-22, or POP-1 localization to puncta is interdependent, as implied by their model.

      RESPONSE: We based our model, shown in Fig. 7E, on known or predicted protein-protein interactions, which we confirmed through yeast two-hybrid analyses (Fig. 7D; Fig. 7-figure supplement 1). It is difficult to test whether localization of these proteins to puncta is interdependent, as a perturbation of UNC-37, LSY-22, and POP-1 result in ectopic ACs. Trying to determine if loss of puncta results in VU-to-AC transdifferentiation or vice versa becomes a chicken-egg argument. It is also possible that UNC-37 and LSY-22 are at least partially redundant in this context.

      4) The evidence that the "repressor condensates" suppress AC fate in VUs is presented in Fig. 4D where the authors deplete the presumed repressor LSY-22. First, the authors do not examine whether NHR-67 forms puncta under these conditions. Second, the authors rely on a single marker (cdh-3p::mCherry::moeABD) to score AC fate: this marker shows weak expression in cells flanking one bright cell (presumably the AC) which the authors interpret as a VU AC transformation. The authors, however, do not identify the cells that express the marker by lineage analyses and dismiss the possibility that the marker-positive cells could arise from the division of an AC-committed cell. Finally, the authors did not test whether marker expression was dependent on NHR-67, as predicted by the model shown in Fig. 7.

      RESPONSE: For the auxin-inducible degron experiments, strains contained labeled AID-tagged proteins, a labeled TIR1 transgene, and a labeled AC marker. Thus, we were limited by the number of fluorescent channels we could covisualize and therefore could not also visualize NHR-67 (to assess for puncta formation) or another AC marker (such as LAG-2). We could have generated an AID-tagged LSY-22 strain without a fluorescent protein, but then we would not be able to quantify its depletion, which this reviewer points out is important to measure. We did visualize NHR-67::GFP expression following RNAi-induced knockdown of POP-1 and observed consistent loss of puncta in ectopic ACs. However, it is unclear whether cell fate change causes loss of puncta or vice-versa.

      5) Interaction between NHR-67 and UNC-37 is shown using Y2H, but not verified in vivo. Furthermore, the functional significance of the NHR-67/UNC-37 interaction is not tested.

      We attempted to remove the intrinsically disordered region found at the C-terminus of the endogenous nhr-67 locus, using CRISPR/Cas9, as this would both confirm the NHR-67/UNC-37 interaction in vivo and allow us to determine the functional significance of this interaction. However, we were unable to recover a viable line after several attempts, suggesting that this region of the protein is vital.

      6) Throughout the manuscript, the authors do not use lineage analysis to confirm fate transformation as is the standard in the field. There are 4 multipotential gonadal cells with the potential to differentiate into VUs or ACs. Which ones contribute to the extra ACs in the different genetic backgrounds examined was not determined, which complicates interpretation. The authors should consider and test the following possibilities: disruption of NHR-67 regulation causes 1) extra pluripotent cells to directly become ACs early in development, 2) causes VU cells to gradually trans-fate to an AC-like fate after VU fate specification (as implied by the authors), or 3) causes an AC to undergo extra cell division(s)? In Fig. 1F, 5 cells are designated as ACs, which is one more that the 4 precursors depicted in Fig. 1A, implying that some of the "ACs" were derived from progenitors that divided.

      The timing between AC/VU cell fate specification and AC invasion (the point at which we look for differentiated ACs) is approximately 10-12 hours at 25 {degree sign}C. With our imaging setup, we are limited to approximately 3-4 hours of live-cell imaging. Therefore, lineage tracing was not feasible for our experiments. Instead, we relied on visualization of established markers of AC and VU cell fate to determine how ectopic ACs arose. In Fig. 6B,C we show that the expression of two AC markers (cdh-3 and lag-2) turn on while a VU marker (lag-1) gets downregulated within the same cell. In our opinion, live-imaging experiments that show in real time changes in cell fate via reporters was the most definitive way to observe the phenotype.

      7) There are 4 multipotential gonadal cells with the potential to differentiate into VUs or ACs. Which ones contribute to the extra ACs in the different genetic backgrounds examined was not determined, which complicates interpretation. The authors should consider and test the following possibilities: disruption of NHR-67 regulation causes 1) extra pluripotent cells to directly become ACs early in development, 2) causes VU cells to gradually trans-fate to an AC-like fate after VU fate specification (as implied by the authors), or 3) causes an AC to undergo extra cell division(s)?? In Fig. 1F, 5 cells are designated as ACs, which is one more that the 4 precursors depicted in Fig. 1A, implying that some of the "ACs" were derived from progenitors that divided.

      RESPONSE: When trying to determine the source of the ectopic ACs, we considered the three possibilities noted by the reviewer: (1) misspecification of AC/VU precursors, (2) VU-to-AC transdifferentiation, or (3) proliferation of the AC. We eliminated option 3 as a possibility, as the ectopic ACs we observed here were invasive and all of our previous work has shown that proliferating ACs cannot invade and that cell cycle exit is necessary for invasion (Matus et al., 2015; MedwigKinney & Smith et al., 2020; Smith et al., 2022). Specifically, NHR-67 is upstream of the cyclin dependent kinase CKI-1 and we found that induced expression of NHR-67 resulted in slow growth and developmental arrest, likely because of inducing cell cycle exit. For our experiment using hsp::NHR-67, we induced heat shock after AC/VU specification. For POP-1 perturbation, we explicitly acknowledged that misspecification of the AC/VU precursors could also contribute to ectopic ACs (Fig. 6A; lines 368-385). We could not achieve robust protein depletion through delayed RNAi treatment, so instead we utilized timelapse microscopy and quantification of AC and VU cell markers (Fig. 6B,C; see response 2.7 above).

    1. Reviewer #2 (Public Review):

      This work clarifies neural mechanisms that can lead to a phenomenology consistent with motor preparation in its broader sense. In this context, motor preparation refers to an activity that occurs before the corresponding movement. Another property often associated with preparatory activity is a correlation with global movement characteristics such as reach speed (Churchland et al., Neuron 2006), reach angle (Sun et al., Nature 2022), or grasp type (Meirhaeghe et al., Cell Reports 2023). Such activity has notably been observed in premotor and primary motor cortices, and it has been hypothesized to serve as an input to a motor execution circuit. The timing and mechanisms by which such 'preparatory' inputs are made available to motor execution circuits remain however unclear in general, especially in light of the presence of a 'trigger-like' signal that appears to relate to the transition from preparatory dynamics to execution activity (Kaufman et al. eNeuron 2016, Iganaki et al., Cell 2022, Zimnik and Churchland, Nature Neuroscience 2021).

      The preparatory inputs have been hypothesized to fulfill one or several (non-mutually-exclusive) possible objectives. Two notable hypotheses are that these inputs could be shaped to maximize output accuracy under regularization of the input magnitude; or that they may help the flexible re-use of the neural machinery involved in the control of movements in different contexts.

      Here, the authors investigate in detail how the former hypothesis may be compatible with the presence of early inputs in recurrent network models driving arm movements, and compare models to data.

      Strengths:

      The authors are able to deploy an in-depth evaluation of inputs that are optimized for producing an accurate output at a pre-defined time while using a regularization term on the input magnitude, in the case of movements that are thought to be controlled in a quasi-open loop fashion such as reaches.

      First, the authors have identified that optimal control theory is a great framework to study this question as it provides methods to find and analyze exact solutions to this cost function in the case of models with linear dynamics. The authors not only use this framework to get an exact assessment of how much activity before movement start happens in large recurrent networks, but also give insight into the mechanisms by which it happens by dissecting in detail low-dimensional networks. The authors find that two key network properties - observability of the readout's nullspace and limited controllability - give rise to optimal inputs that are large before the start of the movement (while the corresponding network activity lies in the nullspace of the readout). Further, the authors numerically investigate the timing of optimized inputs in models with nonlinear dynamics, and find that pre-movement inputs can also arise in these more general networks. Finally, the authors point out some coarse-grained similarities between the pre-movement activity driven by the optimized inputs in some of the models they studied, and the phenomenology of preparation observed in the brain during single reaches and reach sequences. Overall, the authors deploy an impressive arsenal of tools and a very in-depth analysis of their models.

      Limitations:

      1. Though the optimal control theory framework is ideal to determine inputs that minimize output error while regularizing the input norm, it however cannot easily account for some other varied types of objectives - especially those that may lead to a complex optimization landscape. For instance, the reusability of parts of the circuit, sparse use of additional neurons when learning many movements, and ease of planning (especially under uncertainty about when to start the movement), may be alternative or additional reasons that could help explain the preparatory activity observed in the brain. It is interesting to note that inputs that optimize the objective chosen by the authors arguably lead to a trade-off in terms of other desirable objectives. Specifically, the inputs the authors derive are time-dependent, so a recurrent network would be needed to produce them and it may not be easy to interpolate between them to drive new movement variants. In addition, these inputs depend on the desired time of output and therefore make it difficult to plan, e.g. in circumstances when timing should be decided depending on sensory signals. Finally, these inputs are specific to the full movement chain that will unfold, so they do not permit reuse of the inputs e.g. in movement sequences of different orders.

      2. Relatedly, if the motor circuits were to balance different types of objectives, the activity and inputs occurring before each movement may be broken down into different categories that may each specialize into one objective. For instance, previous work (Kaufman et al. eNeuron 2016, Iganaki et al., Cell 2022, Zimnik and Churchland, Nature Neuroscience 2021) has suggested that inputs occurring before the movement could be broken down into preparatory inputs 'stricto sensu' - relating to the planned characteristics of the movement - and a trigger signal, relating to the transition from planning to execution - irrespective of whether the movement is internally timed or triggered by an external event. The current work does not address which type(s) of early input may be labeled as 'preparatory' or may be thought of as a part of 'planning' computations.

      3. While the authors rightly point out some similarities between the inputs that they derive and observed preparatory activity in the brain, notably during motor sequences, there are also some differences. For instance, while both the derived inputs and the data show two peaks during sequences, the data reproduced from Zimnik and Churchland show preparatory inputs that have a very asymmetric shape that really plummets before the start of the next movement, whereas the derived inputs have larger amplitude during the movement period - especially for the second movement of the sequence. In addition, the data show trigger-like signals before each of the two reaches. Finally, while the data show a very high correlation between the pattern of preparatory activity of the second reach in the double reach and compound reach conditions, the derived inputs appear to be more different between the two conditions. Note that the data would be consistent with separate planning of the two reaches even in the compound reach condition, as well as the re-use of the preparatory input between the compound and double reach conditions. Therefore, different motor sequence datasets - notably, those that would show even more coarticulation between submovements - may be more promising to find a tight match between the data and the author's inputs. Further analyses in these datasets could help determine whether the coarticulation could be due to simple filtering by the circuits and muscles downstream of M1, planning of movements with adjusted curvature to mitigate the work performed by the muscles while permitting some amount of re-use across different sequences, or - as suggested by the authors - inputs fully tailored to one specific movement sequence that maximize accuracy and minimize the M1 input magnitude.

      4. Though iLQR is a powerful optimization method to find inputs optimizing the author's cost function, it also has some limitations. First, given that it relies on a linearization of the dynamics at each timestep, it has a limited ability to leverage potential advantages of nonlinearities in the dynamics. Second, the iLQR algorithm is not a biologically plausible learning rule and therefore it might be difficult for the brain to learn to produce the inputs that it finds. It remains unclear whether using alternative algorithms with different limitations - for instance, using variants of BPTT to train a separate RNN to produce the inputs in question - could impact some of the results.

      5. Under the objective considered by the authors, the amount of input occurring before the movement might be impacted by the presence of online sensory signals for closed-loop control. It is therefore an open question whether the objective and network characteristics suggested by the authors could also explain the presence of preparatory activity before e.g. grasping movements that are thought to be more sensory-driven (Meirhaeghe et al., Cell Reports 2023).

    1. Reviewer #2 (Public Review):

      Summary<br /> In this experiment, Voltage Sensitive Dye Imaging (VSDI) was used to measure neural activity in macaque primary visual cortex in monkeys trained to detect an oriented grating target that was presented either alone or against an oriented mask. Monkeys' ability to detect the target (indicated by a saccade to its location) was impaired by the mask, with the greatest impairment observed when the mask was matched in orientation to the target, as is also the case in human observers. VSDI signals were examined to test the hypothesis that the target-evoked response would be maximally suppressed by the mask when it matched the orientation of the target. In each recording session, fixation trials were used to map out the spatial response profile and orientation domains that would then be used to decode the responses on detection trials. VSDI signals were analyzed at two different scales: a coarse scale of the retinotopic response to the target and a finer scale of orientation domains within the stimulus-evoked response. Responses were recorded in three conditions: target alone, mask alone, and target presented with mask. Analyses were focused on the target evoked response in the presence of the mask, defined to be the difference in response evoked by the mask with target (target present) versus the mask alone (target absent). These were computed across five 50 msec bins (total, 250 msec, which was the duration of the mask (target present trials, 50% of trials) / mask + target (target present trials, 50% of trials). Analyses revealed that in an initial (transient) phase the target evoked response increased with similarity between target and mask orientation. As the authors note, this is surprising given that this was the condition where the mask maximally impaired detection of the target in behavior. Target evoked responses in a later ('sustained') phase fell off with orientation similarity, consistent with the behavioral effect. When analyzed at the coarser scale the target evoked response, integrated over the full 250 msec period showed a very modest dependence on mask orientation. The same pattern held when the data were analyzed on the finer orientation domain scale, with the effect of the mask in the transient phase running counter to the perceptual effect of the mask and the sustained response correlating the perceptual effect. The effect of the mask was more pronounced when analyzed at the scale.

      Strengths<br /> The work is on the whole very strong. The experiments are thoughtfully designed, the data collection methods are good, and the results are interesting. The separate analyses of data at a coarse scale that aggregates across orientation domains and a more local scale of orientation domains is a strength and it is reassuring that the effects at the more localized scale are more clearly related to behavior, as one would hope and expect. The results are strengthened by modeling work shown in Figure 8, which provides a sensible account of the population dynamics. The analyses of the relationship between VSDI data and behavior are well thought out and the apparent paradox of the anti-correlation between VSDI and behavior in the initial period of response, followed by a positive correlation in the sustained response period is intriguing.

      Points to Consider / Possible Improvements<br /> The biphasic nature of the relationship between neural and behavioral modulation by the mask and the surprising finding that the two are anticorrelated in the initial phase are left as a mystery. The paper would be more impactful if this mystery could be resolved.

      The finding is based on analyses of the correlation between behavior and neural responses. This appears in the main body of the manuscript and is detailed in Figures S1 and S2, which show the correlation over time between behavior and target response for the retinotopic and columnar scale.

      One possible way of thinking of this transition from anti- to positive correlation with behavior is that it might reflect the dynamics of a competitive interaction between mask and target, with the initial phase reflecting predominantly the mask response, with the target emerging, on some trials, in the latter phase. On trials when the mask response is stronger, the probability of the target emerging in the latter phase, and triggering a hit, might be lower, potentially explaining the anticorrelation in the initial phase. The sustained response may be a mixture of trials on which the target response is or is not strong enough to overcome the effect of the mask sufficiently to trigger target detection.

      It would, I think, be worth examining this by testing whether target dynamics may vary, depending on whether the monkey detected the target (hit trials) or failed to detect the target (miss trials). Unless I missed it I do not think this analysis was done. Consistent with this possibility, the authors do note (lines 226-229) that "The trajectories in the target plus mask conditions are more complex. For example, when mask orientation is at +/- 45 deg to the target, the population response is initially dominated by the mask, but then in mid-flight, the population response changes direction and turns toward the direction of the target orientation." This suggests (to this reviewer, at least) that the emergence of a positive correlation between behavioral and neural effects in the latter phase of the response could reflect either a perceptual decision that the target is present or perhaps deployment of attention to the location of the target.

      It may be that this transition reflected detection, in which it might be more likely on hit trials than miss trials. Given the SNR it would presumably be difficult to do this analysis on a trial-by-trial basis, but the hit and miss trials (which make each make up about 1/2 of all trials) could be averaged separately to see if the mid-flight transition is more prominent on hit trials. If this is so for the +/- 45 degree case it would be good to see the same analysis for other combinations of target and mask. It would also be interesting to separate correct reject trials from false alarms, to determine whether the mid-flight transition tends to occur on false alarm trials.

      If these analyses do not reveal the predicted pattern, they might still merit a supplemental figure, for the sake of completeness.

    1. Reviewer #2 (Public Review):

      The authors of this study investigated the relationship between (under)confidence and the anxious-depressive symptom dimension in a longitudinal intervention design. The aim was to determine whether confidence bias improves in a state-like manner when symptoms improve. The primary focus was on patients receiving internet-based CBT (iCBT; n=649), while secondary aims compared these changes to patients receiving antidepressants (n=82) and a control group (n=88).

      The results support the authors' conclusions, and the authors convincingly demonstrated a weak link between changes in confidence bias and anxious-depressive symptoms (not specific to the intervention arm)

      The major strength and contribution of this study is the use of a longitudinal intervention design, allowing the investigation of how the well-established link between underconfidence and anxious-depressive symptoms changes after treatment. Furthermore, the large sample size of the iCBT group is commendable. The authors employed well-established measures of metacognition and clinical symptoms, used appropriate analyses, and thoroughly examined the specificity of the observed effects.

      However, due to the small expected effect sizes, the comparisons with the antidepressant and control groups were underpowered, reducing comparability between interventions and the generalizability of the results. The lack of interaction effect with treatment makes it harder to interpret the observed differences in confidence.

    1. Reviewer #2 (Public Review):

      This work explored the biological functions of a small family of RNA-binding proteins that was previously studied in animals, but was uncharacterized in plants. Combinatorial T-DNA insertional mutants disrupting the expression of the four Mushashi-like (MSIL) genes in Arabidopsis revealed that only the msil2 msil4 double mutant visibly alters plant development. The msil2/4 plants produced stems that could not stand upright. Transgene complementation, site-directed mutagenesis of MSIL4 conserved RNA-binding motifs, and in vitro RNA binding assays support the conclusion that the loss of MSIL2 and MISL4 function is responsible for the observed morphological defects. MSIL2/4 interact with proteins associated with mRNA 3'UTR binding and translational regulation.

      The authors present compelling biochemical evidence that Mushashi-like2 (MSIL2) and MSIL4 jointly regulate secondary cell wall biosynthesis in the Arabidopsis stem. Quantitative analyses of proteins and transcripts in msil2/4 stems uncovered transcriptional upregulation of several xylan-related enzymes (despite WT-like RNA levels). Consistent with MALDI-TOF data for released xylan oligosaccharides, the authors propose a model in which MSIL2/4 negatively regulate the translation of GXM (glucuronoxylan methyltransferase), a presumed rate-limiting step. The molecular links between overmethylated xylans and the observed stem defects (which include subtle reductions in lignin and increases beta-glucan polymer distribution) warrants further investigation in future studies. Similarly, as the authors point out, it is intriguing that the loss of the broadly expressed MSIL2/4 genes only significantly affects specific cell types in the stem.

    1. Reviewer #2 (Public Review):

      Summary: The authors provide a nice summary on the possibility to study genetic heterogeneity and how to measure the dynamics of stem cells. By combining single cell and bulk sequencing analyses, they aim to use a stochastic process and inform on different aspects of genetic heterogeneity.

      Strengths: Well designed study and strong methods

      Weaknesses: Minor<br /> Further clarification to Figure 3 legend would be good to explain the 'no association' of number of samples and mutational burden estimate as per line 180-182 p.8

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors set out to characterise "trust" in terms of a spatial pattern of neural responses, and then validate whether different tasks, in different datasets, express this pattern or do not express it, according to their hypotheses. They based their approach on linear classifiers (Support Vector Machines), which they trained to distinguish trust from distrust in an investment game, and then applied the classifier to other datasets. Additionally, they performed visualisations of the similarity among participants and among tasks in their neural responses, using dimensionality reduction techniques.

      Strengths:<br /> The key strength of this study is the use of multiple datasets to test whether a single study's characterisation of trust, in terms of a spatial pattern of neural responses, generalises to other tasks and populations. This is a nice use for existing data, which bolsters the interpretation of fMRI results, demonstrating that they are generalisable. While I am not a specialist in decoding methods, the analyses appear to have been performed conscientiously and to a high standard. The manuscript is also clearly written.

      Weaknesses:<br /> It's worth noting an obvious but important statistical point. In this study, the *inability* of a classifier to distinguish between conditions in particular datasets is taken as evidence that those conditions do not differ in terms of the effect of interest (trust). In this case, these results make sense, in that they are consistent with the authors' hypotheses. However, there are various reasons why the classifier may not work well on particular datasets - e.g. differences in noise, or a lack of linear separability between patterns (which might mandate a non-linear classifier or a different SVM kernel). Therefore, any null result obtained with classical statistics should be interpreted with caution.

    1. Reviewer #2 (Public Review):

      Summary:

      This study by Park and Gross investigates the spatiotemporal neural representation of semantic information most pertinent to the gist of speech materials presented to subjects as magnetoencephalography was recorded. Participants heard and saw naturalistic continuous speech recordings (with the auditory component presented to one ear), while also presented with distractor auditory speech (presented in the other ear). Participants were instructed to attend to the speech stream that matched the video of the speaker. The stimuli were semantically parsed to create short segments to which topic probabilities were assigned. These segments were then organized into high and low topic probabilities for each of the four topics (determined using Latent Dirichlet Allocation (LDA) analysis). The results suggest clear differences in the fidelity of neural encoding of the speech envelope during high-topic probability segments, which is interpreted as the brain representing key information for a story whether that information is explicitly attended to.

      Strengths:<br /> The use of LDA analysis makes possible the quantification of whether a particular speech segment is relevant to a particular topic and enables analysis based on this high-temporal resolution of semantic salience. The authors show clear differences between attended and unattended speech conditions, as well as, surprisingly, differences between semantically salient unattended speech and attended, less semantically relevant speech.

      Weaknesses:<br /> Though the effect sizes of the results of this study show clear differences between stimulus conditions, clarification of the experimental methods is needed to appreciate their interpretation. Broadly, I would suggest adding a clearer description of the task during data collection, even though it has been published elsewhere.

      One key piece of information that is missing is how semantically relevant topics are assigned, so that salient semantic information can be compared between attended and unattended stories. It's unclear to me how results are combined across topics and stories. If a particular speech segment is assigned 4 topic probabilities, that segment has both a high probability of belonging to one topic and a low probability of belonging to another. I understand how this can be used to create the experimental conditions for a single topic, but how are results combined across topics?

      I think some discussion of using the encoding and decoding of the speech envelope as a measure of what is semantically relevant is warranted. The fidelity with which the speech envelop is represented has been used as a proxy for how well that speech is attended to, but it is unclear to me whether we should expect to see high-fidelity encoding of speech envelop outside of the primary and secondary auditory regions of the brain, or how it relates to the semantic information contained in the speech signal.

      Additionally, I wonder if it might be more informative to decode the topic labels themselves directly by building a model to predict the topic probabilities from the neural data? This might give a more direct measure of where and when semantically relevant information is represented.

    1. Reviewer #2 (Public Review):

      In the current study, Fischer and colleagues extensively examined the role of parthenolide in inhibiting microtubule detyrosination and making the mechanistic link for the compound to facilitate the role of IL6 and PTEN/KO in promoting neurite outgrowth and axon regeneration. The in vitro and mechanistic work laid the foundation for the authors to reach several key predictions that such detyrosination can be applied for in vivo applications. Thus the authors extended the work to optic nerve regeneration and spinal cord recovery. The in vivo compound that the authors utilized is DMAPT, which plays a synergistic role with existing pro-regeneration therapies, such as Il6 treatment.

      The major strength of the work is the first half of the mechanistic inquiries, where the authors combined cell biology and biochemistry approaches to dissect the mechanistic link from parthenolide to microtube dynamics. The shortcoming is that the in vivo data is limited, and the effects might be considered mild, especially by benchmarking with other established and effective strategies.

      The work is solid and prepares a basis for others to test the role of DMAPT in other settings, especially in the setting of other effective pro-regenerative approaches. With the goal of comprehensive and functional recovery in vivo, the impact of the work and the utilities of the methods remain to be tested broadly in other models in vivo.

    1. Reviewer #2 (Public Review):

      Place cells fire sequentially during hippocampal theta oscillations, forming a spatial representation of behavioral experiences in a temporally-compressed manner. The firing sequences during theta cycles are widely considered as essential assemblies for learning, memory, and planning. Many theoretical studies have investigated the mechanism of hippocampal theta firing sequences; however, they are either entirely extrinsic or intrinsic. In other words, they attribute the theta sequences to external sensorimotor drives or focus exclusively on the inherent firing patterns facilitated by the recurrent network architectures. Both types of theories are inadequate for explaining the complexity of the phenomena, particularly considering the observations in a previous paper by the authors: theta sequences independent of animal movement trajectories may occur simultaneously with sensorimotor inputs (Yiu et al., 2022).

      In this manuscript, the authors concentrate on the CA3 area of the hippocampus and develop a model that accounts for both mechanisms. Specifically, the model generates extrinsic sequences through the short-term facilitation of CA3 cell activities, and intrinsic sequences via recurrent projections from the dentate gyrus. The model demonstrates how the phase precession of place cells in theta sequences is modulated by running direction and the recurrent DG-CA3 network architecture. To evaluate the extent to which firing sequences are induced by sensorimotor inputs and recurrent network architecture, the authors use the Pearson correlation coefficient to measure the "intrinsicity" and "extrinsicity" of spike pairs in their simulations.

      I find this research topic to be both important and interesting, and I appreciate the clarity of the paper. The idea of combining intrinsic and extrinsic mechanisms for theta sequences is novel, and the model effectively incorporates two crucial phenomena: phase precession and directionality of theta sequences. I particularly commend the authors' efforts to integrate previous theories into their model and conduct a systematic comparison. This is exactly what our community needs: not only the development of new models, but also understanding the critical relationships between different models.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This work investigates the effects of various antipsychotic drugs on cortical responses during visuomotor integration. Using wide-field calcium imaging in a virtual reality setup, the researchers compare neuronal responses to self-generated movement during locomotion-congruent (closed loop) or locomotion-incongruent (open loop) visual stimulation. Moreover, they probe responses to unexpected visual events (halt of visual flow, sudden-onset drifting grating). The researchers find that, in contrast to a variety of excitatory and inhibitory cell types, genetically defined layer 5 excitatory neurons distinguish between the closed and the open loop condition and exhibit activity patterns in visual cortex in response to unexpected events, consistent with unsigned prediction error coding. Motivated by the idea that prediction error coding is aberrant in psychosis, the authors then inject the antipsychotic drug clozapine, and observe that this intervention specifically affects closed loop responses of layer 5 excitatory neurons, blunting the distinction between the open and closed loop conditions. Clozapine also leads to a decrease in long-range correlations between L5 activity in different brain regions, and similar effects are observed for two other antipsychotics, aripripazole and haloperidol, but not for saline or the stimulant amphetamine. The authors suggest that altered prediction error coding in layer 5 excitatory neurons due to reduced long-range correlations in L5 neurons might be a major effect of antipsychotic drugs and speculate that this might serve as a new biomarker for drug development.

      Strengths:<br /> - Relevant and interesting research question:<br /> The distinction between expected and unexpected stimuli is blunted in psychosis but the neural mechanisms remain unclear. Therefore, it is critical to understand whether and how antipsychotic drugs used to treat psychosis affect cortical responses to expected and unexpected stimuli. This study provides important insights into this question by identifying a specific cortical cell type and long-range interactions as potential targets. The authors identify layer 5 excitatory neurons as a site where functional effects of antipsychotic drugs manifest. This is particularly interesting as these deep layer neurons have been proposed to play a crucial role in computing the integration of predictions, which is thought to be disrupted in psychosis. This work therefore has the potential to guide future investigations on psychosis and predictive coding towards these layer 5 neurons, and ultimately improve our understanding of the neural basis of psychotic symptoms.

      - Broad investigation of different cell types and cortical regions:<br /> One of the major strengths of this study is quasi-systematic approach towards cell types and cortical regions. By analysing a wide range of genetically defined excitatory and inhibitory cell types, the authors were able to identify layer 5 excitatory neurons as exhibiting the strongest responses to unexpected vs. expected stimuli and being the most affected by antipsychotic drugs. Hence, this quasi-systematic approach provides valuable insights into the functional effects of antipsychotic drugs on the brain, and can guide future investigations towards the mechanisms by which these medications affect cortical neurons.

      - Bridging theory with experiments<br /> Another strength of this study is its theoretical framework, which is grounded in the predictive coding theory. The authors use this theory as a guiding principle to motivate their experimental approach connecting visual responses in different layers with psychosis and antipsychotic drugs. This integration of theory and experimentation is a powerful approach to tie together the various findings the authors present and to contribute to the development of a coherent model of how the brain processes visual information both in health and in disease.

      Weaknesses:<br /> - Unclear relevance for psychosis research<br /> From the study, it remains unclear whether the findings might indeed be able to normalise altered predictive coding in psychosis. Psychosis is characterised by a blunted distinction between predicted and unpredicted stimuli. The main results of this study indicate that antipsychotic drugs further blunt the distinction between predicted and unpredicted stimuli, which would suggest that antipsychotic drugs would deteriorate rather than ameliorate the predictive coding deficit found in psychosis. However, these findings were based on observations in wild-type mice at baseline. Given that antipsychotics are thought to have little effects in health but potent antipsychotic effects in psychosis, it seems possible that the presented results might be different in a condition modelling a psychotic state, for example after a dopamine-agonistic or a NMDA-antagonistic challenge. Therefore, future work in models of psychotic states is needed to further investigate the translational relevance of these findings.

      - Incomplete testing of predictive coding interpretation<br /> While the investigation of neuronal responses to different visual flow stimuli is interesting, it remains open whether these responses indeed reflect internal representations in the framework of predictive coding. While the responses are consistent with internal representation as defined by the researchers, i.e., unsigned prediction error signals, an alternative interpretation might be that responses simply reflect sensory bottom-up signals that are more related to some low-level stimulus characteristics than to prediction errors. Moreover, this interpretational uncertainty is compounded by the fact that the used experimental paradigms were not suited to test whether behaviour is impacted as a function of the visual stimulation which makes it difficult to assess what the internal representation of the animal actually was. For these reasons, the observed effects might reflect simple bottom-up sensory processing alterations and not necessarily have any functional consequences. While this potential alternative explanation does not detract from the value of the study, future work would be needed to explain the effect of antipsychotic drugs on responses to visual flow. For example, experimental designs that systematically vary the predictive strength of coupled events or that include a behavioural readout might be more suited to draw from conclusions about whether antipsychotic drugs indeed alter internal representations.

      Conclusion:<br /> Overall, the results support the idea that antipsychotic drugs affect neural responses to predicted and unpredicted stimuli in deep layers of cortex. Although some future work is required to establish whether this observation can indeed be explained by a drug-specific effect on predictive coding, the study provides important insights into the neural underpinnings of visual processing and antipsychotic drugs, which is expected to guide future investigations on the predictive coding hypothesis of psychosis. This will be of broad interest to neuroscientists working on predictive coding in health and disease.

    1. Reviewer #2 (Public Review):

      Bilgic et al first explored cellular diversity in the developing cerebral cortex of ferret, honing in on progenitor cell diversity by employing FACS sorting of HES5-positive cells. They have generated a novel single cell transcriptomic dataset capturing the diversity of cells in the developing ferret cerebral cortex, including diverse radial glial and excitatory neuron populations. Unexpectedly, this analysis revealed the presence of CRYAB-positive truncated radial glia previously described only in humans. Using bioinformatic analyses, the investigators proposed that truncated radial glia produce ependymal cells, astrocytes, and to a lesser degree, neurons. Of particular interest to the field, they identify enriched expression of FOXJ1 in late truncated radial glia strongly indicating that towards the end of neurogenesis, these cells likely give rise to ependymal cells. This study represents a major advancement in the field of cortical development and a valuable dataset for future studies of ferret cortical development.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this article, the authors provide a method of evaluating the safety of orthopedic implants in relation to radiofrequency-induced heating issues. The authors provide an open-source computational heterogeneous human model and explain computational techniques in a finite element method solver to predict the RF-induced temperature increase due to an orthopedic implant while being exposed to MRI RF fields at 1.5 T.

      Strengths:<br /> The open-access computational human model along with their semiautomatic algorithm to position the implant can help realistically model the implant RF exposure in patients avoiding over- or under-estimation of RF heating measured using rectangular box phantoms such as ASTM phantom. Additionally, using numerical simulation to predict radiofrequency-induced heating will be much easier compared to the experimental measurements in an MRI scanner, especially when the scanner availability is limited.

      Weaknesses:<br /> The proposed method only used radiofrequency (RF) field exposure to evaluate the heating around the implant. However, in the case of bulky implants, the rapidly changing gradient field can also produce significant heating due to large eddy currents. So the gradient-induced heating still remains an issue to be evaluated to decide on the safety of the patient. Moreover, the method is limited to a single human model and might not be representative of patients with different age, sex, and body weights. Additionally, the authors compare the temperature rise predicted by their method to an earlier study. However, there is no information about how they controlled the input power in their simulation testbed compared to the earlier study in showing validation of the method.

    1. Reviewer #2 (Public Review):

      This paper illustrates that PSCs can model myogenesis in vitro by mimicking the in vivo development of the somite and dermomyotome. The advantages of this 3D system include (1) better structural distinctions, (2) the persistence of progenitors, and (3) the spatial distribution (e.g. migration, confinement) of progenitors. The finding is important with the implication in disease modeling. Indeed the authors tried DMD model although it suffered the lack of deeper characterization.

      The differentiation protocol is based on a current understanding of myogenesis and is compelling. They characterized the organoids in depth (e.g. many time points and immunofluorescence). The evidence is solid.

    1. Reviewer #2 (Public Review):

      Catabolic conditions lead to increased formation of ketone bodies in the liver, which under these conditions play an important role in supplying energy to metabolically active organs. In this manuscript, the authors explore the concept of whether and to what extent hepatic formation of acetate might contribute to energy supply under metabolic stress conditions. The authors show that patients with diabetes have increased acetate levels, which is explained as a consequence of the increased fatty acid flux from adipose tissue to the liver. This is confirmed in a preclinical model for type 1 diabetes, where acetate concentrations are in a similar range to ketone bodies. Acetate concentrations also increase under physiological conditions of fasting. Using stable isotopes, the authors show that palmitate is used as the primary source for acetate production in primary hepatocytes. Using cell culture studies and adenoviral-mediated knockdown in mice, it can be shown that the conversion of acetyl-CoA to acetate is catalyzed in peroxisomes by acyl-CoA thioesterase8 (ACOT8) and after transport of citrate from mitochondria and subsequent conversion to acetyl-CoA in the cytosol by ACOT12. Remarkably, ACOT8/12 not only regulates the formation of acetate but plays a crucial role in the maintenance of cellular CoA concentration. Accordingly, depletion of ACOT8/12 activity leads to a reduction of other CoA derivatives such as HMG-CoA, which resulted in the inhibition of ketone body synthesis. In diabetic mice, ACOT 8 or ACOT12 knockdown appears to lead to some limitations in strength and behavior.

      In summary, the authors clearly demonstrate that hepatic release-mediated by ACOT8 and ACOT12-determines the plasma concentration of acetate. This is a very remarkable observation since most studies assume that short-chain fatty acids in plasma are primarily generated by fermentation of dietary fiber by intestinal bacteria. The authors demonstrate in very well performed studies the metabolic changes that result from impaired thiolysis. On the other hand, the ACOT12 phenotype has been demonstrated in a recently published study (PMID: 34285335). In this study, ACOT12 deficiency caused NAFLD, thus it would be worth determining whether deficiency of ACOT12 and/or ACOT8 promotes de novo lipogenesis under the conditions of the present study. As a further limitation, it should be noted that the relevance of acetate production for the energy supply of peripheral organs including the central nervous system could not be clearly demonstrated. For instance, impaired ketone body production due to impaired CoA availability could affect the metabolic activity of various organs. Moreover, the human cohort is not very well described, e.g. it is unclear whether the patients have type 1 or type 2 diabetes.

    1. Reviewer #2 (Public Review):

      The hard work of the authors is much appreciated. With overexpression of a-arrestin Txnip in RPE, cones and the combined respectively, the authors show a potential gene agnostic treatment that can be applied to retinitis pigmentosa. Furthermore, since Txnip is related to multiple intracellular signaling pathway, this study is of value for research in the mechanism of secondary cone dystrophy as well.

      There are a few areas in which the article may be improved through further analysis and application of the data, as well as some adjustments that should be made in to clarify specific points in the article.

    1. Reviewer #2 (Public Review):

      Summary:

      Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness in birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used a generalized linear mixed model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity and fertility and a null model analysis via data randomization for hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite of that hypothesized. While this finding is interesting and worth reporting, there are significant issues with the experimental design and the conclusions that are drawn from the results, which are described below. These issues should be addressed to make the findings trustworthy.

      Strengths:

      1. Using a combination of laboratory feedings and incubators to simulate seasonal environmental conditions is a good, controlled way to assess the potentially interactive impact of host species and seasonality on the fitness of Culex quinquefasciatus in the lab.<br /> 2. The driving hypothesis is an interesting and creative way to think about a potential driver of host switching observed in the field.

      Weaknesses:

      1. There is no replication built into this study. Egg lay is a highly variable trait, even within treatments, so it is important to see replication of the effects of treatment across multiple discrete replicates. It is standard practice to replicate mosquito fitness experiments for this reason. Furthermore, the sample size was particularly small for some groups (e.g. 15 egg rafts for the second gonotrophic cycle of mice in the autumn, which was the only group for which a decrease in fecundity and fertility was detected between 1st and 2nd gonotrophic cycles). Replicates also allow investigators to change around other variables that might impact the results for unknown reasons; for example, the incubators used for fall/summer conditions can be swapped, ensuring that the observed effects are not artifacts of other differences between treatments. While most groups had robust sample sizes, I do not trust the replicability of the results without experimental replication within the study.<br /> 2. Considering the hypothesis is driven by the host switching observed in the field, this phenomenon is discussed very little. I do not believe Cx. quinquefasciatus host switching has been observed in Argentina, only in the northern hemisphere, so it is possible that the species could have an entirely different ecology in Argentina. It would have been helpful to conduct a blood meal analysis prior to this experiment to determine whether using an Argentinian population was appropriate to assess this question. If the Argentinian populations don't experience host switching, then an Argentinian colony would not be the appropriate colony to use to assess this question. Given that this experiment has already been conducted with this population, this possibility should at least be acknowledged in the discussion. Or if a study showing host switching in Argentina has been conducted, it would be helpful to highlight this in the introduction and discussion.<br /> 3. The impacts of certain experimental design decisions are not acknowledged in the manuscript and warrant discussion. For example, the larvae were reared under the same conditions to ensure adults of similar sizes and development timing, but this also prevents mechanisms of action that could occur as a result of seasonality experienced by mothers, eggs, and larvae.<br /> 4. There are aspects of the data analysis that are not fully explained and should be further clarified. For example, there is no explanation of how the levels of categorical variables were compared.<br /> 5. The results show the opposite trend as was predicted by the authors based on observed feeding switches from birds to mammals in the autumn. However, they only state this once at the end of the discussion and never address why they might have observed the opposite trend as was hypothesized.<br /> 6. Generally speaking, the discussion has information that isn't directly related to the results and/or is too detailed in certain parts. Meanwhile, it doesn't dig into the meaning of the results or the ways in which the experimental design could have influenced results.<br /> 7. Beyond the issue of lack of replication limiting trust in the conclusions in general, there is one conclusion reached at the end of the discussion that would not be supported, even if additional replicates are conducted. The results do not show that physiological changes in mosquitoes trigger the selection of new hosts. Host selection is never measured, so this claim cannot be made. The results don't even suggest that fitness might trigger selection because the results show that physiological changes are in the opposite direction as what would be hypothesized to produce observed host switches. Similarly, the last sentence of the abstract is not supported by the results.<br /> 8. Throughout the manuscript, there are grammatical errors that make it difficult to understand certain sentences, especially for the results.

      This study is driven by an interesting question and has the potential to be a valuable contribution to the literature.

    1. Reviewer #2 (Public Review):

      Summary:<br /> 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 cell-specific targeting. 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:<br /> 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:<br /> The study shows the SWIFT approach works in vitro using cell lines, primary human hepatocytes, and human intestinal organoids, but it lacks an in vivo animal model or clinical validation. The applicability of this approach to therapy is still unknown.

      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 or available.

    1. Reviewer #2 (Public Review):

      This paper by Lucas et al follows on from earlier work by the same group. They use high-resolution 2D template matching (2DTM) to find particles of a given target structure in 2D cryo-EM images, either of in vitro single-particle samples or of more complicated samples, such as FIB-milled cells (which would otherwise perhaps be used for 3D electron tomography). One major concern for high-resolution template matching has been the amount of model bias that gets introduced into a reconstruction that is calculated straight from the orientations and positions identified by the projection matching algorithm. This paper assesses the amount of model bias that gets introduced in high-resolution features of such maps.

      For a high-signal-to-noise in vitro single-particle cryo-EM data set, the authors show that their approach does not yield much model bias. This is probably not very surprising, as their method is basically a low false-positive particle picker, which works very well on such data. Still, I guess that is the whole point of it, and it is good to see that they can reconstruct density for a small-molecule compound that was not present in the original template.

      For FIB-milled lamella of yeast cells with stalled ribosomes, the SNR is much lower and the dangers of model bias will be higher. This is also evidenced by the observation that further refinement of initial 2DTM-identified orientations and positions worsens the map. This is obviously a more relevant SNR regime to assess their method. Still, they show convincing density for the GHX compound that was not present in the template but was there in the reconstruction from the identified particles.

      Quantification of the amount of model bias is then performed using omit maps, where every 20th residue is removed from the template and corresponding reconstructions are compared (for those residues) with the full-template reconstructions. As expected, model bias increases with lower thresholds for the picking. Some model bias (Omega=8%) remains even for very high thresholds. The authors state this may be due to overfitting of noise when template-matching true particles, instead of introducing false positives. Probably, that still represents some sort of problem. Especially because the authors then go on to show that their expectation of the number of false positives does not always match the correct number of false positives, probably due to inaccuracies in the noise model for more complicated images. This may warrant further in-depth discussion in a revised manuscript.

      Overall, I think this paper is well written and it has made me think differently (again) about the 2DTM technique and its usefulness in various applications, as outlined in the Discussion. Therefore, it will be a constructive contribution to the field.

    1. Reviewer #2 (Public Review):

      In this study, Koesters et al. investigated whether Rab3A, a small GTPase that regulates synaptic vesicle fusion pore opening, is required for excitatory synaptic scaling in response to TTX-induced activity suppression in dissociated mouse cortical neuronal culture. They first show that, while pyramidal neurons from wild-type (WT) littermates show normal synaptic scaling in response to 48h of TTX treatment (~30% increase in the mean mEPSC amplitude), those from two different mouse lines with either deletion (Rab3A-/-) or loss-of-function mutation of Rab3A (Rab3AEbd/Ebd) fail to engage this homeostatic compensation. They perform cumulative distribution analysis to show that the mEPSC population has gone through divergent scaling in WT neurons. Similarly, this phenomenon is absent in neurons from the two Rab3A mouse lines. They further demonstrate that GluA2-containing AMPARs likely account for the increase in mEPSC amplitudes by comparing measurements before and after washing in blockers specific for GluA2-lacking AMPARs. Subsequently, they perform electrophysiology and immunohistochemistry side by side for WT neurons from the same culture following TTX treatment, and find that both mEPSC amplitudes and GluA2 cluster sizes have shifted towards higher values, while GluA2 cluster intensity remains unchanged. Importantly, all these homeostatic compensations are absent in Rab3A-/- neurons. Finally, they mix neurons and astrocyte feeders either from WT or Rab3A-/- mice, which reveals that neuronal but not astrocytic Rab3A knockout leads to impaired scaling up of mEPSCs. They conclude that Rab3A is required for homeostatic scaling up of mEPSC amplitude in cortical neurons, most likely from the presynaptic side.

      Although the authors have raised an interesting question, their conclusion is not well supported by the data presented. I list my technical and conceptual concerns below.

      Technical concerns:

      1. The culture condition is questionable. The authors saw no NMDAR current present during spontaneous recordings, which is worrisome since NMDARs should be active in cultures with normal network activity (Watt et al., 2000; Sutton et al., 2006). It is important to ensure there is enough spiking activity before doing any activity manipulation. Similarly, it is also unknown whether spiking activity is normal in Rab3A KO/Ebd neurons.

      2. Selection of mEPSC events is not conducted in an unbiased manner. Manually selecting events is insufficient for cumulative distribution analysis, where small biases could skew the entire distribution. Since the authors claim their ratio plot is a better method to detect the uniformity of scaling than the well-established rank-order plot, it is important to use an unbiased population to substantiate this claim.

      3. Immunohistochemistry data analysis is problematic. The authors only labeled dendrites without doing cell-fills to look at morphology, so it is questionable how they differentiate branches from pyramidal neurons and interneurons. Since glutamatergic synapses on these two types of neuron scale in the opposite directions, it is crucial to show that only pyramidal neurons are included for analysis.

      Conceptual concerns:

      The only novel finding here is the implicated role for Rab3A in synaptic scaling, but insights into mechanisms behind this observation are lacking. The author claims that Rab3A likely regulates scaling from the presynaptic side, yet there is no direct evidence from data presented. In its current form, this study's contribution to the field is very limited.

      1. Their major argument for this is that homeostatic effects on mEPSC amplitudes and GluA2 cluster sizes do not match. This is inconsistent with reports from multiple labs showing that upscaling of mEPSC amplitude and GluA2 accumulation occur side by side during scaling (Ibata et al., 2008; Pozo et al., 2012; Tan et al., 2015; Silva et al., 2019). Further, because the acquisition and quantification methods for mEPSC recordings and immunohistochemistry imaging are entirely different (each with its own limitations in signal detection), it is not convincing that the lack of proportional changes must signify a presynaptic component.

      2. The authors also speculate in the discussion that presynaptic Rab3A could be interacting with retrograde BDNF signaling to regulate postsynaptic AMPARs. Without data showing Rab3A-dependent presynaptic changes after TTX treatment, this argument is not compelling. In this retrograde pathway, BDNF is synthesized in and released from dendrites (Jakawich et al., 2010; Thapliyal et al., 2022), and it is entirely possible for postsynaptic Rab3A to interfere with this process cell-autonomously.

      3. The authors propose that a change in AMPAR subunit composition from GluA2-containing ones to GluA1 homomers may account for the distinct changes in mEPSC amplitudes and GluA2 clusters. However, their data from the Naspm wash-in experiments clearly show that GluA1 homomer contributions have not changed before and after TTX treatment.

      Ibata K, Sun Q, Turrigiano GG (2008) Rapid synaptic scaling induced by changes in postsynaptic firing. Neuron 57:819-826.

      Jakawich SK, Nasser HB, Strong MJ, McCartney AJ, Perez AS, Rakesh N, Carruthers CJL, Sutton MA (2010) Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis. Neuron 68:1143-1158.

      Pozo K, Cingolani LA, Bassani S, Laurent F, Passafaro M, Goda Y (2012) β3 integrin interacts directly with GluA2 AMPA receptor subunit and regulates AMPA receptor expression in hippocampal neurons. Proceedings of the National Academy of Sciences 109:1323-1328.

      Silva MM, Rodrigues B, Fernandes J, Santos SD, Carreto L, Santos MAS, Pinheiro P, Carvalho AL (2019) MicroRNA-186-5p controls GluA2 surface expression and synaptic scaling in hippocampal neurons. Proceedings of the National Academy of Sciences 116:5727-5736.

      Sutton MA, Ito HT, Cressy P, Kempf C, Woo JC, Schuman EM (2006) Miniature Neurotransmission Stabilizes Synaptic Function via Tonic Suppression of Local Dendritic Protein Synthesis. Cell 125:785-799.

      Tan HL, Queenan BN, Huganir RL (2015) GRIP1 is required for homeostatic regulation of AMPAR trafficking. Proceedings of the National Academy of Sciences 112:10026-10031.

      Thapliyal S, Arendt KL, Lau AG, Chen L (2022) Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity. eLife 11:e79863.

      Watt AJ, Rossum MCW van, MacLeod KM, Nelson SB, Turrigiano GG (2000) Activity Coregulates Quantal AMPA and NMDA Currents at Neocortical Synapses. Neuron 26:659-670.

    1. Reviewer #2 (Public Review):

      Summary:

      In this manuscript, Mure et al investigated host-microbe interactions in wild-mimicked settings. They analyzed microbiome composition using bananas that had been fed on by wild larvae and found that the microbiota composition shifted from the early stage of feeding to the later stage of the fermentation process. They isolated several yeast and bacterial species from the food, and examined larval growth on banana-based food, mimicking a natural setting where germ-free larvae cannot grow on it. The authors found that a yeast, Hanseniaspora uvarum, can support larval growth sufficiently, and insisted that branched-chain amino acids (BCAAs) provided by the yeast may partly account for the growth support. Interestingly, in other isolated yeast species, some were non-supportive strains in terms of larval growth, which can assist larval development when they are heat-killed. Besides, they showed that acetic acid bacteria, isolated from well-fermented banana (later-stage food), is sufficiently supportive but their presence depended on other microbes, lactic acid bacteria or yeast.

      Strengths:

      So far, host-microbe studies using Drosophila melanogaster have focused relatively less on the roles of fungi, and many studies used only "model" yeasts. In the experimental setting where natural conditions may be well mimicked, the authors successfully isolated wild yeast species and convincingly showed that wild yeast plays a critical role in promoting host growth. In addition, the authors provided intriguing observations that all of the heat-killed yeast promoted larval growth even though some of the yeast never supported the development when they were alive, suggesting that wild yeasts produce the necessary nutrients for larval development, but the nutrients of non-supportive yeasts are not accessible to the host. This might be an interesting indication for further studies revealing host-fungi interactions.

      Weaknesses:

      The experimental setting that, the authors think, reflects host-microbe interactions in nature is one of the key points. However, it is not explicitly mentioned whether isolated microbes are indeed colonized in wild larvae of Drosophila melanogaster who eat bananas. Another matter is that this work is rather descriptive and a few mechanical insights are presented. The evidence that the nutritional role of BCAAs is incomplete, and molecular level explanation is missing in "interspecies interactions" between lactic acid bacteria (or yeast) and acetic acid bacteria that assure their inhabitation. Apart from these matters, the future directions or significance of this work could be discussed more in the manuscript.

    1. Reviewer #3 (Public Review):

      In their study, Purandare & Mehta analyze large-scale single unit recordings from the visual system (LGN, V1, extrastriate regions AM and PM) and hippocampal system (DG, CA3, CA1 and subiculum) while mice monocularly viewed repeats of a 30s movie clip. The data were part of a larger release of publicly available recordings from the Allen Brian Observatory. The authors found that cells in all regions exhibited tuning to specific segments of the movie (i.e. "movie fields") ranging in duration from 20ms to 20s. The largest fractions of movie-responsive cells were in visual regions, though analyses of scrambled movie frames indicated that visual neurons were driven more strongly by visual features of the movie images themselves. Cells in the hippocampal system, on the other hand, tended to exhibit fewer "movie fields", which on average were a few seconds in duration, but could range from >50ms to as long as 20s. Unlike the visual system "movie fields" in the hippocampal system disappeared when the frames of the movie were scrambled, indicating that the cells encoded more complex (episodic) content, rather than merely passively reading out visual input.

      The paper is conceptually novel since it specifically aims to remove any behavioral or task engagement whatsoever in the head-fixed mice, a setup typically used as an open-loop control condition in virtual reality-based navigational or decision making tasks (e.g. Harvey et al., 2012). Because the study specifically addresses this aspect of encoding (i.e. exploring effects of pure visual content rather than something task-related), and because of the widespread use of video-based virtual reality paradigms in different sub-fields, the paper should be of interest to those studying visual processing as well as those studying visual and spatial coding in the hippocampal system.

      Comments on latest version:

      The revised manuscript by Purandare et al. has been improved with the inclusion of additional analyses and discussion, and the changes mainly satisfy the concerns raised in the initial version of the manuscript.

      Regarding the methods, it was particularly helpful that the authors took measures to consider the impact of different states of arousal (pupil diameter), mobility, and SWRs on the expression and significance of movie field tuning, considering the lack of a task structure or behavioral report. Relatedly, the additional metrics applied (information rate and depth of movie field modulation) substantiate the results as based on z-scored sparsity. The explanation of lifetime sparseness as used here vs. in the work of de Vries et al. 2020 was also helpful.

      The addition of more clearly tuned cells also helps the study feel more rooted in solid ground. For clarity, and consistency with the rest of the paper, it would be helpful to add the sparseness metrics above the newly added neural data in the Figure supplements.

      The Discussion also contains elements that help balance both it and the paper as a whole. It draws a clearer distinction between the representation of visual scenes rather than encoding the contents of episodic memory, clarifying that hippocampal neurons were more likely doing the former than the latter. It is also appreciated that the authors added discussion acknowledging that the cortical processing did not quite follow an apparent hierarchical order.

      As a last observation, though the authors assert in their rebuttal that analysis of the visual content encoded in the movie fields is beyond the scope of the study, this would add an interesting dimension to the work. Because, to my awareness, much less is known regarding how the visual and hippocampal systems in rodents encode visual information when the visual input is dynamic and chunked, as with movies. It would prove an interesting addition to the more extensive work on the processing of static visual scenes.

    1. Reviewer #2 (Public Review):

      This paper extends prior work demonstrating the importance of K145 acetylation of TDP-43 as a post-translational modification that impacts its RNA-binding capacity and may contribute to pathology in FTLD-ALS. The main strengths of this paper are the generation of a novel mouse model, using CRISPR gene editing, in which an acetylation-mimetic mutation (K to Q) is introduced at position 145. Behavioral, biochemical, and genetic analyses indicate that these mice display phenotypes relevant to TDP-43-associated disease and will be a valuable contribution to the field.

    1. Reviewer #2 (Public Review):

      Gillespie et al. introduced a novel neurofeedback (NF) procedure to train rats in enhancing their sharp-wave ripple (SWR) rate within a short duration, a key neural mechanism associated with memory consolidation. The training, embedded within a spatial memory task, spanned 20-30 days and utilized food rewards as positive reinforcement upon SWR detection. Rats were categorized into NF and control groups, with the NF group further divided into NF and delay trials for within-subject control. While single trial differences were elusive due to the variability of SWR occurrence, the study revealed that statistically rats in NF trials exhibited a notably higher SWR rate before receiving rewards compared to delay trials. This difference was even more pronounced when juxtaposed with rats not exposed to NF training (control group). The unique design of blending the NF phase with the memory dependent spatial task enabled the authors to analyze whether the NF training influence the task performance and replay content during SWRs across three different conditions (NF trials, delay trials and control group). Interestingly, despite the NF training, there was no significant improvement or decline in the performance of the spatial memory task, and the replay content remained consistent across all three conditions. Hence, the operant conditioning only amplified the SWR rate before reward in NF trials without altering the task performance and the replay content during SWR. Moreover, considering the post-reward period, the total SWR count was consistent across all conditions as well, meaning the NF training also do not affect the total SWR count. The study concludes with the hypothesis of a potential homeostatic mechanism governing the total SWR production in rats. This research significantly extends previous work by Ishikawa et al. (2014), offering insights into the NF training with external reward on the SWR rate/counts, replay content and task performance.

      Strengths:

      - Integration of NF task and spatial memory task in a single trial<br /> The integration of NF training within a spatial memory task poses significant challenges. Gillespie and colleagues overcame this by seamlessly blending the NF task and the spatial memory task into a single trial. Each trial involved a rat undergoing three steps: First, initiating a trial. Second, moving to either the NF port or the delay trial port, as indicated by an LED, and then maintaining a nosepoke at one of the center ports. During this step, the rat had to keep its nose (in the NF port) until a sharp-wave ripple (SWR) exceeding a set threshold was detected, which then triggered a reward, or until a variable time elapsed (in the delay port). Third, the rat would choose one of eight arms to explore before starting the next trial. This integration of the two tasks (step two as the NF task and step three as the spatial memory task) facilitated a direct analysis of the impact of NF training on behaviorally relevant replay content during SWRs and the performance in the spatial memory task.

      - Clear Group Separation<br /> A robust study design necessitates clear distinctions between experimental conditions to ensure that observed differences can be attributed to the variable under investigation. This study meticulously categorized rats into three distinct conditions: NF trials, delay trials (for within-subject control), and a control group (for across-subject control). Furthermore, for each trial, the times of interest (TOI) were separated into pre-reward and post-reward periods. This clear separation ensures that any observed differences in SWR rates and other outcomes can be confidently attributed to the effects of neurofeedback training during specific time periods, minimizing potential confounding factors.

      - Evidence of SWR rate modulation<br /> The study's results offer compelling evidence that rats can be trained to modulate their SWR rates during the pre-reward period. This is evident from the observation that rats in the NF trials consistently displayed a higher SWR rate before receiving rewards compared to those in delay trials or the control group (Fig. 2). Such findings not only validate the efficacy of the NF paradigm but also underscore the potential of operant conditioning in influencing neural mechanisms. The observation that rats were able to produce larger SWR events by modulating their occurrence rate, rather than merely waiting for these events, suggests a learned strategy to generate them more efficiently.

      - Evidence of SWR count homeostasis<br /> A notable finding from the study was the observation of a consistent total SWR count during both pre-reward and post-reward periods across all conditions, despite the evident increase in SWR rates during the pre-reward period in NF trials. This points to a potential homeostatic mechanism governing SWR production in rats. This balance suggests that while NF training can modulate the timing and rate of SWRs over a short duration, it doesn't influence the overall count of SWRs over a longer period. Such a mechanism might be essential in ensuring that the brain neither overcompensates nor depletes its capacity for SWRs, maintaining the overall neural balance and functionality. This discovery deepens our understanding of neural mechanisms and highlights potential avenues for future research into the regulatory processes governing neural activity.

      Weaknesses:

      - Misleading Title<br /> The title, "Neurofeedback training can modulate task-relevant memory replay in rats," implies that through neurofeedback training, rats can learn to modulate the content of their memory replay. However, the study's findings contradict this implication. Particularly, one of the subtitles of this paper is "Neurofeedback training preserves replay content during SWRs," which directly contrasts with the main title's suggestion. The authors conclusively demonstrated that there was no discernible difference in the replay content between animals that underwent NF training and those that did not. The current title easily leads to misinterpretations about the study's primary outcomes, especially for readers who might not delve into the detailed findings.

      - Lack of control analysis baseline for each animal<br /> While the authors meticulously categorized trial types into three distinct conditions: NF trials, delay trials, and control groups, they did not clearly establish a baseline for each animal. The animal could have a total different baseline SWR rates. The paper appears to operate under the assumption that each animal possesses a consistent SWR rate baseline, leading to only the final comparisons being presented.

      - Vagueness of what animal really control during NF trials after training<br /> The authors state that, "Moreover, although we did observe a slightly lower mean speed during the pre-reward period on neurofeedback trials compared to delay trials and trials from the control cohort (Supplementary Figure 2F), movement differences could not explain the difference in SWR rates (Supplementary Figure 2G, H)." This assertion raises questions about the underlying mechanisms at play. In a typical operant conditioning scenario, training could result in direct neural modulation, behavioral changes, or a combination of both. For instance, rats might adopt a more stationary posture during the pre-reward period on NF trials compared to other conditions, or they might actively influence the occurrence rate of SWRs during this period. The paper would benefit from a clearer delineation of what the animals are specifically controlling or modulating during the NF trials, ensuring a more comprehensive understanding of the observed effects.

      - Clinical Implications<br /> The study was conducted on healthy, young animals but suggests potential benefits for older, cognitively impaired animals. However, it's possible that older or deficit animals might not respond to the NF protocol in the same way.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Tian et al. aimed to assess differences in biological motion (BM) perception between children with and without ADHD, as well as relationships to indices of social functioning and possible predictors of BM perception (including demographics, reasoning ability and inattention). In their study, children with ADHD showed poorer performance relative to typically developing children in three tasks measuring local, global, and general BM perception. The authors further observed that across the whole sample, performance in all three BM tasks was negatively correlated with scores on the social responsiveness scale (SRS), whereas within groups a significant relationship to SRS scores was only observed in the ADHD group and for the local BM task. Local and global BM perception showed a dissociation in that global BM processing was predicted by age, while local BM perception was not. Finally, general (local & global combined) BM processing was predicted by age and global BM processing, while reasoning ability mediated the effect of inattention on BM processing.

      Strengths:<br /> Overall, the manuscript is presented in a relatively clear fashion and methods and materials are presented with sufficient detail so the study could be reproduced by independent researchers. The study uses an innovative, albeit not novel, paradigm to investigate two independent processes underlying BM perception. The results are novel and have the potential to have wide-reaching impact on multiple fields.

      Weaknesses:<br /> Except for the main analysis, it is unclear what the authors' specific predictions are regarding the three different tasks they employ. The three BM tasks are used to probe different processes underlying BM perception, but it is difficult to gather from the introduction why these three specific tasks were chosen and what predictions the authors have about the performance of the ADHD group in these tasks. Relatedly, the authors do not report whether (and if so, how) they corrected for multiple comparisons in their analyses. As the number of tests one should control for depends on the theoretical predictions (http://daniellakens.blogspot.com/2016/02/why-you-dont-need-to-adjust-you-alpha.html), both are necessary for the reader to assess the statistical validity of the results and any inferences drawn from them. The same is the case for the secondary analyses exploring relationships between the 3 individual BM tasks and social function measured by the social responsivity scale (SRS).

      In relation to my prior point, the authors could provide more clarity on how the conclusions drawn from the results relate to their predictions. For example, it is unclear what specific conclusions the authors draw based on their findings that ADHD show performance differences in all three BM perception tasks, but only local BM is related to social function within this group. Here, the claim is made that their results support a specific hypothesis, but it is unclear to me what hypothesis they are actually referring to (see line 343 & following). This lack of clarity is aggravated by the fact that throughout the rest of the discussion, in particular when discussing other findings to support their own conclusions, the authors often make no distinction between the two processes of interest. Lastly, some of the authors' conclusions related to their findings on local vs global BM processing are not logically following from the evidence: For instance, the authors conclude that their data supports the idea that social atypicalities are likely to reduce with age in ADHD individuals. However, according to their own account, local BM perception - the only measure that was related to social function in their study - is understood to be age invariant (and was indeed not predicted by age in the present study).

      Results reported are incomplete, making it hard for the reader to comprehensively interpret the findings and assess whether the conclusions drawn are valid. Whenever the authors report negative results (p-values > 0.05), the relevant statistics are not reported, and the data not plotted. In addition, summary statistics (group means) are missing for the main analysis.

      Some of the conclusions/statements in the article are too strong and should be rephrased to indicate hypotheses and speculations rather than facts. For example, in lines 97-99 the authors state that the finding of poor BM performance in TD children in a prior study 'indicated inferior applicability' or 'inapplicable experimental design'. While this is one possibility, a perhaps more plausible interpretation could be that TD children show 'poor' performance due to outstanding maturation of the underlying (global) BM processes (as the authors suggest themselves that BM perception can improve with age). There are several other examples where statements are too strong or misleading, which need attention.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the authors introduced ADSE, a SELEX-based protocol to explore the mechanism of emergency of species. They used DNA hybridization (to the bait pool, "resources") as the driving force for selection and quantitatively investigated the factors that may contribute to the survival during generation evolution (progress of SELEX cycle), revealing that besides individual-resource binding, the inter- and intra-individual interactions were also important features along with mutualism and parasitism.

      Strengths:<br /> The design of using pure biochemical affinity assay to study eco-evolution is interesting, providing an important viewpoint to partly explain the molecular mechanism of evolution.

      Weaknesses:<br /> Though the evidence of the study is somewhat convincing, some aspects still need to be improved, mostly technical issues.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The main conclusion of the manuscript is that the presence of linker Histone H1 protects Arabidopsis pericentromeric heterochromatic regions and longer transposable elements via chromatin compaction from encroachment by other repressive pathways. The manuscript focuses on the RNA-dependent DNA-methylation (RdDM) pathway but indirectly finds that other pathways must also be ectopically enriched.

      Strengths:<br /> The authors present diverse sets of genomic data comparing Arabidopsis wild-type and h1 mutant background allowing an analysis of differential recruitment of RdDM component NPRE1, which is related to changes in DNA methylation and H1 coverage. As an addendum, the manuscript also contains recruitment data for SUVH1 in wild-type and h1 mutant backgrounds.

      Furthermore, the authors make use of a line that recruits NRPE1 ectopically to show that H1 occupancy is not altered because of this recruitment. These are negative data, but well supported.

      Weaknesses:<br /> The manuscript mostly confirms earlier observations but shows very limited novelty. It has already been reported that different classes of TEs show a differential response with respect to DNA methylation in absence of H1. Furthermore, the fact that loss of H1 affect global chromatin accessibility was recently published by Teano et al. in Cell reports (Volume 42, Issue 8, 29 August 2023). The authors have neither cited this report (that had been available since 2021 in BioRxiv), nor set their work in context to this study. The study by Teano showed that for some TEs, loss of H1 is related to a switch from DNA-methylation dependent repressive pathways to Polycomb Group-dependent pathways. The current manuscript could have looked at overlapping classes and integrated information from both studies, which would be particularly interesting for the examples illustrated in Figure 5b, showing examples of TEs that lose NRPE1 targeting and methylation in all contexts in H1 deletion mutants.

      The proposed mechanism is that RdDM along with many other chromatin factors re-distribute to heterochromatic regions in h1 mutants because these regions are more accessible. There is a general problem with measuring the "difference in chromatin compaction" with methods that mostly resolve highly accessible chromatin in contrast to any other chromatin, such as ATAC-seq or DNAse-seq (employed in this manuscript). The changes in the regions of interest are so subtle that they are not easily detected at the level of individual genes, although they become usually more obvious in metagene plots. The general question is if this inadequate method is sufficient to draw strong conclusions on chromatin compaction, but to be fair, the current manuscript is not alone in using this method without pointing out certain caveats.

      As a consequence of redistribution to heterochromatic sites, the authors postulate that there are also sites that lose RdDM coverage in h1, but these sites are not really evidenced in the report.<br /> Unfortunately, another weakness is that it is not possible to make easy use of the analysis from the available material as the current manuscript does not contain supplemental data indicating which TEs were and DMRs were considered in classes such as "long", "short", "heterochromatic", "euchromatic", "Class A", "Class B", "CMT2 dependent hypo-CHH", "DRM2 dependent CHH", "dynamic RdDM" etc. Since the bioinformatics pipelines are poorly documented (absence of dedicated script archive), the analysis cannot be easily recapitulated.

    1. Reviewer #2 (Public Review):

      This study examines the construct of "cognitive spaces" as they relate to neural coding schemes present in response conflict tasks. The authors use a novel experimental design in which different types of response conflict (spatial Stroop, Simon) are parametrically manipulated. These conflict types are hypothesized to be encoded jointly, within an abstract "cognitive space", in which distances between task conditions depend only on the similarity of conflict types (i.e., where conditions with similar relative proportions of spatial-Stroop versus Simon conflicts are represented with similar activity patterns). Authors contrast such a representational scheme for conflict with several other conceptually distinct schemes, including a domain-general, domain-specific, and two task-specific schemes. The authors conduct a behavioral and fMRI study to test which of these coding schemes is used by prefrontal cortex. Replicating the authors' prior work, this study demonstrates that sequential behavioral adjustments (the congruency sequence effect) are modulated as a function of the similarity between conflict types. In fMRI data, univariate analyses identified activation in left prefrontal and dorsomedial frontal cortex that was modulated by the amount of Stroop or Simon conflict present, and representational similarity analyses (RSA) that identified coding of conflict similarity, as predicted under the cognitive space model, in right lateral prefrontal cortex.

      This study tackles an important question regarding how distinct types of conflict might be encoded in the brain within a computationally efficient representational format. The ideas postulated by the authors are interesting ones and the statistical methods are generally rigorous. The evidence supporting the authors claims, however, is limited by confounds in the experimental design and by lack of clarity in reporting the testing of alternative hypotheses within the method and results.

      (1) Model comparison

      The authors commendably performed a model comparison within their study, in which they formalized alternative hypotheses to their cognitive space hypothesis. We greatly appreciate the motivation for this idea and think that it strengthened the manuscript. Nevertheless, some details of this model comparison were difficult for us to understand, which in turn has limited our understanding of the strength of the findings.

      The text indicates the domain-general model was computed by taking the difference in congruency effects per conflict condition. Does this refer to the "absolute difference" between congruency effects? In the rest of this review, we assume that the absolute difference was indeed used, as using a signed difference would not make sense in this setting. Nevertheless, it may help readers to add this information to the text.

      Regarding the Stroop-Only and Simon-Only models, the motivation for using the Jaccard metric was unclear. From our reading, it seems that all of the other models --- the cognitive space model, the domain-general model, and the domain-specific model --- effectively use a Euclidean distance metric. (Although the cognitive space model is parameterized with cosine similarities, these similarity values are proportional to Euclidean distances because the points all lie on a circle. And, although the domain-general model is parameterized with absolute differences, the absolute difference is equivalent to Euclidean distance in 1D.) Given these considerations, the use of Jaccard seems to differ from the other models, in terms of parameterization, and thus potentially also in terms of underlying assumptions. Could authors help us understand why this distance metric was used instead of Euclidean distance? Additionally, if Jaccard must be used because this metric seems to be non-standard in the use of RSA, it would likely be helpful for many readers to give a little more explanation about how it was calculated.

      When considering parameterizing the Stroop-Only and Simon-Only models with Euclidean distances, one concern we had is that the joint inclusion of these models might render the cognitive space model unidentifiable due to collinearity (i.e., the sum of the Stroop-Only and Simon-Only models could be collinear with the cognitive space model). Could the authors determine whether this is the case? This issue seems to be important, as the presence of such collinearity would suggest to us that the design is incapable of discriminating those hypotheses as parameterized.

      (2) Issue of uniquely identifying conflict coding

      We certainly appreciate the efforts that authors have taken to address potential confounders for encoding of conflict in their original submission. We broach this question not because we wish authors to conduct additional control analyses, but because this issue seems to be central to the thesis of the manuscript and we would value reading the authors' thoughts on this issue in the discussion.

      To summarize our concerns, conflict seems to be a difficult variable to isolate within aggregate neural activity, at least relative to other variables typically studied in cognitive control, such as task-set or rule coding. This is because it seems reasonable to expect that many more nuisance factors covary with conflict --- such as univariate activation, level of cortical recruitment, performance measures, arousal --- than in comparison with, for example, a well-designed rule manipulation. Controlling for some of these factors post-hoc through regression is commendable (as authors have done here), but such a method will likely be incomplete and can provide no guarantees on the false positive rate.

      Relatedly, the neural correlates of conflict coding in fMRI and other aggregate measures of neural activity are likely of heterogeneous provenance, potentially including rate coding (Fu et al., 2022), temporal coding (Smith et al., 2019), modulation of coding of other more concrete variables (Ebitz et al., 2020, 10.1101/2020.03.14.991745; see also discussion and reviews of Tang et al., 2016, 10.7554/eLife.12352), or neuromodulatory effects (e.g., Aston-Jones & Cohen, 2005). Some of these origins would seem to be consistent with "explicit" coding of conflict (conflict as a representation), but others would seem to be more consistent with epiphenomenal coding of conflict (i.e., conflict as an emergent process). Again, these concerns could apply to many variables as measured via fMRI, but at the same time, they seem to be more pernicious in the case of conflict. So, if authors consider these issues to be germane, perhaps they could explicitly state in the discussion whether adopting their cognitive space perspective implies a particular stance on these issues, how they interpret their results with respect to these issues, and if relevant, qualify their conclusions with uncertainty on these issues.

      (3) Interpretation of measured geometry in 8C

      We appreciate the inclusion of the measured similarity matrices of area 8C, the key area the results focus on, to the supplemental, as this allows for a relatively model-agnostic look at a portion of the data. Interestingly, the measured similarity matrix seems to mismatch the cognitive space model in a potentially substantive way. Although the model predicts that the "pure" Stroop and Simon conditions will have maximal self-similarity (i.e., the Stroop-Stroop and Simon-Simon cells on the diagonal), these correlations actually seem to be the lowest, by what appears to be a substantial margin (particularly the Stroop-Stroop similarities). What should readers make of this apparent mismatch? Perhaps authors could offer their interpretation on how this mismatch could fit with their conclusions.

    1. 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.

    1. Reviewer #2 (Public Review):

      Summary: The current draft by Deischel et.al., entitled "Inhibition of Notch activity by phosphorylation of CSL in response to parasitization in Drosophila" decribes the role of Pkc53E in the phosphorylation of Su(H) to downregulate its transcriptional activity to mount a successful immune response upon parasitic wasp-infection. Overall, I find the study interesting and relevant especially the identification of Pkc53E in phosphorylation of Su(H) is very nice. However, I have a number of concerns with the manuscript which are central to the idea that link the phosphorylation of Su(H) via Pkc53E to implying its modulation of Notch activity. I enlist them one by one subsequently.

      Strengths: I find the study interesting and relevant especially because of the following:<br /> 1. The identification of Pkc53E in phosphorylation of Su(H) is very interesting.<br /> 2. The role of this interaction in modulating Notch signaling and thereafter its requirement in mounting a strong immune response to wasp infection is also another strong highlight of this study.

      Weaknesses:1. Epistatic interaction with Notch is needed: In the entire draft, the authors claim Pkc53E role in the phosphorylation of Su(H) is down-stream of notch activity. Given the paper title also invokes Notch, I would suggest authors show this in a direct epistatic interaction using a Notch condition. If loss of Notch function makes many more lamellocytes and GOF makes less, then would modulating Pkc53E (and SuH)) in this manifest any change? In homeostasis as well, given gain of Notch function leads to increased crystal cells the same genetic combinations in homeostasis will be nice to see.<br /> While I understand that Su(H) functions downstream of Notch, but it is now increasingly evident that Su(H) also functions independent of Notch. An epistatic relationship between Notch and Pkc will clarify if this phosphorylation event of Su(H) via Pkc is part of the canonical interaction being proposed in the manuscript and not a non-canoncial/Notch pathway independent role of Su(H).

      This is important, as I worry that in the current state, while the data are all discussed inlight of Notch activity, any direct data to show this affirmatively is missing. In our hands we do find Notch independent Su(H) function in immune cells, hence this is a suggestion that stems from our own personal experience.

      2. Temporal regulation of Notch activity in response to wasp-infection and its overlapping dynamics of Su(H) phosphorylation via Pkc is needed: First, I suggest the authors to show how Notch activity post infection in a time course dependent manner is altered. A RT-PCR profile of Notch target genes in hemocytes from infected animals at 6, 12, 24, 48 HPI, to gauge an understanding of dynamics in Notch activity will set the tone for when and how it is being modulated. In parallel, this response in phospho mutant of Su(H) will be good to see and will support the requirement for phosphorylation of Su(H) to manifest a strong immune response. Second, is the dynamics of phosphorylation in a time course experiment is missing. While the increased phosphorylation of Su(H) in response to wasp-infestation shown in Fig.2B is using whole animal, this implies a global down-regulation of Su(H)/Notch activity. The authors need to show this response specifically in immune cells. The reader is left to the assumption that this is also true in immune cells. Given the authors have a good antibody, characterizing this same in circulating immune cells in response to infection will be needed. A time course of the phosphorylation state at 6, 12, 24, 48 HPI, to guage an understanding of this dynamics is needed. The authors suggest, this mechanism may be a quick way to down-regulate Notch, hence a side by side comparison of the dynamics of Notch down-regulation (such as by doing RT-PCR of Notch target genes following different time point post infection) alongside the levels of pS269 will strengthen the central point being proposed. Last, in Fig7. the authors show Co-immuno-precipitation of Pkc53EHA with Su(H)gwt-mCh 994 protein from Hml-gal4 hemocytes. I understand this is in homeostasis but since this interaction is proposed to be sensitive to infection, then a Co-IP of the two in immune cells, upon infection should be incorporated to strengthen their point.

      3. In Fig 5B, the authors show the change in crystal cell numbers as read out of PMA induced activation of Pkc53E and subsequent inhibition of Su(H) transcriptional activity, I would suggest the authors use more direct measures of this read out. RT-PCR of Su(H) target genes, in circulating immune cells, will strengthen this point. Formation of crystal cells is not just limited to Notch, I am not convinced that this treatment or the conditions have other affect on immune cells, such as any impact on Hif expression may also lead to lowering of CC numbers. Hence, the authors need to strengthen this point by showing that effects are direct to Notch and Su(H) and not non-specific to any other pathway also shown to be important for CC development.

      4. In addition to the above mentioned points, the data needs to be strengthened to further support the main conclusions of the manuscript. I would suggest the authors present the infection response with details on the timing of the immune response. Characterization of the immune responses at respective time points (as above or at least 24 and 48 HPI, as norms in the field) will be important. Also, any change in overall cell numbers, other immune cells, plasmatocytes or CC post infection is missing and is needed to present the specificity of the impact. The addition of these will present the data with more rigor in their analysis.

      5. Finally, what is the view of the authors on what leads to activation of Pkc53E, any upstream input is not presented. It will be good to see if wasp infection leads to increased Pkc53 kinase activity.

      Overall, I think the findings in the current state are interesting and fill an important gap, but the authors will need to strengthen the point with more detailed analysis that includes generating new data and also presenting the current data with more rigor in their approach. The data have to showcase the relationship with Notch pathway modulation upon phosphorylation of CSL in a much more comprehensive way, both in homeostasis and in response to infection which is entirely missing in the current draft.

    1. Reviewer #2 (Public Review):

      Breast cancer is the most common malignant tumor in women. One of subtypes in breast cancer is so called triple-negative breast cancer (TNBC), which represents the most difficult subtype to treat and cure in the clinic. Chemotherapy drugs including epirubicin and cisplatin are widely used for TNBC treatment. However, drug resistance remains as a challenge in the clinic. The authors uncovered a molecular pathway involved in chemotherapy drug resistance, and molecular players in this pathway represent as potential drug targets to overcome drug resistance. The experiments are well designed and the conclusions drawn mostly were supported by the data. The findings have potential to be translated into the clinic.

    1. Reviewer #2 (Public Review):

      Pheochromocytoma (PCC), a rare neuroendocrine tumor, is currently considered malignant, but non-surgical treatment options are very limited and there is an urgent need for more basic research to support the development of new therapeutic approaches. In the present work, the authors described the intra- and inter-tumor heterogeneity by performing scRNA-seq on tumor samples from five patients with PCC, and evaluated the corresponding PASS scores.

      Strengths: The tumor microenvironment of PCC was characterized and potential molecular classification criteria based on single-cell transcriptomics were proposed, offering new theoretical possibilities for the treatment of PCC. The article is logically written and the results are clearly presented.

      Weaknesses: I still have concerns about some of the article's content. My main concerns are: In this study, the authors seem to have demonstrated the inaccuracy of a subjective score (PASS) by another objective means (scRNA-seq). In fact, the multiparametric scoring systems such as PASS are no longer endorsed in the 2022 WHO guidelines. The PASS scoring system does not have a high positive predictive value for risk stratification of PCC metastasis, but "rule-out" of metastasis risk with a PASS score of <4 seems to be fairly reliable. Could the authors please explain why the PASS scores were chosen rather than the GAPP, m-GAPP, or COPPS scoring systems? If possible, please try to emphasize the importance and necessity of using the PASS scoring system, either by replacing it with a more acceptable scoring system or by deleting the relevant part, which does not seem to be very relevant to the subject of the article.

      Moreover, I noted the following statement in the text "There are no studies reporting the composition of immune cells in PCCs. The few published studies investigating the immune microenvironment of PCCs have been limited to the expression of PDL1 at the histological level and to assessment of the tumor mutation burden (TMB) at the genomic level, and these results only seem to suggest that PCCs are immune-cold (Bratslavsky et al, 2019; Guo et al, 2019; Pinato et al, 2017)." This statement is very wrong. The reason for this error may be that the authors did not adequately search and read the relevant literature. I noticed that almost all references in this paper are dated 2021 and earlier, which is surprising. Please update the references cited in this paper in a comprehensive and detailed manner; referring to literature published too early may lead to inadequate discussion or even one-sided or incorrect conclusions and conjectures.

      For example, the text statement "Combined with previously reported negative regulatory effects of kinases (such as RET, ALK, and MEK) on HLA-I expression on tumor cells (Brea et al., 2016; Oh et al., 2019), we speculate that the possible reason for inability in recruiting CD8+ T cells of kinase-type PCCs is the downregulation of HLA-I in tumor cells regulated by RET, while the mechanism of immune escape in metabolism-type PCCs (with antigen presentation ability) needs to be further explored. Our results also indicate that the application of immunotherapy to metabolism-type PCCs is likely unsuitable, while kinase-type PCCs may have the potential of combined therapy with kinase inhibitors and immunotherapy." is rather one-sided; in fact, the presence of immune escape in PCC, as the malignancy with the lowest tumor mutation compliance, has been well characterized, and the low number of infiltrating T cells in tumor tissue may be influenced by a variety of factors, such as the release of catecholamines, the expression of inhibitory receptors on the surface of T cells, and so on, although genetic mutation still plays the most crucial role. The Discussion section also has a lot of information that needs to be updated or corrected and expanded, so please rewrite the above section with sufficiently updated references.

      Below I have listed some references for the authors to read:<br /> Tufton N, Hearnden RJ, Berney DM, et al. The immune cell infiltrate in the tumour microenvironment of phaeochromocytomas and paragangliomas. Endocr Relat Cancer. 2022;29(11):589-598. Published 2022 Sep 19. doi:10.1530/ERC-22-0020<br /> Jin B, Han W, Guo J, et al. Initial characterization of immune microenvironment in pheochromocytoma and paraganglioma. Front Genet. 2022;13:1022131. Published 2022 Dec 7. doi:10.3389/fgene.2022.1022131<br /> Celada L, Cubiella T, San-Juan-Guardado J, et al. Pseudohypoxia in paraganglioma and pheochromocytoma is associated with an immunosuppressive phenotype. J Pathol. 2023;259(1):103-114. doi:10.1002/path.6026<br /> Calsina B, Piñeiro-Yáñez E, Martínez-Montes ÁM, et al. Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma. Nat Commun. 2023;14(1):1122. Published 2023 Feb 28. doi:10.1038/s41467-023-36769-6

    1. Reviewer #2 (Public Review):

      In this manuscript by Kang et. al., the authors investigated the mechanisms of K+-efflux-coupled SOCE in NLRP3 inflammasome activation by LP(LPS+PA, and identified an essential role of TRPM2-mediated lysosomal Ca2+ release and subsequent IP3Rs-mediated ER Ca2+ release and store depletion in the process. K+ efflux is shown to be mediated by a Ca2+-activated K+ channel (KCa3.1). LP-induced cytosolic Ca2+ elevation also induced a delayed activation of ASK1 and JNK, leading to ASC oligomerization and NLRP3 inflammasome activation. Overall, this is an interesting and comprehensive study that has identified several novel molecular players in metabolic inflammation. The manuscript can benefit if the following concerns could be addressed:

      1. The expression of TRPM2 in the lysosomes of macrophages needs to be more definitively established. For instance, the cADPR-induced TRPM2 currents should be abolished in the TRPM2 KO macrophages. Can you show the lysosomal expression of TRPM2, either with an antibody if available or with a fluorescently-tagged TRPM2 overexpression construct?

      2. Can you use your TRPM2 inhibitor ACA to pharmacologically phenocopy some results, e.g., about [Ca2+]ER, [Ca2+]LY, and [Ca2+]i from the TRPM2 knockout?

      3. In Fig. S4A, bathing the cells in zero Ca2+ for three hours might not be ideal. Can you use a SOCE inhibitor, e.g, YM-58483, to make the point?

      4. In Fig. 1A, you need a positive control, e.g., ionomycin, to show that the GPN response was selectively reduced upon LP treatment.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors identified a new chloride-conducting Channelrhodopsin (MsACR1) that can be activated at low light intensities and within the red part of the visible spectrum. Additional engineering of MsACR1 yielded a variant (raACR1) with increased current amplitudes, accelerated kinetics, and a 20nm red-shifted peak excitation wavelength. Stimulation of MsACR1 and raACR1 expressing neurons with 635nm in mice's primary motor cortices inhibited the animals' locomotion.

      Strengths:<br /> The in vitro characterization of the newly identified ACRs is very detailed and confirms the biophysical properties as described by the authors. Notably, the ACRs are very light sensitive and allow for efficient in vitro inhibition of neurons in the nano Watt/mm^2 range. These new ACRs give neuroscientists and cell biologists a new tool to control chloride flux over biological membranes with high temporal and spatial precision. The red-shifted excitation peaks of these ACRs could allow for multiplexed application with blue-light excited optogenetic tools such as cation-conducting channelrhodopsins or green-fluorescent calcium indicators such as GCaMP.

      Weaknesses:<br /> The in-vivo characterization of MsACR1 and raACR1 lacks critical control experiments and is, therefore, too preliminary. The experimental conditions differ fundamentally between in vitro and in vivo characterizations. For example, chloride gradients differ within neurons which can weaken inhibition or even cause excitation at synapses, as pointed out by the authors. Notably, the patch pipettes for the in vitro characterization contained low chloride concentrations that might not reflect possible conditions found in the in vivo preparations, i.e., increasing chloride gradients from dendrites to synapses.

      Interestingly, the authors used soma-targeted (st) MsACR1 and raACR1 for some of their in vitro characterization yielding more efficient inhibition and reduction of co-incidental "on-set" spiking. Still, the authors do not seem to have utilized st-variants in vivo.

      Most importantly, critical in vivo control experiments, such as negative controls like GFP or positive controls like NpHR, are missing. These controls would exclude potential behavioral effects due to experimental artifacts. Moreover, in vivo electrophysiology could have confirmed whether targeted neurons were inhibited under optogenetic stimulations.

      Some of these concerns stem from the fact that the pulsed raACR stimulation at 635 nm at 10Hz (Fig. 3E) was far less efficient compared to MsACR1, yet the in vivo comparison yielded very similar results (Fig. 4D).

      Also, the cortex is highly heterogeneous and comprises excitatory and inhibitory neurons. Using the synapsin promoter, the viral expression paradigm could target both types and cause differential effects, which has not been investigated further, for example, by immunohistochemistry. An alternative expression system, for example, under VGLUT1 control, could have mitigated some of these concerns.

      Furthermore, the authors applied different light intensities, wavelengths, and stimulation frequencies during the in vitro characterization, causing varying spike inhibition efficiencies. The in vivo characterization is notably lacking this type of control. Thus, it is unclear why the 635nm, 2s at 20Hz every 5s stimulation protocol, which has no equivalent in the in vitro characterization, was chosen.

      In summary, the in vivo experiments did not confirm whether the observed inhibition of mouse locomotion occurred due to the inhibition of neurons or experimental artifacts.

      In addition, the author's main claim of more efficient neuronal inhibition would require them to threshold MsACR1 and raACR1 against alternative methods such as the red-shifted NpHR variant Jaws or other ACRs to give readers meaningful guidance when choosing an inhibitory tool.

      The light sensitivity of MsACR1 and raACR1 are impressive and well characterized in vitro. However, the authors only reported the overall light output at the fiber tip for the in vivo experiments: 0.5 mW. Without context, it is difficult to evaluate this value. Calculating the light power density at certain distances from the light fiber or thresholding against alternative tools such as NpHR, Jaws, or other ACRs would allow for a more meaningful evaluation.

    1. Reviewer #2 (Public Review):

      This paper presents a novel measure of complexity that can be applied to recorded neurophysiological time series. The paper first introduces an existing measure, Lempel-Ziv complexity, reviewing its computation, application, and potential issues. They then present their new metric: CSER. They show CSER values change similarly to LZ under psychedelics, sleep, and general anaesthesia. A key advantage of CSER is that it can be decomposed in both time and frequency. They give example applications for each of these. They show the differences in CSER in the previous examples are mostly located in the gamma band. For a temporal example, they consider monkey ecog in an oddball task and so CSER changes between oddballs and deviants.

      Major comments<br /> Most of the technical details are rightly in the methods, but it would be nice as a reader to have more of a concrete idea of the type of state space model used in the main text, the assumptions underlying this, and typical orders used perhaps with a schematic diagram etc. I appreciate they have written the paper to appeal to a broad general audience, but it seems like this is an important part of the method that anyone using the method should understand in more detail.

      It might be nice to cover some other methods of signal variation e.g. as reviewed in Washke et al. Neuron 2021 and how CSER fits into the broader taxonomy of measures of neural variability (even if restricted to information-theoretic ones e.g. multi-scale entropy and permutation entropy, which have also been linked to prediction in the brain Washke et al. elife 2019).

      While the examples are clear and well-motivated, the novel parts could be more developed in terms of interpretation, or linking to existing measures. For example, the frequency results show the complexity changes in "gamma" which is defined as >25Hz. From a biological point of view, it would be nice to understand this better, perhaps splitting low gamma (including 40Hz oscillations) from high gamma (ie MUA). How is the frequency measure affected by the width of the frequency band considered? I understand the sum of the shown terms equals the broadband result but e.g. in Figure 3 if the values were normalised by the bandwidth of each band, gamma might not stand out so much (as it is by far the widest band, 75Hz vs 3Hz for the delta). So if gamma is not contributing more per-unit of frequency, the interpretation might be different. What is it about the gamma band activity that is changing between the conditions: autocorrelation of power, more variability in phase procession? What would this measure give for simulated systems with known changes (for example, changes in oscillatory power, or changes in 1/f slope). What sort of system would give the profiles in Figure 3?

      For the temporal example, the result is a nice proof of concept. It looks quite reminiscent of "novel mutual information" time-course (e.g. compare the absolute value of CSER difference to Figure 13, Ince et al HBM 2017, which also showed two peaks of novel information at the time where the gradient of the ERP starts to change, 20-30ms prior to the ERP peak, but in a task with no predictive component). It might be nice to explicitly compare the statistical power to this existing method (conditional mutual information between signal+gradient and experimental condition, conditioning out the selection of previous time points with peak conditional MI). Deviant stimuli initially seem to decrease entropy - by eye, it's surprising this isn't significant (stands out a lot from baseline). Was a two-sided or one-sided (matching the prior hypothesis) test performed here? Could it be that the change in entropy rate is a property of any ERP signal (ie it looks like the change in CSER reflects the following difference in peak ERP - for the first negative peak, the deviant amplitude is lower, for the second positive peak the deviant amplitude is higher), and a lower level signal interpretation (ie amplitude of CSER difference is related to the difference in ERP amplitude, rather than directly reflecting neural mechanisms of prediction).

    1. Reviewer #3 (Public Review):

      Summary:<br /> In the current manuscript, Dekraker and colleagues have demonstrated the ability to align hippocampal subfield parcellations across disparate 3D histology samples that differ in contrast, resolution, and processing/staining methods. In doing so, they validated the previously generated Big-Brain atlas by comparing across seven different ground-truth subfield definitions. This is an impressive effort that provides important groundwork for future in vivo multi-atlas methods.

      Strengths:<br /> DeKraker and colleagues have provided novel evidence for the tremendously complicated curvature/gyrification of the hippocampus. This work underscores the challenge that this complicated anatomy presents in our ability to co-register other types of hippocampal data (e.g. MRI data) to appropriately align and study a structure in which the curvature varies considerably across individuals.

      This paper is also important in that it highlights the utility of using post-mortem histological datasets, where ground truth histology is available, to inform our rigorous study of the in vivo brain.

      This work may encourage readers to consider the limitations of the current methods that they currently use to co-register and normalize their MRI data and to question whether these methods are adequate for the examination of subfield activity, microstructure, or perfusion in the hippocampal head, for example. Thus the implications of this work could have a broad impact on the study of hippocampal subfield function in humans.

      Weaknesses:<br /> As the authors are well aware, hippocampal subfield definitions vary considerably across laboratories. For example, some neuroanatomists (Ding, Palomero-Gallagher, Augustinack) recognize that the prosubiculum is a distinct region from subiculum and CA1 but others (e.g. Insausti, Duvernoy) do not include this as a distinct subregion. Readers should be aware that there is no universal consensus about the definition of certain subfields and that there is still disagreement about some of the boundaries even among the agreed upon regions.

    1. Reviewer #2 (Public Review):

      In this paper, the authors utilize optogenetic stimulation and imaging techniques with fluorescent reporters for pH and membrane voltage to examine the extent of intracellular acidification produced by different ion-conducting opsins. The commonly used opsin CheRiff is found to conduct enough protons to alter intracellular pH in soma and dendrites of targeted neurons and in monolayers of HEK293T cells, whereas opsins ChR2-3M and PsCatCh2.0 are shown to produce negligible changes in intracellular pH as their photocurrents are mostly carried by metal cations. The conclusion that ChR2-3M and PsCatCh2.0 are more suited than proton conducting opsins for optogenetic applications is well supported by the data.

    1. Reviewer #2 (Public Review):

      In this study, Moore et al. utilise resting-state fMRI data from the Developing Human Connectome Project, applying a recently developed technique ("connectopic mapping") to identify gradients of functional connectivity within resting-state networks in the human foetal brain. Whilst such gradients have previously been identified in adults, this is the first study to explore the topographic organisation of functional connectivity in the foetal brain. Furthermore, the authors describe localised changes within these gradients over the course of gestation, particularly in brain regions implicated in multisensory processing. Together, these results imply that topographic gradients of brain function are present within the developing foetal brain, and continue to develop through gestation. However, the study does not consider critical confounds inherent in the connectopic mapping technique, and as such I do not believe that the data as presented are sufficient to support the conclusions.

      Recent evidence (Watson & Andrews, 2023, Neuroimage) has indicated that the connectopic mapping technique employed here can be substantially confounded by spatial autocorrelations present within the data (for instance, occurring naturally due to the inherent smoothness of the BOLD response, and/or introduced artificially during standard data pre-processing steps such as spatial smoothing or interpolation between co-ordinate spaces). These confounds allow connectopic gradients to be obtained even from random data, and which appear highly similar to those obtained from real data, suggesting that these gradients are strongly influenced by such confounds. Consequently, the resulting gradients may be an inevitability of the way the connectopic mapping technique works, rather than reflecting underlying brain functions per se.

      In the current study, all of the gradients flow smoothly and continuously along a single axis within every network region, typically oriented relative to the long axis of the region. To put it another way - the connectopic mapping gives fundamentally the same answer in every network region. Such an organisation does feel a bit biologically implausible, and could be more consistent with the gradients representing an inevitable solution of the analysis technique, rather than necessarily reflecting brain function. Indeed, in some cases the gradients do not correspond well to known organisational principles of the regions. For instance, the primary gradient in the principal visual network flows smoothly along a superior to inferior axis, which the authors suggest corresponds to retinotopic polar angle maps - however, polar angle maps would be expected to reverse direction between each visual region, yet such reversals are not present in this connectopic map. The authors note that the foetal gradients appear highly similar to those previously obtained within similar regions in adult participants - this could be indicative of a consistent organisation across development, but would also be consistent with the same confound affecting foetal and adult participants. The reported changes in the gradients across gestation could reflect changes in the extent of these spatial autocorrelations or in the shape of the regions of interest (perhaps in turn resulting from changes in the underlying brain geometry) rather than necessarily reflecting development of brain function or specialisation. None of this precludes the possibility that these connectopic gradients may (at least partially) also reflect genuine brain functions, but it does obfuscate the extent to which they do so. It would be useful for the authors to give some consideration to this issue.

      On a different note, could the authors comment on their reason for studying these gradients at the network level. The authors argue (and I agree) that brain function is likely to be organised topographically, rather than split into discrete parcellated regions. Nevertheless, the brain networks the authors choose to use are themselves discrete regions of interest (albeit fairly large ones). Other groups (e.g., Margulies et al, 2016, PNAS) have described coarser-scale connectopic gradients spanning the whole brain. Is there a reason that the authors have chosen to extract network-level gradients, rather than say coarser-scale whole-brain gradients? Have the authors considered examining how whole-brain gradients change over gestation?

      Lastly, the correlated changes between gradients and gestation week appear to occur within small localised clusters. Does this reflect local perturbations of the gradient, or is there perhaps a wider change in the gradient as a whole and these clusters reflect extreme points within this that have changed the most (for instance corresponding to an expansion/contraction of the gradient)?

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this study, the authors sought to understand how the receptive fields of bipolar cells contribute to direction selectivity in starburst amacrine cell (SAC) dendrites, their post synaptic partners. In previous literature, this contribution is primarily conceptualized as the 'space-time wiring model', whereby bipolar cells with slow-release kinetics synapse onto proximal dendrites while bipolar cells with faster kinetics synapse more distally, leading to maximal summation of the slow proximal and fast distal depolarizations in response to motion away from the soma. The space-time wiring contribution to SAC direction selectivity has been extensively tested in previous literature using connectomic, functional, and modeling approaches. However, the authors argue that previous functional studies of bipolar cell kinetics have focused on static stimuli, which may not accurately represent the spatiotemporal properties of the bipolar cell receptive field in response to movement. Moreover, this group and others have recently shown that bipolar cell signal processing can change directionally when visual stimuli starts within the receptive field rather than passing through it, complicating the interpretation of moving stimuli that start within a bipolar cell of interest's receptive field (e.g. stimulating only one branch of a SAC or expanding/contracting rings). Thus, the authors choose to focus on modeling and functionally mapping bipolar cell kinetics in response to moving stimuli across the entire SAC dendritic field.

      General Comments<br /> There have been several studies that have addressed the contribution of space-time wiring to SAC process direction selectivity. The impact of this project is to show that this contribution is limited. First, the optimal solution obtained by the evolutionary algorithm to generate DS processes is slow proximal and fast distal inputs - exactly what is predicted by space-time wiring, which is exactly what is required of the HRC model. Hence, this result seems expected and it's not clear what the alternative hypothesis is. Second, the experimental results based on glutamate imaging to assess the kinetics of glutamate release under conditions of visual stimulation across a large region of retina confirm previous observations but were important to test. Third, by combining their model model with this experiment data, they conclude that even the optimal space-time wiring is not sufficient to explain the SAC process DS. The results of this approach might be more impactful if the authors come to some conclusion as to what factors do determine the direction selectivity of the SAC process since they have argued that all the current models are not sufficient.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this paper by de Guglielmo and colleagues, the authors were interested in analyzing addiction-like behaviors using a very large number of heterogeneous outbred rats in order to determine the relationships among these behaviors. The paper used both males and females on the order of hundreds of rats, allowing for detailed and complex statistical analyses of the behaviors. The rats underwent cocaine self-administration, first via 2-hour access and then via 6-hour access. The rats also underwent a test of punishment resistance in which footshocks were administered a portion of the time a lever was pressed. The authors also conducted a progressive ratio test to determine the break point for "giving up" pressing the lever and a bottle-brush test to determine the rats' "irritability". Ultimately, principal component analysis revealed that escalation of intake during 6-hour access, punishment resistance, and breakpoint all loaded onto the same principal component. Moreover, the authors also identified a subgroup of "resilient" rats that qualitatively differed from the "vulnerable" rats and also identified sex differences in their work.

      Strengths:<br /> The use of heterogeneous rats and the use of so many rats are major strengths of this paper. Moreover, the statistical analyses are particular strengths as they enabled the identification of the three measures as likely reflecting a single underlying construct. The behavioral methods themselves are also strong, as the authors used behavioral measures commonly used in the field that will enable comparison with the field at large. In general, the results support most of the conclusions and provide a wealth of data to the field.

      Weaknesses:<br /> Because the authors used so many rats (~600), it is not clear how strong the effects are. That is, a large n makes it easy to identify small effect sizes, but no effect sizes are presented regarding the findings.

      The Discussion includes parts that argue that the extended access model is a better model of addiction than short access and suggests that this paper provides support for that. However, there were no rats given short-access for the same period of time as the rats in this paper - i.e., no comparison group. Rather, the only comparison that can be made is as the rats transition from short to long access. The data in Figure 1B appear to show that the rats continue their increase in cocaine intake when they transition from short access to long access. The authors do not provide any statistical analyses about this escalation of intake during short access. However, they claim that "measures related to short-term cocaine intake" were orthogonal to those collected during longer access periods, yet it is not clear to me what measures those are. Nonetheless, as indicated in Figure 1H, it appears that the rats consistently shift from PC1 to PC2 across self-administration, regardless of whether they are in the short or long access period. That is, the long-access measures appear to simply be a continuation of the pattern begun during short access. As a result, notwithstanding the lack of a true short-access control group, it is difficult to see how the authors can draw conclusions about short vs. long access in this paper.

      Moreover, as illustrated in Figure 3A, the resilient vs. vulnerable subtypes are apparent during short access self-administration (i.e., they do not require long-access self-administration to develop or be revealed). This suggests, if anything, that short access would be sufficient for identifying such groups. Similarly, Figure 5 shows that short access would be sufficient to identify the "low" vulnerability quartile vs. the other three groups.

      During the discussion, the authors briefly discuss gender differences with regard to cocaine use disorder, with the authors trying to claim that women may be more vulnerable to cocaine use disorder. However, the two papers cited do not support that, as they are papers with rodents. A recent comprehensive review on humans with regard to cocaine craving and relapse noted no reliable gender differences (Nicolas et al., 2022, Pharmacological Reviews) and, as the authors themselves noted, men suffer from cocaine use disorder at higher rates than women.

      The authors noted that the rats received 0.5 mg/kg/infusion of cocaine but provided no explanation for how this dosing was maintained (or whether it was maintained) across the length of the study. Considering that rats, especially males, increase in size quite a bit during this stage, this could affect measures like intake as well as skew sex difference results. Likewise, the data are presented strictly in the number of cocaine infusions, which does not allow for consideration of body weight.

      In the Introduction, the authors make a number of arguments in the second paragraph that have no citations and, therefore, are unsupported.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The idea of harnessing small molecules that may affect protein-protein interactions to promote axon regeneration is interesting and worthy of study. In this manuscript, Liu et al. explore a 14-3-3-Spastin complex and its role in axon regeneration.

      Strengths:<br /> Some of the effects of FC-A on locomotor recovery after spinal cord contusion look interesting.

      Weaknesses:<br /> The manuscript falls short of establishing that a 14-3-3-Spastin complex is important for any FC-A-dependent effects and there are several issues with data quality that make it difficult to interpret the results. Importantly, the effects of the Spastin inhibitor have a major impact on neurite outgrowth suggesting that cells simply cannot grow in the presence of the inhibitor and raising serious questions about any selectivity for FC-A - dependent growth. Aspects of the histology following spinal cord injury were not convincing.

    1. Reviewer #2 (Public Review):

      Summary: Franke et al. characterize the representation of color in the primary visual cortex of mice and how it changes across the visual field, with a particular focus on how this may influence the ability to detect aerial predators. Using calcium imaging in awake, head-fixed mice, they characterize the properties of V1 neurons (layer 2/3) using a large center-surround stimulation where green and ultra-violet were presented in random combinations. Using a clustering approach, a set of functional cell-types were identified based on their preference to different combinations of green and UV in their center and surround. These functional types were demonstrated to have varying spatial distributions in V1, including one neuronal type (Green-ON/UV-OFF) that was much more prominent in the posterior V1 (i.e. upper visual field). Modelling work suggests that these neurons likely support the detection of predator-like objects in the sky.

      Strengths:<br /> The large-scale single-cell resolution imaging used in this work allows the authors to map the responses of individual neurons across large regions of the visual cortex. Combining this large dataset with clustering analysis enabled the authors to group V1 neurons into distinct functional cell types and demonstrate their relative distribution in the upper and lower visual fields. Modelling work demonstrated the different capacity of each functional type to detect objects in the sky, providing insight into the ethological relevance of color opponent neurons in V1.

      Weaknesses:<br /> While the study presents solid evidence a few weaknesses exist, including the size of the dataset, clarity regarding details of data included in each step of the analysis and discussion of caveats of the work. The results presented here are based on recordings of 3 mice. While the number of neurons recorded is reasonably large (n > 3000) an analysis that tests for consistency across animals is missing. Related to this, it is unclear how many neurons at each stage of the analysis come from the 3 different mice (except for Suppl. Fig 4). Finally, the paper would greatly benefit from a more in depth discussion of the caveats related to the conclusion drawn at each stage of the analysis. This is particularly relevant regarding the caveats related to using spike triggered averages to assess the response preferences of ON-OFF neurons, and the conclusions drawn about the contribution of retinal color opponency.

      The authors provide solid evidence to support an asymmetric distribution of color opponent cells in V1 and a reduced color contrast representation in lower light levels. Some statements would benefit from more direct evidence such as the integration of upstream visual signals for color opponency in V1.

      Overall, this study will be a valuable resource for researchers studying color vision, cortical processing, and the processing of ethologically relevant information. It provides a useful basis for future work on the origin of color opponency in V1 and its ethological relevance.

    1. Reviewer #2 (Public Review):

      In this work, Dasgupta et al. investigates the role of Sema7a in the formation of peripheral sensory circuit in the lateral line system of zebrafish. They show that Sema7a protein is present during neuromast maturation and localized, in part, to the base of hair cells (HCs). This would be consistent with pre-synaptic Sema7a mediating formation and/or stabilization of the synapse. They use sema7a loss-of-function strain to show that lateral line sensory terminals display abnormal arborization. They provide highly quantitative analysis of the lateral line terminal arborization to show that a number of specific topological parameters are affected in mutants. Next, they ectopically express a secreted form of Sema7a to show that lateral line terminals can be ectopically attracted to the source. Finally, they also demonstrate that the synaptic assembly is impaired in the sema7a mutant. Overall, the data are of high quality and properly controlled. The availability of Sema7a antibody is a big plus, as it allows to address the endogenous protein localization as well to show the signal absence in the sema7a mutant. The quantification of the arbor topology should be useful to people in the field who are looking at the lateral line as well as other axonal terminals. I think some results are overinterpreted though. The authors state: "Our findings demonstrate that Sema7A functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development." However, they have not actually demonstrated which isoform functions in HCs (also see comments below). In addition, they have to be careful in interpreting their topology analysis, as they cannot separate individual axons. Thus, such analysis can generate artifacts. They can perform additional experiments to address these issues or adjust their interpretations.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In an fMRI study requiring participants to attend to one or another object category, either when the object was presented in isolation or with another object superimposed, the authors compared measured univariate and multivariate activation from object-selective and early visual cortex to predictions derived from response gain and tuning sharpening models. They observed a consistent result across higher-level visual cortex that more-divergent responses to isolated stimuli from category pairs predicted a greater modulation by attention when attending to a single stimulus from the category pair presented simultaneously, and argue via simulations that this must be explained by tuning sharpening for object categories.

      Strengths:<br /> - Interesting experiment design & approach - testing how category similarity impacts neural modulations induced by attention is an important question, and the experimental approach is principled and clever.

      - Examination of both univariate and multivariate signals is an important analysis strategy.

      - The acquired dataset will be useful for future modeling studies.

      Weaknesses:<br /> - The experimental design does not allow for a neutral 'baseline' estimate of neural responses to stimulus categories absent attention (e.g., attend fixation), nor of the combination of the stimulus categories. This seems critical for interpreting results (e.g., how should readers understand univariate results like that plotted in Fig. 4C-D, where the univariate response is greater for 2 stimuli than one, but the analyses are based on a shift between each extreme activation level?).

      - Related, simulations assume there exists some non-attended baseline state of each individual object representation, yet this isn't measured, and the way it's inferred to drive the simulations isn't clearly described.

      - Some of the simulation results seem to be algebraic (univariate; Fig. 7; multivariate, gain model; Fig. 8).

      - Cross-validation does not seem to be employed - strong/weak categories seem to be assigned based on the same data used for computing DVs of interest - to minimize the potential for circularity in analyses, it would be better to define preferred categories using separate data from that used to quantify - perhaps using a cross-validation scheme? This appears to be implemented in Reddy et al. (2009), a paper implementing a similar multivariate method and cited by the authors (their ref 6).

      - Multivariate distance metric - why is correlation/cosine similarity used instead of something like Euclidean or Mahalanobis distance? Correlation/cosine similarity is scale-invariant, so changes in the magnitude of the vector would not change distance, despite this likely being an important data attribute to consider.

      - Details about simulations implemented (and their algebraic results in some cases) make it challenging to interpret or understand these results. E.g., the noise properties of the simulated data aren't disclosed, nor are precise (or approximate) values used for simulating attentional modulations.

      - Eye movements do not seem to be controlled nor measured. Could it be possible that some stimulus pairs result in more discriminable patterns of eye movements? Could this be ruled out by some aspect of the results?

      - A central, and untested/verified, assumption is that the multivariate activation pattern associated with 2 overlapping stimuli (with one attended) can be modeled as a weighted combination of the activation pattern associated with the individual stimuli. There are hints in the univariate data (e.g., Fig. 4C; 4D) that this might not be justified, which somewhat calls into question the interpretability of the multivariate results.

      - Throughout the manuscript, the authors consistently refer to "tuning sharpening", an idea that's almost always used to reference changes in the width of tuning curves for specific feature dimensions (e.g., motion direction; hue; orientation; spatial position). Here, the authors are assaying tuning to the category (across exemplars of the category). The link between these concepts could be strengthened to improve the clarity of the manuscript.

    1. Reviewer #2 (Public Review):

      This study used coarse-grained molecular dynamics simulation to explain how the binding of polyPR might interfere with distinct stages of the transport cycle. This finding shows that the interaction between polyPR and transport components is driven by electrostatic interactions and is correlated with the salt concentration and the length of polyPR, providing an important basis for subsequent exploration of the impact of C9orf72 R-DPRs on NCT disruption.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study builds upon previous work that demonstrated that brain injury results in leakage of albumin across the blood-brain barrier, resulting in activation of TGF-beta in astrocytes. Consequently, this leads to decreased glutamate uptake, reduced buffering of extracellular potassium, and hyperexcitability. This study asks whether such a process can play a physiological role in cortical plasticity. They first show that stimulation of a forelimb for 30 minutes in a rat results in leakage of the blood-brain barrier and extravasation of albumin on the contralateral but not ipsilateral cortex. The authors propose that the leakage is dependent upon neuronal excitability and is associated with an enhancement of excitatory transmission. Inhibiting the transport of albumin or the activation of TGF-beta prevents the enhancement of excitatory transmission. In addition, gene expression associated with TGF-beta activation, synaptic plasticity, and extracellular matrix are enhanced on the "stimulated" hemisphere. That this may translate to humans is demonstrated by a breakdown in the blood-brain barrier following activation of brain areas through a motor task.

      Strengths:<br /> This study is novel and the results are potentially important as they demonstrate an unexpected breakdown of the blood-brain barrier with physiological activity and this may serve a physiological purpose, affecting synaptic plasticity.

      The strengths of the study are:<br /> 1) The use of an in vivo model with multiple methods to investigate the blood-brain barrier response to a forelimb stimulation.<br /> 2) The determination of a potential functional role for the observed leakage of the blood-brain barrier from both a genetic and electrophysiological viewpoint.<br /> 3) The demonstration that inhibiting different points in the putative pathway from activation of the cortex to transport of albumin and activation of the TGF-beta pathway, the effect on synaptic enhancement could be prevented.<br /> 4) Preliminary experiments demonstrating a similar observation of activity-dependent breakdown of the blood-brain barrier in humans.

      Weaknesses:<br /> There are both conceptual and experimental weaknesses.

      1) The stimulation is in an animal anesthetized with ketamine, which can affect critical receptors (ie NMDA receptors) in synaptic plasticity.

      2) The stimulation protocol is prolonged and it would be helpful to know if briefer stimulations have the same effect or if longer stimulations have a greater effect ie does the leakage give a "readout" of the stimulation intensity/length.

      3) For some of the experiments (see below), the numbers of animals are low and the statistical tests used may not be the most appropriate, making the results less clear cut.

      4) The experimental paradigms are not entirely clear, especially the length of time of drug application and the authors seem to try to detect enhancement of a blocked SEP.

      4) It is not clear how long the enhancement lasts. There is a remark that it lasts longer than 5 hours but there is no presentation of data to support this.

      5) It is not clear if this enhancement of synaptic transmission has any physiological role.

      6) The spatial and temporal specificity of this effect is unclear (other than hemispheric in rats) and even less clear in humans.

      7) It is not clear to what extent the experimenters and those doing the analysis were blinded to group. If neither were blind to group, then considerable biases could be introduced.

      8) The experimenters rightly use separate controls for most of the experiments but this is not always the case, also raising the possibility that the application of drugs was not done randomly or interleaved, but possibly performed in blocks of animals, which can also affect results.

      9) Methyl-beta-cyclodextrin clears cholesterol so the effect on albumin transport is not specific, it could be mediating its effect through some other pathway.

      10) Since the breakdown of the blood-brain barrier can be inhibited by a TGF-beta inhibitor, then this implies that TGF-beta is necessary for the breakdown of the blood-brain barrier. This does not sit well with the hypothesis that TGF-beta activation depends upon blood-brain barrier leakage.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The paper entitled "PAK3 downregulation induces cognitive 1 impairment following cranial irradiation" by Lee et al. aimed at investigating the functional impact of cranial irradiation in mouse and propose PAK3 as molecular element involved in radiation-induced cognitive decrement. The results provided in this paper are problematic as both the irradiation paradigm (5X2 Gy) as well as the timing of investigation (3 to 8 days post-IR) are completely irrelevant to investigate radiation induced neurocognitive impairment. This testifies to the team's lack of knowledge in radiobiology/radiotherapy and the methodology to explore radiation induced neurocognitive damages. It precludes any further relevance of the molecular results.

      Weaknesses:<br /> First and according to the BED equation a single dose of 10 Gy cannot not be approximated by 5 fractions of 2 Gy, as fractionation is known to decrease normal tissue toxicity. Note that in radiobiology/radio-oncology, the BED stands for "Biologically Effective Dose." This equation is used to compare the effects of different radiation treatments on biological tissues, taking into account the dose, fractionation, and the overall biological response of the tissue to radiation.<br /> The BED equation is commonly used to calculate the equivalent dose of a fractionated radiation treatment, which is the dose that would produce the same biological effect as a single, higher dose delivered in a single fraction.<br /> The general formula for BED is:BED = D * (1 + d / α/β)<br /> D is the total physical dose of radiation delivered in Grays (Gy)<br /> d is the dose per fraction in Gy<br /> α/β is the tissue-specific ratio of the linear (α) and quadratic (β) components of the radiation response. It is measured in Gy and describes how the tissue responds to different fractionation schedules (usually equal to 3 for the normal brain).<br /> Please refers to radiobiology/radiotherapy textbooks by Hall or Joiner.

      Second, the brain is a late responding organ. GBM patients treated with 60 Gy exhibit progressive and debilitating impairments in memory, attention and executive function several month post-irradiation. In mice, neurocognitive decrements after a single dose of 10 Gy delivered to the whole brain does occur at late time point, usually > 2 months post-exposure. Multiple publications such as the one by Limoli C lab, Rossi S lab, Britten R lab or earlier Fike J lab and Robin M lab support this. Next, 5 fractions of 2 Gy will be more protective than a single dose of 10 Gy and neurocognitive decrements will require at least 5-6 months to occur if they ever occur. In Figure 1, the decrement reported is marginal, the number of animals included (4 to 5 at most?) The number of animals is not specified) is too low to draw any significant conclusions. In addition to the timing issue, the strategy described for NOR analysis shows methodological issues with the habituation period being too short and exploration level being very low.

    1. Reviewer #2 (Public Review):

      Summary:

      Spargo and colleagues present an analysis of the shared genetic architectures of Schizoprehnia and several late-onset neurological disorders. In contrast to many polygenic traits for which global genetic correlation estimates are substantial, global genetic correlation estimates for neurological conditions are relatively small, likely for several reasons. One is that assortative mating, which will spuriously inflate genetic correlation estimates, is likely to be less salient for late-onset conditions. Another, which the authors explore in the current manuscript, is that some loci affecting two or more conditions (i.e., pleiotropic loci) may have effects in opposite directions, or shared loci are sparse, such that the global genetic correlation signal washes out.

      The authors apply a local genetic correlation approach that assesses the presence and direction of pleiotropy in much smaller spatial windows across the genome. Then, within regions evidencing local genetic correlations for a given trait pair, they apply fine-mapping and colocalization methods to attempt to differentiate between two scenarios: that the two traits share the same causal variant in the region or that distinct loci within the region influence the traits. Interestingly, the authors only discover one instance of the former: an SNP in the HLA region appearing to confer risk for both AD and ALS. This is in contrast to six regions with distinct causal loci, and twenty regions with no clear shared loci.

      Finally, the authors have published their analysis pipeline such that other researchers might easily apply the same techniques to other collections of traits.

      Strengths:<br /> - All such analysis pipelines involve many decision points where there is often no clear correct option. Nonetheless, the authors clearly present their reasoning behind each such decision.<br /> - The authors have published their analytic pipeline such that future researchers might easily replicate and extend their findings.

      Weaknesses:<br /> - The majority of regions display no clear candidate causal variants for the traits, whether shared or distinct. Further, despite the potential of local genetic correlation analysis to identify regions with effects in opposing directions, all of the regions for causal variants were identified for both traits evidenced positive correlations. The reasons for this aren't clear and the authors would do well to explore this in greater detail.<br /> - The authors very briefly discuss how their findings differ from previous analyses because of their strict inclusion for "high-quality" variants. This might be the case, but the authors do not attempt to demonstrate this via simulation or otherwise, making it difficult to evaluate their explanation.

    1. Reviewer #2 (Public Review):

      The authors characterized the recombinase-based cumulative fate maps for vesicular glutamate transporters (Vglut1, Vglut2 and Vglut3) expression and compared those maps to their real-time expression profiles in central NA neurons by RNA in situ hybridization in adult mice. Authors have revealed a new and intriguing expression pattern for Vglut2, along with an entirely uncharted co-expression domain for Vglut3 within central noradrenergic neurons. Interestingly, and in contrast to previous studies, the authors demonstrated that glutamatergic signaling in central noradrenergic neurons does not exert any influence on breathing and metabolic control either under normoxic/normocapnic conditions or after chemoreflex stimulation. Also, they showed for the first-time the Vglut3-expressing NA population in C2/A2 nuclei. In addition, they were also able to demonstrate Vglut2 expression in anterior NA populations, such as LC neurons, by using more refined techniques, unlike previous studies.

      A major strength of the study is the use of a set of techniques to investigate the participation of NA-based glutamatergic signaling in breathing and metabolic control. The authors provided a full characterization of the recombinase-based cumulative fate maps for Vglut transporters. They performed real-time mRNA expression of Vglut transporters in central NA neurons of adult mice. Further, they evaluated the effect of knocking down Vglut2 expression in NA neurons using a DBH-Cre; Vglut2cKO mice on breathing and control in unanesthetized mice. Finally, they injected the AAV virus containing Cre-dependent Td tomato into LC of v-Glut2 Cre mice to verify the VGlut2 expression in LC-NA neurons. A very positive aspect of the article is that the authors combined ventilation with metabolic measurements. This integration holds particular significance, especially when delving into the exploration of respiratory chemosensitivity. Furthermore, the sample size of the experiments is excellent.

      Despite the clear strengths of the paper, some weaknesses exist. It is not clear in the manuscript if the experiments were performed in males and females and if the data were combined. I believe that the study would have benefited from a more comprehensive analysis exploring the sex specific differences. The reason I think this is particularly relevant is the developmental disorders mentioned by the authors, such as SIDS and Rett syndrome, which could potentially arise from disruptions in central noradrenergic (NA) function, exhibit varying degrees of sex predominance. Moreover, some of the noradrenergic cell groups are sexually dimorphic. For instance, female Wistar rats exhibit a larger LC size and more LC-NA neurons than male subjects (Pinos et al., 2001; Garcia-Falgueras et al., 2005). More recently, a detailed transcriptional profiling investigation has unveiled the identities of over 3,000 genes in the LC. This revelation has highlighted significant sexual dimorphisms, with more than 100 genes exhibiting differential expression within LC-NA neurons at the transcript level. Furthermore, this investigation has convincingly showcased that these distinct gene expression patterns have the capacity to elicit disparate behavioral responses between sexes (Mulvey et al., 2018). Therefore, the authors should compare the fate maps, Vglut transporters in males and females, at least considering LC-NA neurons. Even in the absence of identified sex differences, this information retains significant importance.

      An important point well raised by the authors is that although suggestive, these experiments do not definitively rule out that NA-Vglut2 based glutamatergic signaling has a role in breathing control. Subsequent experiments will be necessary to validate this hypothesis.

      An improvement could be made in terms of measuring body temperature. Opting for implanted sensors over rectal probes would circumvent the need to open the chamber, thereby preventing alterations in gas composition during respiratory measurements. Further, what happens to body temperature phenotype in these animals under different gas exposures? These data should be included in the Tables.

      Is it plausible that another neurotransmitter within NA neurons might be released in higher amounts in DBH-Cre; Vglut2 cKO mice to compensate for the deficiency in glutamate and prevent changes in ventilation?

      Continuing along the same line of inquiry is there a possibility that Vglut2 cKO from NA neurons not only eliminates glutamate release but also reduces NA release? A similar mechanism was previously found in VGLUT2 cKO from DA neurons in previous studies (Alsio et al., 2011; Fortin et al., 2012; Hnasko et al., 2010). Additionally, does glutamate play a role in the vesicular loading of NA? Therefore, could the lack of effect on breathing be explained by the lack of noradrenaline and not glutamate?

    1. Reviewer #2 (Public Review):

      In this work, the authors found in the mouse line of GABA a1 subunit KO in thalamic neurons, which was previously reported lacking ocular dominance (OD) plasticity in juvenile V1 and dLGN (Sommeijer et al., 2017), the adult V1 and dLGN OD plasticity was also missing. Through muscimol inhibiting the V1 feedback, thalamic OD plasticity was unaffected in both WT and KO adult mice. However, during the critical period, the thalamic OD plasticity was dependent on V1 feedback in WT mice.

      Strengths:

      1. The experiments were well designed. The authors used both MD and No MD controls with both WT and KO mice. The authors used in vivo SU recording, which is broadly accepted as the major method for evaluating OD plasticity.

      2. The data analysis was solid. The authors used proper statistical tests for non-parametric data set.

      Weaknesses:

      1. In my previous review I pointed out that an alternative interpretation of the results is that the lack of OD plasticity in adult V1 and dLGN was caused by an early blockade of the development of the inhibitory circuit in dLGN, which causes life-long deficits in the functional connection of dLGN. The best way to rule out this possibility is by using conditional KO mice that dLGN synaptic inhibition was only interfered in adulthood. In response to my concern, the authors replied with a long text of reasoning why the current results are solid enough and the proposed experiment was unnecessary. I agree with most of the explanation that the current conclusion is solid, but I still think that the cKO experiment will be a good supplement to the current study, and if we do see a similar result in the cKO mice, the conclusion that the adult perturbation of thalamic inhibitory circuit interfere with the OD plasticity will be more convincing. However, I do understand that repeating the experiments again in another mouse line will be difficult and time-consuming, so the authors could choose if they want to perform the experiment or not.

      2. Now the discussion part is very long and complex. Rearranging the discussion with sub-sections will make it easy to read.

    1. Reviewer #2 (Public Review):

      This manuscript by Xu et al. explores the potential joint storage/retrieval of associated signals in learning/memory and how that is encoded by some associative memory neurons using a mouse model. The authors examined mouse associative learning by pairing multimodal mouse learning including olfactory, tactile, gustatory, and pain/tail heating signals. The key finding is that after associative learning, barrel neurons respond to other multi-model stimulations. They found these barrel cortical neurons interconnect with other structures including piriform cortex, S1-Tr and gustatory cortical neurons. Further studies showed that Neuroligin 3 mediated the recruitment of associative memory neurons during paired stimulation group. The authors found that knockdown Neuroligin 3 in the barrel cortex suppressed the associative memory cell recruitment in the paired stimulation learning. Overall, while the findings of this study are interesting, the concept of associative learning involving multiple functionally connective cortical regions is not that novel. While some data presented are convincing, the other seems to lack rigor. In addition, more details and clarification of the experimental methods are needed.

    1. Reviewer #2 (Public Review):

      Starting from the observation that difficulty estimation lies at the core of human cognition, the authors acknowledge that despite extensive work focusing on the computational mechanisms of decision-making, little is known about how subjective judgments of task difficulty are made. Instantiating the question with a perceptual decision-making task, the authors found that how humans pick the easiest of two stimuli, and how quickly these difficulty judgments are made, are best described by a simple evidence accumulation model. In this model, perceptual evidence of concurrent stimuli is accumulated and difficulty is determined by the difference between the absolute values of decision variables corresponding to each stimulus, combined with a threshold crossing mechanism. Altogether, these results strengthen the success of evidence accumulation models in describing human decision-making, now extending it to judgments of difficulty.

      The manuscript addresses a timely question and is very well written, with its goals, methods and findings clearly explained and directly relating to each other. The authors are specialists of evidence accumulation tasks and models. Their modelling of human behaviour within this framework is state-of-the-art. In particular, their model comparison is guided by qualitative signatures which are diagnostic to tease apart different models (e.g., the RT criss-cross pattern). Human behaviour is then inspected for these signatures, instead of relying exclusively on quantitative comparison of goodness-of-fit metrics.

      The study has potential limitations well flagged by the authors after the revision process. The main limitation pertains to the (dis)similarity between the behavioural task used in the study and difficulty judgments people actually do in real world (and which are well illustrated in the introduction). First, difficulty judgments made in the task never impact the participant (a new trial simply follows) while difficulty judgments in the wild often determine whether to pursue or quit the corresponding task, which can have consequences years after the difficulty estimation (e.g., deciding to engage in a particular academic path as a function of the estimated difficulty). Second, while trial-by-trial feedback is delivered in the task, difficulty estimation in the wild has to be made with partial information and feedback is either absent or delayed. How much these differences are key in providing an accurate computational description of human difficulty judgments will likely require further research.

      Another limitation is the absence of models based on computational principles other than evidence accumulation. Although there are good reasons to favour evidence accumulation models in these settings (as mentioned by the authors in their manuscript), showing that evidence accumulation models would have won against competitors would have further strengthened the authors' claim that difficulty judgment about perceptual information are firmly anchored in the principles of evidence accumulation.

      These limitations should not distract the reader from the impact of the present work, which will likely be wide, spanning the whole field of decision-making, and this across species. It will echo in particular with the many other seminal studies that have relied on a similar theoretical account of behaviour and brain activity (evidence accumulation). In addition, this study will hopefully inspire novel task designs aiming at addressing difficulty judgment estimations in controlled lab experiments, possibly with features closer to real world difficulty estimation (e.g., long-term consequences of difficulty estimation and absence of feedback).

    1. Reviewer #2 (Public Review):

      Summary:<br /> In the present study, van Gerwen et al. perform deep phosphoproteomics on muscle from saline or insulin-injected mice from 5 distinct strains fed a chow or HF/HS diet. The authors follow these data by defining a variety of intriguing genetic, dietary, or gene-by-diet phosphor-sites that respond to insulin accomplished through the application of correlation analyses, linear mixed models, and a module-based approach (WGCNA). These findings are supported by validation experiments by intersecting results with a previous profile of insulin-responsive sites (Humphrey et al, 2013) and importantly, mechanistic validation of Pfkfb3 where overexpression in L6 myotubes was sufficient to alter fatty acid-induced impairments in insulin-stimulated glucose uptake. To my knowledge, this resource provides the most comprehensive quantification of muscle phospho-proteins which occur as a result of diet in strains of mice where genetic and dietary effects can be quantifiably attributed in an accurate manner. Utilization of this resource is strongly supported by the analyses provided highlighting the complexity of insulin signaling in muscle, exemplified by contrasts to the "classically-used" C57BL6/J strain. As it stands, I view this exceptional resource as comprehensive with compelling strength of evidence behind the mechanism explored. Therefore, most of my comments stem from curiosity about pathways within this resource, many of which are likely well beyond the scope of incorporation in the current manuscript. These include the integration of previous studies investigating these strains for changes in transcriptional or proteomic profiles and intersections with available human phospho-protein data, many of which have been generated by this group.

      Strengths:<br /> Generation of a novel resource to explore genetic and dietary interactions influencing the phospho-proteome in muscle. This is accompanied by the elegant application of in silico tools to highlight the utility.

      Weaknesses:<br /> Some specific aspects of integration with other data among the same fixed strains could be strengthened and/or discussed.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript provides microprobe serial oxygen isotope data from thin-sectioned modern and fossil orangutan teeth in an effort to reconstruct the seasonality of rainfall in Borneo and Sumatra. The authors also explore the hypothesis that nursing could affect early tooth (first molar) isotope values. They find that all molars yield similar oxygen isotope values and therefore conclude that future research need not exclude the use of first molars. With regard to seasonality, the modern orangutans yield similar results from both islands. The authors suggest differences between modern and fossil orangutan teeth, but the comparisons could be more fully explored.

      Strengths:<br /> The study employs a sampling method that captures serial isotope values within thin sections of teeth using a microprobe that provides a much higher resolution than traditional hand-held drilling.

      Weaknesses:<br /> The study only examines six modern and six fossil orangutan individuals. Of those, only four modern individuals were samples across multiple molars. The comparisons between modern and fossil teeth are difficult to follow, making unclear the conclusion that climate has changed.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Radial spokes are evolutionarily conserved protein complexes that are important for cilia motility. So far, the composition of certain radial spokes was investigated in the algae Chlamydomonas, mice, and humans. This work by Bicka et al. investigated the composition of radial spokes in the ciliate Tetrahymena by analyzing knockouts and strains that express tagged radial spoke proteins, using mass spectrometry and cryo-electron tomography. While three specific types of radial spokes have been reported thus far, this study suggests that in Tetrahymena, there is another layer to the variability in radial spokes. Additionally, many proteins with predicted enzymatic folds have now been assigned to radial spokes. The comparison of ciliary complexes between species is important to define the basic principles that govern cilia motility, as well as to reveal the differences that enable cilia of various organisms to beat in diverse environments.

      Strengths:<br /> The manuscript includes a thorough bioinformatic analysis of radial spoke proteins in Tetrahymena and reveals the presence of multiple orthologs to certain algae and mammalian radial spoke proteins. The mass spectrometry analysis and cryo-electron tomography experiments are solid and informative. This work provides a lot of important data and thus, opens the door to resolve the exact composition and structures of radial spokes in Tetrahymena and perhaps other species.

      Weaknesses:<br /> The assignment of the three RSP3 orthologs to RS1, RS2, and RS3 is based only on missing structures in the knockouts. Although this method is informative, it is not sufficient to draw conclusions regarding the positions of the missing proteins. There are numerous examples where a structure was missing, but the absent protein was localized elsewhere (i.e., absence of central pair protrusions in patients with mutations in radial spoke proteins). To directly demonstrate the position of an RSP3 ortholog in a certain radial spoke, the protein can be labeled with a tag that is visualized in subtomogram averages (as was done in Oda et al., 2014 and other studies). Relying on the data from knockouts alone, the model for radial spoke composition in Tetrahymena (Fig. 6) may be incomplete.

      The control for the bio-ID experiment was WT cells. Since there are many hits in the experiment, a better control would have been a strain with free BirA, or BirA fused to a protein that is distant from the radial spokes, such as one of the outer-dynein arm proteins, or a ciliary membrane protein.

    1. Reviewer #2 (Public Review):

      It is well known that introducing clusters in balanced random networks leads to metastable dynamics that potentially span long time scales. The authors build on their previous work (Stern et al. 2014) and here show that the lifetime of metastable states depends on the size of the individual activated clusters. Showing qualitative similarities between clustered spiking networks and networks of bistable rate units, the authors further derive dynamic mean-field predictions for the separation of time scales of the dynamics in relation to differences in the strength of self-couplings in rate networks. Further, they confirm these results in simulations of spiking networks and compare them to time scales observed in the orbitofrontal cortex. Finally, the authors show that assemblies of a particular size (and thus time scale) get entrained by specific external input frequencies, allowing the network to demix temporal signals in a spatial manner.

      The manuscript is in general well written and addresses a timely and important topic in neuroscience. However, there are concerns related to the discussion of alternative mechanisms for a large repertoire of time scales as well as the relation between the spiking and rate network model.

    1. Reviewer #2 (Public Review):

      The work is very clearly designed, executed, and written. The transcription output data is rigorous and well quantified, and the fit of the TF binding model clearly shows agreement with experiments in the case of cooperativity, but not in its absence, making a strong case for the authors' conclusion.

      How the Hidden Markov Model fit results (promoter kon and koff values) lead to the observed effects on transcription output is less clear. For instance, Dl1 deletion results in a small increase in kon and a moderate increase in koff, which seems at odds with the other variants. Yet all variants exhibit similar transcription output profiles. One other intriguing observation is that the promoter states in Fig. 4C&D do not look dramatically different in their kinetics, yet the input transcription traces exhibit a 3-fold amplitude difference. Maybe the authors can clarify these apparent discrepancies.

      The authors observe cooperativity between TF binding sites and transcription output, which their model suggests is driven by TF binding cooperativity ("We propose that the cooperativity allows TF binding sites with moderate or weak affinities to recruit more TFs to the enhancer"). This is plausible and likely, but not rigorously demonstrated; another possibility could be cooperativity at the step of transcription activation. One could verify that the binding step is the cooperative one via ChIP-qPCR in the different variants, but given the cautious wording of the paper, this is not absolutely necessary.

    1. Reviewer #2 (Public Review):

      In this paper, the authors carry out neural circuit modeling to theoretically elucidate the mechanism underlying the empirically observed (in a previous study by some of the current authors) reduction in neural synchrony in the monkey prefrontal cortex (PFC), as a result of NMDAR blockade. Empirically it was previously found that in monkeys performing a cognitive control task, PFC neurons exhibit precisely timed synchronous firing, especially in the short period before the monkey's response, leading to "0-lag" (zero in the 1-2 millisecond timescale) spiking correlations. This signature of synchrony was then found to be extinguished or diminished with the systemic administration of an NMDAR antagonist.

      In the current study, the authors simulate and analyze a network of excitatory and inhibitory spiking neurons as a model of a local PFC circuit, to elucidate the mechanism underlying this effect. The model network is composed of leaky integrate-and-fire neurons with conductance-based synaptic inputs and is sparsely and randomly connected as in the classic studies of balanced networks in which neurons fire irregularly as observed in the cortex. Using mean-field theory, the authors start by mapping out the phase boundary between the asynchronous irregular and synchronous irregular states in the network as a function of network parameters controlling synaptic connectivity and external background inputs (which they parametrize as ratios of recurrent or external currents mediated by AMPAR, NMDAR or GABAA). The transition between the two phases corresponds to a Hopf-like bifurcation above which synchronous oscillations with frequency in the gamma-band (or above) emerge. It is found that with an increase in external inputs, a network in the asynchronous state (but close to criticality) can switch to the synchronous state. Based on this, the authors hypothesize that an increase in the external drive is the mechanism underlying the empirically observed increase in synchrony before the behavioral response. It is then shown that a reduction in NMDAR conductance (keeping AMPAR or GABAR conductances fixed) has the opposite effect, and pushes the network towards the asynchronous state, and can counteract or weaken the effect of increased external input. In both cases increase or decrease in synchrony is quantified by an increase or decrease in 0-lag pairwise correlations; transition to synchrony is shown to also lead to the development of nonzero-lag peaks in the average spiking correlation reflecting gamma-band oscillations. The authors then show that (with the appropriate choice of primary network parameters) their proposed mechanisms for the (natural) increase in synchrony via an increase in external inputs and the weakening of this effect with the weakening of NMDA conductances do semi-quantitatively match the observed changes in 0-lag synchrony and nonzero lag peaks in spiking correlations. Finally, they discuss the effect of the balance between average NMDA and GABA currents in the primary (baseline) network on the above effects.

      Strengths:<br /> - The modeling and analysis are solid and overall this work succeeds in providing a convincing mechanistic explanation for the specific empirically observed effects in monkey PFC: the natural task-dependent modulation of 0-lag synchrony and its extinction with NMDA blockage.

      - The manuscript is very readable and the figures and plots are clearly described.

      - The mathematical mean-field analysis in the Methods section is also sound and well written and does/can (see below) provide a sufficient mathematical explanation of the simulation results.

      Weaknesses:<br /> 1) I found the intuitive explanation of the effects of external input or NMDAR conductance on synchrony incomplete. While simulations and mean-field analysis both predict this effect, the mean-field theory and the linearization analysis and stability analysis can be used to further shed light on the precise mechanism by which external input and NMDAR conductance promote synchrony (or destabilization of the asynchronous state).

      2) An important natural question (which is relevant to the connection with schizophrenia) is what are the distinct roles of AMPAR-based and NMDAR-based excitation on the transition to synchrony, and this is not addressed in this study. It would be important to clarify what is special/distinct about NMDAR in the current findings.

      3) In the Introduction and Discussion, the authors speculate on the possible connection between their empirical and theoretical findings (on the effect of NMDAR hypofunction on synchronous spiking) and the pathogenesis of schizophrenia. While this is not central to the findings of the paper, because it is relevant to the broader significance and impact of this work I will note the following. Their proposed specific link to pathogenesis is as follows: the reduction in precisely timed synchrony resulting from NMDAR hypofunction can disrupt spike-timing dependent plasticity (STDP) and lead to "disconnection" of cortical circuits as observed in schizophrenia. Letting aside the fact that observations in schizophrenia relate to functional connectivity and not synaptic connectivity, previous theoretical studies of STDP in spiking networks do not support the claim that lack of synchronous activity would lead to disconnection of the circuit.

    1. Reviewer #2 (Public Review):

      This study is impressive in several ways and will be of interest to behavioral and brain scientists working on diverse topics.

      First, from a theoretical point of view, it very convincingly integrates several lines of research (confidence, interpersonal alignment, psychophysical, and neural evidence accumulation) into a mechanistic computational framework that explains the existing data and makes novel predictions that can inspire further research. It is impressive to read that the corresponding model can account for rather non-intuitive findings, such as that information about high confidence by your collaborators means people are faster but not more accurate in their judgements.

      Second, from a methodical point of view, it combines several sophisticated approaches (psychophysical measurements, psychophysical and neural modelling, electrophysiological and pupil measurements) in a manner that draws on their complementary strengths and that is most compelling (but see further below for some open questions). The appeal of the study in that respect is that it combines these methods in creative ways that allow it to answer its specific questions in a much more convincing manner than if it had used just either of these approaches alone.

      Third, from a computational point of view, it proposes several interesting ways by which biologically realistic models of perceptual decision-making can incorporate socially communicated information about other's confidence, to explain and predict the effects of such interpersonal alignment on behavior, confidence, and neural measurements of the processes related to both. It is nice to see that explicit model comparison favor one of these ways (top-down driving inputs to the competing accumulators) over others that may a priori have seemed more plausible but mechanistically less interesting and impactful (e.g., effects on response boundaries, no-decision times, or evidence accumulation).

      Fourth, the manuscript is very well written and provides just the right amount of theoretical introduction and balanced discussion for the reader to understand the approach, the conclusions, and the strengths and limitations.

      Finally, the manuscript takes open science practices seriously and employed preregistration, a replication sample, and data sharing in line with good scientific practice.

      Having said all these positive things, there are some points where the manuscript is unclear or leaves some open questions. While the conclusions of the manuscript are not overstated, there are unclarities in the conceptual interpretation, the descriptions of the methods, some procedures of the methods themselves, and the interpretation of the results that make the reader wonder just how reliable and trustworthy some of the many findings are that together provide this integrated perspective.

      First, the study employs rather small sample sizes of N=12 and N=15 and some of the effects are rather weak (e.g., the non-significant CPP effects in study 1). This is somewhat ameliorated by the fact that a replication sample was used, but the robustness of the findings and their replicability in larger samples can be questioned.

      Second, the manuscript interprets the effects of low-confidence partners as an impact of the partner's communicated "beliefs about uncertainty". However, it appears that the experimental setup also leads to greater outcome uncertainty (because the trial outcome is determined by the joint performance of both partners, which is normally reduced for low-confidence partners) and response uncertainty (because subjects need to consider not only their own confidence but also how that will impact on the low-confidence partner). While none of these other possible effects is conceptually unrelated to communicated confidence and the basic conclusions of the manuscript are therefore valid, the reader would like to understand to what degree the reported effects relate to slightly different types of uncertainty that can be elicited by communicated low confidence in this setup.

      Third, the methods used for measurement, signal processing, and statistical inference in the pupil analysis are questionable. For a start, the methods do not give enough details as to how the stimuli were calibrated in terms of luminance etc so that the pupil signals are interpretable. Moreover, while the authors state that the traces were normalized to a value of 0 at the start of the ITI period, the data displayed in Figure 2 do not show this normalization but different non-zero values. Are these data not normalized, or was a different procedure used? Finally, the authors analyze the pupil signal averaged across a wide temporal ITI interval that may contain stimulus-locked responses (there is not enough information in the manuscript to clearly determine which temporal interval was chosen and averaged across, and how it was made sure that this signal was not contaminated by stimulus effects).

      Fourth, while the EEG analysis in general provides interesting data, the link to the well-established CPP signal is not entirely convincing. CPP signals are usually identified and analyzed in a response-locked fashion, to distinguish them from other types of stimulus-locked potentials. One crucial feature here is that the CPPs in the different conditions reach a similar level just prior to the response. This is either not the case here, or the data are not shown in a format that allows the reader to identify these crucial features of the CPP. It is therefore questionable whether the reported signals indeed fully correspond to this decision-linked signal.

      Fifth, the authors present some effective connectivity analysis to identify the neural mechanisms underlying the possible top-down drive due to communicated confidence. It is completely unclear how they select the "prefrontal cortex" signals here that are used for the transfer entropy estimations, and it is in fact even unclear whether the signals they employ originate in this brain structure. In the absence of clear methodical details about how these signals were identified and why the authors think they originate in the prefrontal cortex, these conclusions cannot be maintained based on the data that are presented.

      Sixth, the description of the model fitting procedures and the parameter settings are missing, leaving it unclear for the reader how the models were "calibrated" to the data. Moreover, for many parameters of the biophysical model, the authors seem to employ fixed parameter values that may have been picked based on any criteria. This leaves the impression that the authors may even have manually changed parameter values until they found a set of values that produced the desired effects. The model would be even more convincing if the authors could for every parameter give the procedures that were used for fitting it to the data, or the exact criteria that were used to fix the parameter to a specific value.

      Seventh, on a related note, the reader wonders about some of the decisions the authors took in the specification of their model. For example, why was it assumed that the parameters of interest in the three competing models could only be modulated by the partner's confidence in a linear fashion? A non-linear modulation appears highly plausible, so extreme values of confidence may have much more pronounced effects. Moreover, why were the confidence computations assumed to be finished at the end of the stimulus presentation, given that for trials with RTs longer than the stimulus presentation, the sensory information almost certainly reverberated in the brain network and continued to be accumulated (in line with the known timing lags in cortical areas relative to objective stimulus onset)? It would help if these model specification choices were better justified and possibly even backed up with robustness checks.

      Eight, the fake interaction partners showed several properties that were highly unnatural (they did not react to the participant's confidence communications, and their response times were random and thus unrelated to confidence and accuracy). This questions how much the findings from this specific experimental setting would transfer to other real-life settings, and whether participants showed any behavioral reactions to the random response time variations as well (since several studies have shown that for binary choices like here, response times also systematically communicate uncertainty to others). Moreover, it is also unclear how the confidence convergence simulated in Figure 3d can conceptually apply to the data, given that the fake subjects did not react to the subject's communicated confidence as in the simulation.

    1. Reviewer #2 (Public Review):

      This study looks at how optomotor turning in fruit flies varies with stimulus conditions. Although the response has usually been observed in the same direction of rotation as the stimulus, they find that in many situations the flies turn strongly in the opposite direction to the stimulus. This 'anti-directional' turning increases with stimulus brightness, contrast, and duration of the stimulus, and also varies with many factors such as rearing temperature, lab, strain, and developmental stage. They show that the anti-directional response depends on neurons in the visual system that are also important for the more standard response, but they don't find clear changes in the activity of these neurons that could explain the directional switch. The main conclusion is that supposedly simple behaviors may be more complicated than they first appear, and careful consideration needs to be given to the precise stimulus conditions and the response dynamics when measuring such behaviors, and especially when comparing data across labs.

    1. Reviewer #2 (Public Review):

      This is a very interesting study with a potential impact on understanding the 3D mechanics of cells in epithelia. The assay that the authors developed is novel and quite useful for future studies. However, I was hoping to see more experimental results in the manuscript. For example, there is a zoo of mutants that the community speculates about possible mechanical changes in cells. I was hoping to see if the authors can settle some of these arguments by using their novel technique and analysis.

    1. Reviewer #2 (Public Review):

      Plasmodium falciparum RH5 (PfRH5) is an integral membrane protein of P. falciparum merozoites that acts as an essential ligand involved in host erythrocyte invasion, functioning by binding to the erythrocyte surface protein basigin. Previous work by the authors of this study and other groups has demonstrated that antibodies to PfRH5 can block invasion and can be protective in in vivo challenge studies, so PfRH5 is a promising malaria vaccine candidate. This study by Jamwal et al addresses the paradoxical observation, made in earlier work by these authors, that certain antibodies to PfRH5 efficiently inhibit parasite invasion of erythrocytes yet does not block the binding of PfRH5 to recombinant basigin ectodomain. The authors first demonstrate through a range of approaches that most native erythrocyte basigin is expressed in the form of detergent-stable complexes with one of two distinct erythrocyte membrane proteins, plasma membrane calcium ATPase (PMCA) or monocarboxylate transporter (MCT). Using in vitro biophysical techniques, they then show that recombinant PfRH5 binds more tightly (and with slower off-rates) to the native basigin-PMCA or basigin-MCT1 complexes than to the isolated recombinant basigin ectodomain. Finally and crucially, the authors then show that 2 of these known invasion-inhibitory anti-PfRH5 antibodies (called R5.016 and 9AD4) that do not block the interaction between recombinant basigin and PfRH5 do in contrast block the interaction between PfRH5 and basigin-PMCA and basigin-MCT1 complexes. By docking known atomic structures of the R5.016 and 9AD4 Fab-basigin structures onto the known or modelled basigin complex structures, the authors present a convincing argument that the invasion-inhibitory antibodies function through steric hindrance, preventing PfRH5 binding to the basigin-PMCA or basigin-MCT1 complexes. The work provides a rational explanation for the invasion-inhibitory activity of this class of PfRH5-specific antibodies and demonstrates the potential complexity underlying the mode of action of invasion-inhibitory anti-malarial antibodies.

    1. Reviewer #2 (Public Review):

      This is a review of "Effect of an enhanced public health contact tracing intervention on the secondary transmission of SARS-CoV-2 in educational settings: the four-way decomposition analysis", by Djuric et al.

      In late 2020, a province in northern Italy implemented a new testing regimen for all contacts of people known to have COVID-19, offering them SARS-CoV-2 testing immediately after the detection of the index case instead of at the end of a quarantine period. The authors of this study investigated whether this policy change reduced secondary transmission of SARS-CoV-2 in schools. In addition to studying this primary outcome, they examined two "process" outcomes; whether this policy of testing earlier enabled public health officials to more successfully identify the source of infection of the index case, and if the time interval from detection of the index case to testing of contacts in the educational setting reduced.

      They concluded that the time between detection of the index case and testing of contacts did reduce before and after the policy change. Similarly, the proportion of cases for which the source of infection was identified also increased after the policy change. Both of these "process" indicators correlated with reduced secondary transmission, though only identifying the source of infection was associated with a statistically significant (at the 5% level) reduction in secondary transmission.

      Strengths of this paper

      Educational settings experienced significant disruption during the COVID-19 pandemic, and efforts to better understand the spread of SARS-CoV-2 in schools - and how to mitigate this spread - are of significant public health importance. This paper, therefore, addresses an important topic.

      Additionally, the authors describe a detailed dataset comprising case and contact tracing data from over 1,600 index cases with in-school contacts. The richness of the data described in Table 1 provides a good opportunity to conduct a natural experiment on the potential impact of testing contacts immediately after exposure on secondary transmission. The authors also appropriately acknowledge that this interrupted time series study would be insufficient to provide causal information, given the potential for confounders.

      Finally, the primary statistical method (a four-way decomposition analysis) was new to me, but - from the references cited - seems appropriate. Given the relative novelty of this method, more space could be dedicated to explaining it in the methods.

      Weakness of this paper

      Although the paper tackles an important topic with an appropriate dataset, the analyses feel insufficient to fully support the authors' conclusions.

      First and most critically, it is difficult to understand exactly what the primary outcome of the study is. Both the median number of secondary cases per class and the proportion of classes that experienced any secondary transmission are presented in Table 1, but - at least in the unadjusted analyses - point in different directions regarding the impact of the effect of the intervention (albeit neither strongly). For example, before the policy change, the median number of secondary cases per index case is 2, while after the policy change, it has reduced to 1. In contrast, before the policy change 37% of classes experienced any secondary transmission, but after the policy change, this had increased to 39% of classes. In some of the adjusted analyses, "number of secondary cases" is stated as the outcome variable, but that is not fully defined. The "attack rate", which is well defined in the methods, could be one option for use as a consistent primary outcome, however, it is only provided for the total study population and the attack rates pre- or post-policy change are not presented or compared.

      Additionally, although using a "process measure" as a secondary outcome could be valuable - especially in a natural experiment like this, where identifying a causal relationship with a complex outcome like secondary transmission will be difficult - it was somewhat unclear how the process measures described in this study were measured, or their validity. For example, the reduced time between detection of the index case and testing of contacts seems unsurprising, since the intervention itself is to test contacts immediately after the index case is identified. Additionally, the results describe reductions in median testing delay and median tracing delay, but only testing delay is defined in the methods.

      Finally, there is existing published literature that provides additional context on the impact of testing on secondary transmission within schools that arguably provides a higher level of evidence than the current study, but is not cited by the authors. A key limitation of this study - which the authors acknowledge - is the interrupted time series nature of their study, which is open to confounding by other important factors that happened at the same time, including but not limited to: changes in overall incidence of COVID-19; viral evolution (e.g. the emergence of the Alpha variant (B.1.1.7) which occurred during this study and which significantly altered the risk of secondary transmission); the efficiency of the contact tracing system (including skill and size of the contact tracing workforce); and the availability of non-molecular diagnostic tests (e.g. lateral flow devices) that might allow individuals to change their behaviors even without enrolling in this study. Examples of alternative studies which might reduce some of this potential confounding include around 400 schools in Los Angeles County, California, USA, that implemented "test to stay" in 2021 and were compared to 1,600 schools that did not implement "test to stay" [https://www.cdc.gov/mmwr/volumes/70/wr/mm705152e1.htm] and a cluster-randomized trial of daily testing of exposed contacts to study in-school transmission in England, UK, also in 2021 [https://www.sciencedirect.com/science/article/pii/S0140673621019085]. Although these examples describe slightly different interventions involving enhanced testing of exposed contacts, they both compared educational settings with and without the intervention across the same time periods; and the UK study in particular has methodological advantages over this current paper, including randomization. While the findings in the current paper did not contradict these earlier, stronger papers, the example from this province should be placed in context with the totality of evidence around testing in schools.

    1. Reviewer #2 (Public Review):

      In their present work, Briggs et al. combine biophysical simulations and experimental recordings of beta cell activity with analyses of functional network parameters to determine the role played by gap-junctional coupling, metabolism, and KATP conductance in defining the functional roles that the cells play in the functional networks, assess the structure-function relationship, and to resolve an important current open question in the field on the role of so-called hub cells in islets of Langerhans.

      Combining differential equation-based simulations on 1000 coupled cells with demanding calcium, NAPDH, and FRAP imaging, as well as with advanced network analyses, and then comparing the network metrics with simulated and experimentally determined properties is an achievement in its own right and a major methodological strength. The findings have the potential to help resolve the issue of the importance of hub cells in beta cell networks, and the methodological pipeline and data may prove invaluable for other researchers in the community.<br /> However, methodologically functional networks may be based on different types of calcium oscillations present in beta cells, i.e., fast oscillations produced by bursts of electrical activity, slow oscillations produced by metabolic/glycolytic oscillations, or a mixture of both. At present, the authors base the network analyses on fast oscillations only in the case of simulated traces and on a mixture of fast and slow oscillations in the case of experimental traces. Since different networks may depend on the studied beta cell properties to a different extent (e.g., fast oscillation-based networks may, more importantly, depend on electrical properties and slow oscillation-based networks may more strongly depend on metabolic properties), it is important that in drawing the conclusions the authors separately address the influence of a cell's electrical and metabolic properties on its functional role in the network based on fast oscillations, slow oscillations, or a mixture of both.

    1. Reviewer #2 (Public Review):

      Using fluorescent-TFEB fusion proteins and mutants thereof for live-cell imaging single cells, the authors investigated how mTORC1 responds to amino acids and growth factors. First, they demonstrated that the stably expressed fusion protein behaves as endogenous TFEB with regards to mTORC1 activation. Next, using the phosphodeficient TFEB mutant, they showed that GSK3 phosphorylation amplifies the C/N ratio, supporting the role of GSK3 and mTORC1 in co-regulating TFEB. When amino acids or insulin were added to starved cells, they found a graded response depending on amounts of AA or insulin, respectively, thus suggesting an incremental response. When multiple inputs were assessed, they found that TFEB C/N ratio also increased in increments when nutrients were added first followed by insulin. But when insulin was added first before nutrients, a minimal response occurred although this could be subsequently increased upon addition of the nutrients. Lastly, by tracking down TFEB C/N in response to different amounts of nutrients over longer periods (12 hr), they observed that a new steady state is achieved, indicating adaptation of mTORC1 activity and that this correlates with signal inputs from Akt and AMPK. Based on these findings, the authors conclude that the mTORC1-TFEB signaling continuously adjust to nutrient availability rather than just behave in "AND" gate logic fashion.

      Overall, the results are robust and supportive of their conclusion. The use of fluorescent fusion proteins/mutants is nicely done. The authors have created useful tools to further analyze mTOR signaling at the single-cell level. However, the findings that mTORC1 signaling behaves like a rheostat is not really new and rather more confirmatory of previous studies. The current studies further support this model with their use of TFEB as mTORC1 target in single cells.

    1. Reviewer #2 (Public Review):

      This paper presents an extensive numerical study of microbial evolution using a model of fitness inspired by spin glass physics. It places special emphasis on elucidating the combined effects of microscopic epistasis, which dictates how the fitness effect of a mutation depends on the genetic background on which it occurs, and clonal interference, which describes the proliferation of and competition between multiple strains. Both microscopic epistasis and clonal interference have been observed in microbial evolution experiments, and are chief contributors to the complexity of evolutionary dynamics. Correlations between random mutations and nonlinearities associated with interactions between sub-populations consisting of competing strains make it extremely challenging to make quantitative theoretical predictions for evolutionary dynamics and associated observables such as the mean fitness. While the body of theoretical and computational research on modeling evolutionary dynamics is extensive, most theoretical efforts rely on making simplifications such as the strong selection weak mutation (SSWM) limit, which neglects clonal interference, or assumptions about the distribution of fitness effects that are not experimentally verifiable.

      The authors have addressed this challenge by running a numerical microbial evolution experiment over realistic population sizes (~ 100 million cells) and timescales (~ 10,000 generations) using a spin glass model of fitness that considers pairwise interactions between mutations on distinct genetic loci. By independently tuning mutation rate as well as the strength of epistasis, the authors have shown that epistasis generically slows down the growth of fitness trajectories regardless of the amount of clonal interference. On the other hand, in the absence of epistasis, clonal interference speeds up the growth of fitness trajectories, but leaves the growth unchanged in the presence of epistasis. The authors quantitatively characterize these observations using asymptotic power law fits to the mean fitness trajectories. Further, the authors employ more simplified macroscopic models that are informed by their empirical findings, to reveal the mechanistic origins of the epistasis mediated slowing down of fitness growth. Specifically, they show that epistasis leads to a broadening of the distribution of fitness increments, leading to the fixation of a large number of mutations that confer small benefits. Effectively, this leads to an increase in the number of fixed mutations required to climb the fitness peak. This increased number of required beneficial mutations together with the decreasing availability of beneficial mutations at high fitness lead to the slowdown of fitness growth. The authors' data analysis is quite solid and their conclusions are well supported by quantitative macroscopic models. The paper also includes an interesting analysis of dynamical correlations between mutations, using tools developed in the spin glass literature.

      One of the highlights of this paper is the author's astute choice of model, which strikes an impressive balance between complexity, flexibility, and numerical accessibility. In particular, the authors were able to achieve results over realistic population sizes and timescales largely because of the amenability of the model to the implementation of an efficient simulation algorithm. At the same time, the strength of epistasis and clonal interference can be tuned in a facile manner, enabling the authors to map out a phase diagram spanning these two axes. One could argue that the numerical scheme employed here would only work for a specific class of models, and is therefore not generalizable to all models of evolutionary dynamics. While this is likely true, the model is capable of recapitulating several complex aspects of microbial evolution, and is therefore not unduly restrictive.

      Spin glass physics has already provided significant insights into a wide range of topics in the life sciences including protein folding, neuroscience, ecology and evolution. The present work carries this approach forward, with immediate implications for microbial evolution, and potential implications in related areas of research such as microbial ecology. In addition to the theoretical value of spin glass physics, the high performance algorithm developed in this work lays the foundation for formulating data driven approaches aimed at understanding evolutionary dynamics. In the future, there is considerable scope for utilizing data generated by such models to train machine learning algorithms for quantifying parameters associated with epistasis, clonal interference, and the distribution of fitness effects in laboratory experiments.

    1. Reviewer #2 (Public Review):

      The manuscript by Seah and Saranathan investigates the cell-based growth mechanism of so called honeycomb-structures in the upper lamina of papilionid wing scales by investigating a number of different species. The authors chose Parides eurimedes as a focus species with the developmental pathway of five other papilionid as a comparative backup. Through state-of-the-art microscopy images of different developmental steps, the authors find that the intricate f-actin filaments reorganise, support cuticular discs that template the air holes that form the honeycomb lattice.

      The revised manuscript is well written and easy to follow, yet based on a somewhat limited sample size for their focus species, limiting attempts to suppress expression and alter structure shape. I have no further comments.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors are interested in large-scale cell flow during gastrulation and in particular in the polonaise movement. This movement corresponds to a bilateral vortex-like counter-rotating cell flow and transport the mesendodermal cells allowing ingression of cells through the primitive streak and ultimately the formation of the mesoderm and endoderm. The authors specifically wanted to investigate the coupling of the polonaise movement and primitive streak to understand whether the polonaise movement is a consequence of the formation of the primitive streak or the other way around. They propose a model where the primitive streak elongation is not required for the cell flow but rather for its maintenance and that robust cell flow is not required for primitive streak extension.

      Strengths:<br /> Overall, the manuscript is well written with clear experimental designs. The authors have used live imaging and cell flow analysis in different conditions, where either the formation of the primitive streak or the cell flow was perturbed.<br /> Their live imaging and PIV-based analyses convincingly support their conclusions that primitive streak deformation or mitotic arrest do not impact the initiation of the polonaise movement but rather the location or maintenance of these rotations. They additionally showed that disruption of the polonaise movement in the authentic primitive streak by elegant addition of an ectopic primitive streak does not impact the original primitive streak elongation.

      Weaknesses:<br /> - When using the delta-DEP-GFP construct, the authors showed that they can manipulate the shape of the primitive streak without affecting the identity and number of primitive streak cells. It is not clear however how this can affect the shape, volume or adhesion of the cells. Some mechanistic insights would strengthen the paper.<br /> - Overall, frequencies of observation are missing for a better view of the phenomenon. For example, do Vg1/Cos cells always disrupt the flow at the authentic primitive streak? Can replicate vector fields be integrated to reflect quantification?<br /> - Since myosin cables have been shown to be instrumental for the polonaise movement, it would be interesting to better investigate how the manipulations by the delta-DEP-GFP construct, or Vg1/Cos affect the myosin cables (as shown in preliminary form for the aphidicolin-treated embryos).

    1. 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 specific therapeutic use of the medicine. For example, simple but intense tastes were correlated with the 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 of 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 /> While I find this study very interesting and potentially insightful into the development and classification of certain botanical drugs for specific medicinal use, I would encourage the authors to revise the manuscript and the accompanying figures significantly to improve the reader's understanding of the methods, analyses, and findings. A more thorough discussion of the limitations of this particular study and this general type of approach would also be very important to include.

      The metric of versatility seems somewhat arbitrary. It is not well explained why versatility is important and/or its relationship with taste complexity or intensity. Similarly, the rationale for examining the relationships between individual therapeutic uses and taste intensity/complexity is not well explained, and given that a similar high intensity/low complexity relationship is common for most of the therapeutic uses, it restates the same concepts that were covered by the initial versatility comparison. There are multiple issues with the figures - the use of icons is in many cases counterproductive and other representations are not clear or cause confusion (especially Figure 3). The phylogenetic information about the botanicals is missing. Also missing is any reference/discussion about how that analysis was able to disambiguate the confounding effects of shared uses and tastes of drugs from closely related species.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This manuscript applies a mutational scanning analysis to identify the secondary structure of two previously suggested self-cleaving ribozyme candidates in the human genome. Through this analysis, minimal structured and conserved regions with imminent importance for the ribozyme's activity are suggested and further biochemical evidence for cleavage activity are presented. Additionally, the study reveals a close resemblance of these human ribozyme candidates to the known self-cleaving ribozyme class of twister sister RNAs. Despite the high conservation of the catalytic core between these RNAs, it is suggested that the human ribozyme examples constitute a new ribozyme class. Evidence for this however is not conclusive.

      Strengths:<br /> The deep mutational scanning performed in this study allowed the elucidation of important regions within the proposed LINE-1 and OR4K15 ribozyme sequences. Part of the ribozyme sequences could be assigned a secondary structure supported by covariation and highly conserved nucleotides were uncovered. This enabled the identification of LINE-1 and OR4K15 core regions that are in essence identical to previously described twister sister self-cleaving RNAs.

      Weaknesses:<br /> I am skeptical of the claim that the described catalytic RNAs are indeed a new ribozyme class. The studied LINE-1 and OR4K15 ribozymes share striking features with the known twister sister ribozyme class (e.g. Figure 3A) and where there are differences they could be explained by having tested only a partial sequence of the full RNA motif. It appears plausible, that not the entire "functional region" was captured and experimentally assessed by the authors.

      They identify three twister sister ribozymes by pattern-based similarity searches using RNA-Bob. Also comparing the consensus sequence of the relevant region in twister sister and the two ribozymes in this paper underlines the striking similarity between these RNAs. Given that the authors only assessed partial sequences of LINE-1 and OR4K15, I find it highly plausible that further accessory sequences have been missed that would clearly reveal that "lantern ribozymes" actually belong to the twister sister ribozyme class. This is also the reason I do not find the modeled structural data and biochemical data results convincing, as the differences observed could always be due to some accessory sequences and parts of the ribozyme structure that are missing.

      Highly conserved nucleotides in the catalytic core, the need for direct contacts to divalent metal ions for catalysis, the preference of Mn2+ oder Mg2+ for cleavage, the plateau in observed rate constants at ~100mM Mg2+, are all characteristics that are identical between the proposed lantern ribozymes and the known twister sister class.

      The difference in cleavage speed between twister sister (~5 min-1) and proposed lantern ribozymes could be due to experimental set-up (true single-turnover kinetics?) or could be explained by testing LINE-1 or OR4K15 ribozymes without needed accessory sequences. In the case of the minimal hammerhead ribozyme, it has been previously observed that missing important tertiary contacts can lead to drastically reduced cleavage speeds.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors investigated the expression profile of enterochromaffin (EC) cells after creating a new tryptophan hydroxylase 1 (Tph1) GFP-reporter mouse using scRNAseq and confirmative RNAscope analysis. They distinguish 14 clusters of Tph1+ cells found along the gut axis. The manuscript focuses on two of these, (i) a multihormonal cell type shown to express markers of pathogen/toxin and nutrient detection in the proximal small intestine, and (ii) on a EC-cluster in the distal colon, which expresses Piezo2, rendering these cells mechanosensitive. In- and ex- vivo data explore the role of the mechanosensitive EC population for intestinal/colonic transit, using chemogenetic activation, diptheria-toxin receptor dependent cell ablation and conditional gut epithelial specific Piezo2 knock-out. Whilst some of these data are confirmative of previous reports - Piezo2 has been implicated in mechanosensitive serotonin release previously, as referred to by the authors - the data are solid and emphasize the importance of mechanosensitive serotonin release for colonic propulsion. The transcriptomic data will guide future research.

      Strengths:<br /> The transcriptomic data, whilst confirmative, is more granular than previous data sets. Employing new tools to establish a role of mechanosensitive EC cells for colonic and thus total intestinal transit.

      Weaknesses:<br /> 1) The proposed villus/crypt distribution of the 14 cell types is not verified adequately. The RNAscope and immunohistochemistry samples presented do not allow assessment of whether this interpretation is correct - spatial transcriptomics, now approaching single-cell resolution, would be likely to help verify this claim.

      2) The physiological function and/or functionality of most of the transcriptomically enriched gene products has not been assessed. Whilst a role for Piezo2 expressing cells for colonic transit is convincingly demonstrated, the nature of the mechanical stimulus or the stimulus-secretion coupling downstream of Piezo2 activation is not clear.

    1. Reviewer #2 (Public Review):

      Thawornwattana et al. reconstruct a species tree of the genus Heliconius using the full-likelihood multispecies coalescent, an exciting approach for genera with a history of extensive gene flow and introgression. With this, they obtain a species tree with H. aoede as the earliest diverging lineage, in sync with ecological and morphological characters. They also add resolution to the species relationships of the melpomene-silvaniform clade and quantify introgression events. Finally, they trace the origins of an inversion on chromosome 15 that exists as a polymorphism in H. numata, but is fixed in other species. Overall, obtaining better species tree resolutions and estimates of gene flow in groups with extensive histories of hybridization and introgression is an exciting avenue. Being able to control for ILS and get estimates between sister species are excellent perks. One overall quibble is that the paper seems to be best suited to a Heliconius audience, where past trees are easily recalled, or members of the different clades are well known.

      Overall, applying approaches such as these to gain greater insight into species relationships with extensive gene flow could be of interest to many researchers. However, the conclusions could be strengthened with a bit more clarity on a few points.

      1) The biggest point of concern was the choice of species to use for each analysis. In particular the omission of H. ismenius in the resolution of the BNM clade species tree. The analysis of the chromosome 15 inversion seems to rely on the knowledge that H. ismenius is sister to H. numata, so without that demonstrated in the BNM section the resulting conclusions of the origin of that inversion are less interruptible.

      2) An argument they make in support of the branching scenario where H. aoede is the earliest diverging branch is based on which chromosomes support that scenario and the key observation that less introgression is detected in regions of low recombination. Yet, they go no further to understand the relationship between recombination rate and species trees produced.

      3) How the loci were defined could use more clarity. From the methods, it seems like each loci could vary quite a bit in total bp length and number of informative sites. Understanding the data processing would make this paper a better resource for others looking to apply similar approaches.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This paper described the role of BRCT repeat 5 in TOPBP1, a DNA damage response protein, in the maintenance of meiotic sex chromosome inactivation (MSCI). By analyzing a Topbp1 mutant mouse with amino acid substitutions in BRCT repeat 5, the authors found reduced phosphorylation of a DNA/RNA helicase, Sentaxin, and decreased localization of the protein to the X-Y sex body in pachynema. Moreover, the authors also found decreased repression of several genes on the sex chromosomes in the male mice.

      Strengths:<br /> The works including phospho-proteomics and single-cell RNA sequencing with lots of data have been done with great care and most of the results are convincing.

      Weaknesses:<br /> One concern is that, although the Topbp1 mutant spermatocytes show very severe defects after the stage of late pachynema, the defect in the gene silencing in the sex body is relatively weak. It is a bit difficult to explain how such a weak misregulation of the gene silencing in mice causes the complete loss of cells in the late stage of spermatogenesis.

    1. Reviewer #2 (Public Review):

      In the manuscript by Kahraman et al. the authors tested a recently developed Zn2+ indicator fluorogenic sensor as a tool to sort and purify human alpha cells from cadaveric donor islets, for downstream transcriptional and functional analysis. They demonstrate that their previously published sensor DA-ZP1, which was used to sort adult human islet beta cells in their previous work (Lee et al. 2020) they have now adapted for sorting alpha cells based on the 'intermediate' fluorescence intensity of these cells during staining. FACS purification of DA-ZP1-intermediate cells reveals they are strongly enriched for GCG+ cells (alpha cells). The sorted alpha cells can be reaggregated into alpha-pseudoislets for further studies. They carry out a variety of assays to characterize the viability, proliferation, apoptosis, glucagon secretion and transcriptomic changes in their sort purified alpha cells as compared with unsorted islet cells and intact islets. They conclude that sorting alpha cells with DA-ZP1 staining does not alter their function or transcriptome and allows stable maintenance of alpha-pseudoislets in culture for up to 10 days with no deleterious effects.

      Strengths:<br /> 1. The study is a nice resource for the field, particularly with the ongoing interest in studying alpha cell biology and function relevant to health and diabetes. The probe that they have previously published can now be used to simultaneously sort alpha and beta cells, which would be a great approach for the field. The results are generally supportive of the conclusions.

      2. The study used several human cadaveric donor islet preparations (four in total) representing different ancestries, limiting bias and inter-donor variation. A variety of cellular/molecular assays are employed to provide detailed phenotypic information.

      3. The transcriptomic profiling are very strong and provide solid evidence that the reaggregated alpha-pseudoislets are not dedifferentiating or losing function during prolonged (10 day) culture times.

      4. Visual presentation is clear and easy to follow for non-specialists.

      Weaknesses:

      1. The authors are presenting a previously developed probe/tool and also mention that other probes have been developed that can perform a very similar function, so the overall novelty is limited. They did not provide experimental evidence of how their probe is comparable or superior to other probes (e.g. ZIGIR, Newport Green).

      2. The authors performed glucagon secretion assays to monitor the function of the sort purified and reaggregated alpha-pseudoislets, but this was only done on 1 of the 4 human islet donors, limiting the generalizability of the conclusions. Also very few experiments were performed to examine alpha cell function in the sort purified cells.

    1. Reviewer #2 (Public Review):

      Summary:

      Preeclampsia is a disorder of pregnancy that affects 4-5% of pregnancies worldwide. Identifying this condition early is clinically relevant as it will help clinicians to make management decisions to prevent adverse outcomes. The placenta holds a key to many pregnancy-related pathologies including preeclampsia and studies have shown many differences in the placenta of women with preeclampsia as compared to controls. However as the placenta cannot be collected directly during pregnancy, the exosomes secreted by it are considered a good alternative to tissue biopsy. In this study, the authors have compared the proteins in different sizes of exosomes from the placenta of women with and without preeclampsia. The idea is to eventually use these as biomarkers for early detection of preeclampsia.

      Strengths:

      The novelty factor of this study is the use of two different-sized exosomes which has not been achieved earlier.

      Weaknesses:

      There is already enough information about the differences in exosome contents from the placentas of women with and without preeclampsia. There are some issues with the methods which may influence the outcomes of the data.

      The patient population described in the methods section is of HELLP syndrome while the title and the manuscript describe preeclampsia. While it is an important life-threatening condition to address, it is extremely rare and needs careful assessment by clinicians in terms of patient characteristics and outcomes measured.

      The study measured the proteins at only a single time point after the disease has already occurred. However, the placenta is an ever-changing tissue throughout pregnancy and different proteins can come up at different times in pregnancy. Thus serial measurements are necessary and a single time point measurement like that done here does little value addition. Unfortunately, this site has not validated the identified biomarkers in plasma or circulating placental exosomes from women with and without preeclampsia. Thus the validity of these findings in real-life situations can not be judged.

    1. Reviewer #2 (Public Review):

      Summary:

      The paper provides evidence that CPK3 plays a role in plant virus infection, and reports that viral infection is accompanied by changes in the dynamics of CPK3 and REM1.2, the phosphorylation substrate of CPK3, in the plasma membrane. In addition, the dynamics of the two proteins in the PM are shown to be interdependent.

      Strengths:

      The paper contains novel, important information.

      Weaknesses:

      The interpretation of some experimental data is not justified, and the proposed model is not fully based on the available data.

    1. Reviewer #2 (Public Review):

      Summary: Shotgun data have been analysed to obtain fungal and bacterial organisms' abundance. Through their metabolic functions and through co-occurrence networks, a functional relationship between the two types of organisms can be inferred. By means of metabolomics, function-related metabolites are studied in order to deepen the fungus-bacteria synergy.

      Strengths:<br /> Data obtained from bacteria correlate with data from other authors.<br /> The study of metabolic "interactions" between fungi and bacteria is quite new.<br /> The inclusion of metabolomics data to support the results is a great contribution.

      Weaknesses: Methodological descriptions are minimal.

      Some example:<br /> *The CON group (line 147) has not been defined. I supposed it is the control group.<br /> * There are no statistics related to shotgun sequencing. How many reads have been sequenced? How many have been removed from the host? How many are left to study bacteria and fungi? Are these reads proportional among the 48 samples? If not, what method has been used to normalise the data?<br /> * ggClusterNet has numerous algorithms to better display the modules of the microbiome network. Which one has been used?

    1. Reviewer #2 (Public Review):

      A high fraction of cells in early embryos carry aneuploid karyotypes, yet even chromosomally mosaic human blastocysts can implant and lead to healthy newborns with diploid karyotypes. Previous studies in other models have shown that genotoxic and proteotoxic stresses arising from aneuploidy lead to the activation of the p53 pathway and autophagy, which helps eliminate cells with aberrant karyotypes. These observations have been here evaluated and confirmed in human blastocysts. The study also demonstrates that the second lineage and formation of primitive endoderm are particularly impaired by aneuploidy.

      This is a timely and potentially important study. Aneuploidy is common in early embryos and has a negative impact on their development, but the reasons behind this are poorly understood. Furthermore, how mosaic aneuploid embryos with a fraction of euploidy greater than 50 % can undergo healthy development remains a mystery. Most of our current information comes from studies on murine embryos, making a substantial study on human embryos of great importance. However, there are only very few new findings or insights provided by this study. Some of the previous findings were reproduced, but it is difficult to say whether this is a real finding, or whether it is a consequence of a low sample number. The authors could get much more insight with their data.

    1. Reviewer #2 (Public Review):

      Antibody-dependent enhancement (ADE) of Dengue is largely driven by cross-reactive antibodies that target the DENV fusion loop or pre-membrane protein. Screening polyclonal sera for antibodies that bind to these cross-reactive epitopes could increase the successful implementation of a safe DENV vaccine that does not lead to ADE. However, there are few reliable tools to rapidly assess the polyclonal sera for epitope targets and ADE potential. Here the authors develop a live viral tool to rapidly screen polyclonal sera for binding to fusion loop and pre-membrane epitopes. The authors performed a deep mutational scan for viable viruses with mutations in the fusion loop (FL). The authors identified two mutations functionally tolerable in insect C6/36 cells, but lead to defective replication in mammalian Vero cells. These mutant viruses, D2-FL and D2-FLM, were tested for epitope presentation with a panel of monoclonal antibodies and polyclonal sera. The D2-FL and D2-FLM viruses were not neutralized by FL-specific monoclonal antibodies demonstrating that the FL epitope has been ablated.

      Overall the central conclusion that the engineered viruses can predict epitopes targeted by antibodies is supported by the data and the D2-FL and D2-FLM viruses represent a valuable tool to the DENV research community.

    1. Reviewer #2 (Public Review):

      Miller et al. take a variety of measurements and analytical techniques to assess the ecology of various species of the enantiornithine clade Bohaiornithidae. From this they suggest that the ancestral enantiornithine was a generalist and that the descendant clades occupied a breadth of niches similar to that of the radiation of derived birds after the K-Pg extinction.

      I am not a statistician so I found much of the paper to be outside my ability to review. I also am not an expert on enantiornithines or cranial morphology of birds, so these areas I also am not the best reviewer.

      However, I have published on bird foot functional morphology, notably that of birds of prey. This area thus is where I concentrated my efforts in the review.

      Overall, I find the idea that enantiornithines had occupied a similar niche breadth to post-K-Pg derived birds to be a curious, thought provoking proposal. On methodology, I have a few questions about bird feet comparisons. Whether my comments require minor or major edits is not really possible to say since I am not commenting on e.g. the skull-based analyses.

      STRENGTHS<br /> The paper uses a multi-proxy approach to assess ecological categories. This is broader than in previous works and is to be commended. I am not well placed to comment on the specifics of the statistical methods however.

      LANGUAGE<br /> The manuscript is very well written. I don't recall seeing many or possibly any grammatical issues. That's rare these days and I commend the authors on checking their manuscript and making it readable. This said, I found the extensive use of acronyms and abbreviations to be difficult to follow. This is not much of a criticism but in a general-readership journal, perhaps not having everything abbreviated might be preferential.

      The manuscript uses phrases like "superficially resembles" and "is similar to" a lot. I'm trying not to be picky, but very often these phrasings don't say how the features are similar (or not). Is it the curvature etc? Could these be expanded upon a bit more in the text please? It isn't very easy to assess similarity r dissimilarity without some point of reference.

      FIGURES<br /> The figures are generally very good, and the captions are generously descriptive. However, all figures are graphs, tables, etc. It would be nice, somewhere, to have an image or group of images showing us what a bohaiornithine is.. especially since this is a general-readership journal. I wasn't aware of the details of enantiornithine clades before reading this manuscript, and I suspect other readers would be in the same place. Can we get some images of fossils, a skeletal diagram, or something?

      RAPTOR CLAWS<br /> This is my main criticism.

      The foot morphometrics suggest that there is a morphological difference between claws of raptors that feed on large prey, and those of raptors that feed on small prey. I am curious what these morphological differences are.

      In our paper(s) (Fowler et al., 2009; 2011), we looked at the feet (especially the claws) of various birds of prey, and studied foot functional morphology compared with prey choice, capture and immobilization strategy. We devised a behavioural categorization that separated the behavior (mainly in subduing the prey) between "small" and "large" prey, that being whether they can be fully contained within the foot of the raptor. Most if not all raptors take small prey, and these are typically killed using constriction. Some raptors have specialized in small prey/constriction (e.g. most owls). Some raptors might also take large prey, but since (by definition) large prey cannot be fully contained within the foot then the prey item cannot be constricted and a different immobilization (kill) mechanism must be employed (which differs among clades).

      We never made a morphological distinction between small and large prey specialists largely because all raptors take small prey. I am thus interested in what taxa are designated small vs large prey specialists in this study. Perhaps these authors have found characters that distinguish primarily small-prey-specialist raptors, but I do not know what they are and maybe this should be included in the text somewhere.

      Owls are mainly small prey specialists. Compared with other raptors, they have a unusual foot that has (I am generalising here) short non-ungual phalanges contrasting with long ungual phalanges which are relatively low curvature. We (Fowler et al 2009) suggest that this gives owls a more tightly closable foot (short non-ungual phalanges), but maintains reach of each toe (long claw). This could be seen as indicative of small -prey specialization, but again, other raptor clades take small prey without this very specialized foot. If the "small prey specialist" category here is really just owls then it might be slightly misleading.

      This is my main criticism. I would at least like some explanation of what is in this category.

      Otherwise I must leave assessment of cranial functional morphology, and general statistical analysis to other reviewers.

      IMPACT<br /> As I have already stated, the idea that Enantiornithines occupied a similar breadth of niches to post K-Pg birds is thought provoking, moreso than upon initial reading. The authors note that this raises questions about the adaptations or survivorship of derived birds, and this is what I find most intriguing, and is what I think will appeal to most readers.

    1. Reviewer #2 (Public Review):

      This study builds upon the team's recent discovery that antibiotic treatment and other disturbances favour the persistence of bacteria with genomes that encode complete modules for the synthesis of essential metabolites (Watson et al. 2023). Veseli and collaborators now provide an in-depth analysis of metabolic pathway completeness within microbiomes, finding strong evidence for an enrichment of bacteria with high metabolic independence in the microbiomes associated with IBD and other gastrointestinal disorders. Importantly, this study provides new open-source software to facilitate the reconstruction of metabolic pathways, estimate their completeness and normalize their results according to species diversity. Finally, this study also shows that the metabolic independence of microbial communities can be used as a marker of dysbiosis. The function-based health index proposed here is more robust to individuals' lifestyles and geographic origin than previously proposed methods based on bacterial taxonomy.

      The implications of this study have the potential to spur a paradigm shift in the field. It shows that certain bacterial taxa that have been consistently associated with disease might not be harmful to their host as previously thought. These bacteria seem to be the only species that are able to survive in a stressed gut environment. They might even be important to rebuild a healthy microbiome (although the authors are careful not to make this speculation).

      This paper provides an in-depth discussion of the results, and limitations are clearly addressed throughout the manuscript. Some of the potential limitations relate to the use of large publicly available datasets, where sample processing and the definition of healthy status varies between studies. The authors have recognised these issues and their results were robust to analyses performed on a per-cohort basis. These potential limitations, therefore, are unlikely to have affected the conclusions of this study.

      Overall, this manuscript is a magnificent contribution to the field, likely to inspire many other studies to come.

    1. Reviewer #2 (Public Review):

      This manuscript reports on the role of Rho-associated coiled-coil kinase (ROCK) in biomineralization of sea urchin larval skeletons. A number of experiments examine the initiation, growth, and patterning of the skeleton in an effort to determine if, and how, ROCK participates in skeletal formation. The authors conclude that ROCK controls the formation, growth, and morphology (patterning) of the skeleton based on a number of inhibition studies. The main target of the experiments is the actomyosin cytoskeleton which has been the focus of many ROCK studies in vertebrates. Based on similar experimental outcomes when comparing the results here with published data from vertebrates, they suggest that ROCK and the actomyosin network operate in a similar way in biomineralization despite independent evolutionary origins of the sea urchin larval skeletons and the skeletons of vertebrates.

      My concerns are the interpretation of the experiments. The main overriding concern is a possible over-interpretation of the role of ROCK. In the literature that ROCK participates in many biological processes with a major contribution to the actin cytoskeleton. And when a function is attributed to ROCK, it is usually based on the determination of a protein that is phosphorylated by this kinase. Here that is not the case. The observation here is in most cases stunted growth of the spicule skeleton and some mis-patterning occurs or there is an absence of skeleton if the inhibitor is added prior to initiation of skeletal growth. They state in the abstract that ROCK impairs the organization of F-actin around the spicules. The evidence for that as a direct role is absent. They use morpholino data and ROCK inhibitor data to draw their conclusion. My main concern is the concentration of the inhibitor used since at the high concentrations used, the inhibitor chosen is known to inhibit other kinases as well as ROCK (PKA and PKC). They indicate that this inhibition is specifically in the skeletogenic cells based on the isolation of skeletogenic cells in culture and spicule production either under control or ROCK inhibition and they observe the same - stunting and branching or absence of skeletons if treated before skeletogenesis commences. Again, however, the high concentrations are known to inhibit the other kinases. They use blebbistatin and latrunculin and show that these known inhibitors of actin cytoskeleton lead to abnormal spiculogenesis, This coincidence is suggestive but is not proof that it is ROCK acts on the actomyosin cytoskeleton given the specificity concerns.

    1. Reviewer #2 (Public Review):

      In this manuscript, Birkbak and colleagues use a novel approach to transform multi-omics datasets in images and apply Deep Learning methods for image analysis. Interestingly they find that the spatial representation of genes on chromosomes and the order of chromosomes based on 3D contacts leads to best performance. This supports that both 1D proximity and 3D proximity could be important for predicting different phenotypes. I appreciate that the code is made available as a github repository. The authors use their method to investigate different cancers and identify novel genes potentially involved in these cancers. Overall, I found this study important for the field.

      In the original submission there were several major points with this manuscript could be grouped in three parts:

      1. While the authors have provided validation for their model, it is not always clear that best approaches have been used. This has now been addressed in the revised version of the manuscript.

      2. Potential improvement to the method

      a. It is very encouraging the use of HiC data, but the authors used a very coarse approach to integrate it (by computing the chromosome order based on interaction score). We know that genes that are located far away on the same chromosome can interact more in 3D space than genes that are relatively close in 1D space. Did the authors consider this aspect? Why not group genes based on them being located in the same TAD? In the revised version of the manuscript, the authors discussed this possibility but did not do any new additional analysis.

      b. Authors claim that "given that methylation negatively correlates with gene expression, these were considered together". This is clearly not always the case. See for example https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02728-5. In the revised version of the manuscript, the authors addressed fully this comment.

      3. Interesting results that were not explained.

      a. In Figure 3A methylation seems to be most important omics data, but in 3B, mutations and expression are dominating. The authors need to explain why this is the case. In the revised version of the manuscript, the authors have clarified this.

    1. Reviewer #2 (Public Review):

      Summary: The goal of this study is to clarify how the brain simultaneously represents item-specific temporal information and item-independent boundary information. The authors report spectral EEG data from intracranial patients performing a delayed free recall task. They perform cosine similarity analyses on principal components derived from gamma band power across stimulus duration. The authors find that similarity between items in serial position 1 (SP1) and all other within-list items decreases as a function of serial position, consistent with temporal context models. The authors find that across-list item similarity to SP1 is greatest for SP1 items relative to items from other serial positions, an effect that is greater in medial parietal lobe compared to lateral temporal cortex and hippocampus. The authors conclude that their findings suggest that perceptual boundary information is represented in medial parietal lobe. Despite a robust dataset, the methodological limitations of the study design prevent strong interpretations from being made from these data. The same-serial position across-list similarity may be driven by attentional mechanisms that are distinct from boundary information.

      Strengths:<br /> 1. The motivation of the study is strong as how both temporal contextual drift and event boundaries contribute to memory mechanisms is an important open question.

      2. The dataset of spectral EEG data from 99 intracranial patients provides the opportunity for precise spatiotemporal investigation of neural memory mechanisms.

      Weaknesses:<br /> 1. Because this is not a traditional event boundary study, the data are not ideally positioned to demonstrate boundary specific effects. In a typical study investigating event boundary effects, a series of stimuli are presented and within that series occurs an event boundary -- for instance, a change in background color. The power of this design is that all aspects between stimuli are strictly controlled -- in particular, the timing -- meaning that the only difference between boundary-bridging items is the boundary itself. The current study was not designed in this manner, thus it is not possible to fully control for effects of time or that multiple boundaries occur between study lists (study to distractor, distractor to recall, recall to study). Each list in a free recall study can be considered its own "mini" experiment such that the same mechanisms should theoretically be recruited across any/all lists. There are multiple possible processes engaged at the start of a free recall study list which may not be specific to event boundaries per se. For example, and as cited by the authors, neural fatigue/attentional decline (and concurrent gamma power decline) may account for serial position effects. Thus, SP1 on all lists will be similar by virtue of the fact that attention/gamma decrease across serial position, which may or may not be a boundary-specific effect. In an extreme example, the analyses currently reported could be performed on an independent dataset with the same design (e.g. 12 word delayed free recall) and such analyses could potentially reveal high similarity between SP1-list1 in the current study and SP1-list1 in the second dataset, effects which could not be specifically attributed to boundaries.

      2. Comparisons of recalled "pairs" does not account for the lag between those items during study or recall, which based on retrieved context theory and prior findings (e.g. Manning et al., 2011), should modulate similarity between item representations. Although the GLM will capture a linear trend, it will not reveal serial position specific effects. It appears that the betas reported for the SP12 analyses are driven by the fact that similarity with SP12 generally increases across serial position, rather a specific effect of "high similarity to SP12 in adjacent lists" (Page 5, excluding perhaps the comparison with list x+1). It is also unclear how the SP12 similarity analyses support the statement that "end-list items are represented more distinctly, or less similarly, to all succeeding items" (Page 5). It is not clear how the authors account for the fact that the same participants do not contribute equally to all ROIs or if the effects are consistent if only participants who have electrodes in all ROIs are included.

      3. The authors use the term "perceptual" boundary which is confusing. First, "perceptual boundary" seems to be a specific subset of the broader term "event boundary," and it is unclear why/how the current study is investigating "perceptual" boundaries specifically. Second and relatedly, the current study does not have a sole "perceptual" boundary (as discussed in point 1 above), it is really a combination of perceptual and conceptual since the task is changing (from recalling the words in the previous list to studying the words in the current list OR studying the words in the current list to solving math problems in the current list) in addition to changes in stimulus presentation.

      4. Although the results show that item-item similarity in the gamma band decreases across serial position, it is unclear how the present findings further describe "how gamma activity facilitates contextual associations" (Page 5). As mentioned in point 1 above, such effects could be driven by attentional declines across serial position -- and a concurrent decline in gamma power -- which may be unrelated to, and actually potentially impair, the formation of contextual associations, given evidence from the literature that increased gamma power facilitates binding processes.

      5. Some of the logic and interpretations are inconsistent with the literature. For example, the authors state that "The temporal context model (TCM) suggests that gradual drift in item similarity provides context information to support recovery of individual items" however, this does not seem like an accurate characterization of TCM. According to TCM, context is a recency-weighted average of previous experience. Context "drifts" insofar as information is added to/removed from context. Context drift thus influences item similarity -- it is not that item similarity itself drifts, but that any change in item-item similarity is due to context drift. The current findings do not appear at odds with the conceptualization of drift and context in current version of the context maintenance and retrieval model. Furthermore, the context representation is posited to include information beyond basic item representations. Two items, regardless of their temporal distance, can be associated with similar contexts if related information is included in both context representations, as predicted and shown for multiple forms of relatedness including semantic relatedness (Manning & Kahana, 2012) and task relatedness (Polyn et al., 2012).

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, Basson et al. study the representation of women in "high-impact" journals through the lens of gendered submission behavior. This work is clear and thorough, and it provides new insights into gender disparities in submissions, such as that women were more likely to avoid submitting to one of these journals based on advice from a colleague/mentor. The results have broad implications for all academic communities and may help toward reducing gender disparities in "high-impact" journal submissions. I enjoyed reading this article, and I have several recommendations regarding the methodology/reporting details that could help to enhance this work.

      Strengths:<br /> This is an important area of investigation that is often overlooked in the study of gender bias in publishing. Several strengths of the paper include:<br /> 1) A comprehensive survey of thousands of academics. It is admirable that the authors retroactively reached out to other researchers and collected an extensive amount of data.<br /> 2) Overall, the modeling procedures appear thorough, and many different questions are modeled.<br /> 3) There are interesting new results, as well as a thoughtful discussion. This work will likely spark further investigation into gender bias in submission behavior, particularly regarding the possible gendered effect of mentorship on article submission.

      Weaknesses:<br /> 1) The GitHub page should be further clarified. A detailed description of how to run the analysis and the location of the data would be helpful. For example, although the paper says that "Aggregated and de-identified data by gender, discipline, and rank for analyses are available on GitHub," I was unable to find such data.<br /> 2) Why is desk rejection rate defined as "the number of manuscripts that did not go out for peer review divided by the number of manuscripts rejected for each survey respondent"? For example, in your Grossman 2020 reference, it appears that manuscripts are categorized as "reviewed" or "desk-rejected" (Grossman Figure 2). If there are gender differences in the denominator, then this could affect the results.<br /> 3) Have you considered correcting for multiple comparisons? Alternatively, you could consider reporting P-values and effect sizes in the main text. Otherwise, sometimes the conclusions can be misleading. For example, in Figure 3 (and Table S28), the effect is described as significant in Social Sciences (p=0.04) but not in Medical Sciences (p=0.07).<br /> 4) More detail about the models could be included. It may be helpful to include this in each table caption so that it is clear what all the terms of the model were. For instance, I was wondering if journal or discipline are included in the models.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The aster, consisting of microtubules, plays important roles in spindle positioning and the determination of the cleavage site in animals. The mechanics of aster movement and positioning have been extensively studied in several cell types. However, there is no unified biophysical model, as different mechanisms appear to predominate in different model systems. In the present manuscript, the authors studied aster positioning mechanics in the Drosophila syncytial embryo, in which short-ranged aster repulsion generates a separation force. Taking advantage of the ex vivo system developed by the group and the fly gnu mutant, in which the nuclear number can be minimized, the authors performed time-lapse observations of single asters and multiple asters in the explant. The observed aster dynamics were interpreted by building a mathematical model dealing with forces. They found that aster dissociation from the boundary depends on the microtubule pushing force. Additionally, laser ablation targeting two separating asters showed that aster-aster separation is also mediated by the microtubule pushing force. Furthermore, they built a simulation model based on the experimental results, which reproduced aster movement in the explant under various conditions. Notably, the actual aster dynamics were best reproduced in the model by including a short-ranged inhibitory term when asters are close to the boundary or each other.

      Strengths:<br /> This study reveals a unique aster positioning mechanics in the syncytial embryo explant, which leads to an understanding of the mechanism underlying the positioning of multiple asters associated with nuclei in the embryo. The use of explants enabled accurate measurement of aster motility and, therefore, the construction of a quantitative model. This is a notable achievement.

      Weaknesses:<br /> The main conclusion that aster repulsion predominates in this system has already been drawn by the same authors in their recent study (de-Carvalho et al., Development, 2022). As the present work provides additional support to the previous study using different experimental system, the authors should emphasize that the present manuscripts adds to it (but the conceptual novelty is limited). The molecular mechanisms underlying aster repulsion remain unexplored since the authors were unable to identify specific factor(s) responsible for aster repulsion in the explant.

      Specific suggestions:<br /> Microtubules should be visualized more clearly (either in live or fixed samples). This is particularly important in Figure 4E and Video 4 (laser ablation experiment to create asymmetric asters).

    1. Reviewer #2 (Public Review):

      Summary:<br /> Sharninghausen et al use a generic screening platform to search for short (5 amino acid) degrons that function in the lumen of the endoplasmic reticulum (ER) of budding yeast. The screen did indeed identify a number of sequences which increased the rate of degradation of their test proteins. Although the effect of the single degron was rather modest the authors could show that by mutimerising the sequence (4x) they obtained degrons that functioned fairly efficiently. Further characterisation indicated that the degrons only functioned when placed at the N-terminus of the target protein and, were dependent on both the proteasome and the segregase Cdc48 (p97) for degradation. The authors also demonstrated that degradation was via the ERAD pathway.

      Strengths:<br /> In general, the data presented is supportive of the conclusions drawn and the authors have thus identified a sequence that can be appended onto other ER targeted proteins to mediate their degradation within the lumen of the ER. How useful this will be to the community remains to be seen.

      Weaknesses:<br /> While the observation that such mutimerised sequences can act as degrons is an interesting curiosity, it is not clear that such sequences function in vivo. In fact the DegV1 sequence used throughout the paper is not present in any yeast or fungal proteins and the fact that it has to be located at the N-terminus of the protein to induce degradation is at odds with the idea that proteins to be degraded need to be unfolded. Thus, the role of such sequences in vivo is questionable.

    1. Reviewer #2 (Public Review):

      This paper tried to assess the link between genetic and environmental factors on psychotic-like experiences, and the potential mediation through cognitive ability. This study was based on data from the ABCD cohort, including 6,602 children aged 9-10y. The authors report a mediating effect, suggesting that cognitive ability is a key mediating pathway in the link between several genetic and environmental (risk and protective) factors on PLEs.

      Strengths of the methods<br /> The authors use a wide range of validated (genetic, self- and parent-reported, as well as cognitive) measures in a large dataset with a 2-year follow-up period. The statistical methods have potential to address key limitations of previous research.

      Weaknesses of the methods<br /> The methodological advantage of the method (Integrated generalized structured component analysis, IGSCA) over the standard method (Structural equation modeling, SEM) is not fully clear.<br /> Not all methods are fully explained (how genetic components were derived; how cognition was assessed in Lee et al., 2018).<br /> Not the largest or most recent GWAS (Genome-wide association studies) were used to generate PGS.

      Strengths of the results<br /> The authors included a comprehensive array of analyses.

      Weaknesses of the results<br /> Some factor loadings presented in Figure 3 seem counterintuitive/inconsistent.<br /> Supplementary tables are difficult to assess. Unclear significance statement / p-values in Table 2.

      Appraisal<br /> The authors suggest that their findings provide evidence for policy reforms (e.g., targeting residential environment, family SES (social economic status), parenting, and schooling).

      Impact<br /> Immediate impact is limited given the short follow-up period (2y), possibly concerns for selection bias and attrition in the data, and some methodological concerns. The authors are transparent about most of these limitations.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The study builds on the work of the Pan group and others which has described the existence of core Hippo pathway proteins in Capsaspora and, more recently, described a role for a Yorkie/YAP homologue in regulation of cell shape and actin, as opposed to proliferation. For this recent study, they developed genetic techniques to mutate genes in Capsaspora, and this technology has been leveraged again in this study. Using loss of genetic approaches, the authors find that loss of either of the two major kinases in the Hippo pathway core kinase cassette (Warts and Hippo) impact Capsaspora morphology and the actin cytoskeleton. This is phenocopied by overexpression of Capsaspora Yorkie/YAP. In addition, Capsaspora Yorkie/YAP accumulates in the nucleus of organisms lacking Warts or Hippo, as it does in metazoans. While these experiments are not overly surprising, they still provide important verification that core Hippo signaling events are conserved in Capsaspora.

      Subsequently, they show that Capsaspora lacking Warts or Hippo do not overproliferate, which contrasts with many studies in animals, particularly in epithelial tissues where loss of Warts or Hippo often causes overproliferation. Rather, the authors show that Capsaspora Warts and Hippo regulate cell morphology and actomyosin-dependent contractile behaviour. They speculate from these findings that Hippo signalling could regulate the density of Capsaspora when they grow in aggregates and draw parallels to the known role of the Hippo pathway in contact inhibition of mammalian cells grown in culture.

      Strengths:<br /> Together with their 2022 paper, this study paints an emerging picture that the ancestral function of the Hippo pathway is to regulate the actin cytoskeleton, not proliferation, which is a significant finding. This also suggests that the ability to control proliferation was something that the Hippo pathway was re-purposed to do at some stage during the evolution of metazoans. These findings are important for the Hippo field, and our understanding of cellular signalling and evolution more broadly.

      Weaknesses:<br /> Further biochemical and genetic experiments would allow the authors to more convincingly prove that core features of Hippo signalling are conserved in Capsaspora - e.g., that Capsaspora Hippo/MST activates Warts/LATS by phosphorylation and Warts/LATS represses Yorkie/YAP by phosphorylation hey serine residues. Additional genetic studies would also allow one to determine whether Capsaspora Yki/YAP controls actomyosin contractility by transcription (with the Scalloped/TEAD homologue) and/or by non-transcriptional mechanisms, as have been reported for Yki in Drosophila. Higher resolution imaging approaches such as electron microscopy would likely give further mechanistic insights into how Hpo, Wts and Yki modulate actomyosin contractility in Capsaspora.

    1. Reviewer #2 (Public Review):

      This manuscript describes colony-growth phenotypes to measure the fitness of deletion mutants for 3509 non-essential S. pombe genes in 131 conditions. 3492 mutants, including 124 mutants of 'priority unstudied' proteins conserved in humans, providing varied functional clues.

      Phenotype-correlation networks provide evidence for the roles of poorly characterized proteins through guilt by association with known proteins. Gene Ontology (GO) terms were predicted using machine learning methods that take advantage of protein-network and protein-homology data.

      Integrated analyses produced 1,675 novel GO predictions for 783 genes, including 47 predictions for 23 priority unstudied proteins. Experimental validation for genes involved in cellular ageing were obtained.

      A method called NET-FF, which combines network embeddings and protein homology data to predict GO annotations, was developed. The authors demonstrate NET-FF predicts GO terms better than random and compare the information content of the predicted terms with the PomBase GO annotations. The phenotypic data was used to filter the GO annotation predictions made by NET-FF and then explore specific biological examples supported by both datasets

      This is a very impressive and rich resource of phenotypic data and it will be particularly useful for the S. pombe research community and generally useful for the functional characterization of highly conserved eukaryotic genes. Overall, the analysis is powerful and sound.

    1. Reviewer #2 (Public Review):

      Summary:

      Here Jeong et al., use a combination of theoretical and experimental approaches to define molecular contexts that support specific chromatin conformations. They seek to define features that are associated with TADs that are retained after cohesin depletion (the authors refer to these TADs as P-TADs). They were motivated by differences between single cell data, which suggest that some TADs can be maintained in the absence of cohesin, whereas ensemble HiC data suggest complete loss of TADs. By reananalyzing a number of HiC datasets from different cell types, the authors observe that in ensemble methods, a significant subset of TADs are retained. They observe that P-TADs are associated with mismatches in epigenetic state across TAD boundaries. They further observe that "physical boundaries" are associated with P-TAD maintenance. Their structure/simulation based approach appears to be a powerful means to generate 3D structures from ensemble HiC data, and provide chromosome conformations that mimic the data from single-cell based experiments. Their results also challenge current dogma in the field about epigenetic state being more related to compartment formation rather than TAD boundaries. Their analysis is particularly important because limited amounts of imaging data are presently available for defining chromosome structure at the single-molecule level, however, vast amounts of HiC and ChIP-seq data are available. By using HiC data to generate high quality simulated structural data, they overcome this limitation. Overall, this manuscript is important for understanding chromosome organization, particularly for contacts that do not require cohesin for their maintenance, and for understanding how different levels of chromosome organization may be interconnected. I cannot comment on the validity of the provided simulation methods and hope that another reviewer is qualified to do this.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors analyze the functions and regulation of Bon, the sole Drosophila ortholog of the TIF1 family of mammalian transcriptional regulators. Bon has been implicated in several developmental programs, however the molecular details of its regulation have not been well understood. Here, the authors reveal the requirement of Bon in oogenesis, thus establishing a previously unknown biological function for this protein. Furthermore, careful molecular analysis convincingly established the role of Bon in transcriptional repression. This repressor function requires interactions with the NuRD complex and histone methyltransferase SetDB1, as well as sumoylation of Bon by the E3 SUMO ligase Su(var)2-10. Overall, this work represents a significant advance in our understanding of the functions and regulation of Bon and, more generally, the TIF1 family. Since Bon is the only TIF1 family member in Drosophila, the regulatory mechanisms delineated in this study may represent the prototypical and important modes of regulation of this protein family. The presented data are rigorous and convincing. As discussed below, this study can be strengthened by a demonstration of a direct association of Bon with its target genes, and by analysis of the biological consequences of the K20R mutation.

      Strengths:<br /> 1. This study identified the requirement for Bon in oogenesis, a previously unknown function for this protein.<br /> 2. Identified Bon target genes that are normally repressed in the ovary, and showed that the repression mechanism involves the repressive histone modification mark H3K9me3 deposition on at least some targets.<br /> 3. Showed that Bon physically interacts with the components of the NuRD complex and SetDB1. These protein complexes are likely mediating Bon-dependent repression.<br /> 4. Identified Bon sumoylation site (K20) that is conserved in insects. This site is required for repression in a tethering transcriptional reporter assay, and SUMO itself is required for repression and interaction with SetDB1. Interestingly, the K20-mutant Bon is mislocalized in the nucleus in distinct puncta.<br /> 5. Showed that Su(var)2-10 is a SUMO E3 ligase for Bon and that Su(var)2-10 is required for Bon-mediated repression.

      Weaknesses:<br /> The study would be strengthened by demonstrating a direct recruitment of Bon to the target genes identified by RNA-seq. Given that the global ChIP-seq was not successful, a few possibilities could be explored. First, Bon ChIP-qPCR could be performed on the individual targets that were functionally confirmed (e.g. rbp6, pst). Second, a global Bon ChIP-seq has been reported in PMID: 21430782 - these data could be used to see if Bon is associated with specific targets identified in this study. In addition, it would be interesting to see if there is any overlap with the repressed target genes identified in Bon overexpression conditions in PMID: 36868234.

      The second area where the manuscript can be improved is to analyze the biological function of the K20R mutant Bonus protein. The molecular data suggest that this residue is important for function, and it would be important to confirm this in vivo.

    1. Reviewer #2 (Public Review):

      In this study, the authors explore the structure/function of the DCLK kinases, most specifically DCLK1 as it is the most studied to date. Recently, the C-terminal domain has garnered attention as it was found to regulate the kinase domain, however, the different isoforms retain additional amino acid sequences with as-yet-undefined functions. The authors provide an evolutionary and biochemical characterization of these regions and provide evidence for some functionality for these additional C-terminal sequences. While these experiments are informative they do require that the protein is soluble and not membrane-bound as has been suggested to be important for functionality in other studies. Still, this is a major contribution to understanding the structure/function of these proteins that will be important in future experimental designs.

    1. Reviewer #2 (Public Review):

      In this study, the authors take a multipronged approach to identify the substrate repertoire of calcium-dependent protein kinase, CDPK1 in Toxoplasma that includes quantitative phosphoproteomics, myristoylation, thiophosphorylation, immunoprecipitation as well as proximity-based labeling. Their finding also reveals that CDPK1 functions in parasite invasion and egress by phosphorylating different protein candidates. More importantly, the authors successfully determine one branch of the CDPK1 signaling pathway that regulates invasion through the phosphorylation of the HOOK protein involved in the translocation and secretion of micronemal proteins.

    1. Reviewer #2 (Public Review):

      This manuscript addresses an important question: what is the role of the gene Clock in the control of circadian rhythms in a very primitive group of animals: Cnidaria. Clock has been found to be essential for circadian rhythms in several animals, but its function outside of Bilaterian animals is unknown. The authors successfully generated a severe loss-of-function mutant in Nematostella. This is an important achievement that should help in understanding the early evolution of circadian clocks. Unfortunately, this study currently suffers from several important weaknesses. In particular, the authors do not present their work in a clear fashion, neither for a general audience nor for more expert readers, and there is a lack of attention to detail. There are also important methodological issues that weaken the study, and I have questions about the robustness of the data and their analysis. I am hoping that the authors will be able to address my concerns, as this work should prove important for the chronobiology field and beyond. I have highlighted below the most important issues, but the manuscript needs editing throughout to be accessible to a broad audience, and referencing could be improved.

      Major issues:<br /> 1) Why do the authors make the claim in the abstract that CLOCK function is conserved with other animals when their data suggest that it is not essential for circadian rhythms? dCLK is strictly required in Drosophila for circadian rhythms. In mammals, there are two paralogs, CLOCK and NPAS2, but without them, there are no circadian rhythms either. Note also that the recent claim of BMAL1-independent rhythms in mammals by Ray et al., quoted in the discussion to support the idea that rhythms can be observed in the absence of the positive elements of the circadian core clock, had to be corrected substantially, and its main conclusions have been disputed by both Abruzzi et al. and Ness-Cohn et al. This should be mentioned.

      2) The discussion of CIPC on line 222 is hard to follow as well. How does mRNA rhythm inform the function of CIPC, and why would it function as a "dampening factor"? Given that it is "the only core clock member included in the Clock-dependent CCGs," (220) more discussion seems warranted. Discussing work done on this protein in mammals and flies might provide more insight.

      3) The behavioral arrhythmicity seen with their Clock mutation is really interesting. However, what is shown is only an averaged behavior trace and a single periodogram for the entire population. This leaves open the possibility that individual animals are poorly synchronized with each other, rather than arrhythmic. I also note that in DD there seem to be some residual rhythms, though they do not reach significance. Thus, it is also possible that at least some individual animals retain weak rhythms. The authors should analyze behavioral rhythms in individual animals to determine whether behavioral rhythmicity is really lost. This is important for the solidity of their main conclusions.

      4) There is no mention in the results section of the behavior of heterozygotes. Based on supplement figure 2A, there is a clear reduction in amplitude in the heterozygous animals. Perhaps this might be because there is only half a dose of Clock, but perhaps this could be because of a dominant-negative activity of the truncated protein. There is no direct functional evidence to support the claim that the mutant allele is nonfunctional, so it is important to discuss carefully studies in other species that would support this claim, and the heterozygous behavior since it raises the possibility that the mutant allele acts as a dominant negative.

      5) I do not understand what the bar graphs in Figure 2E and 3B represent - what does the y-axis label refer to?

      6a. I note that RAIN was used, with a p<0.05 cut-off. I believe RAIN is quite generous in calling genes rhythmic, and the p-value cut-off is also quite high. What happens if the stringency is increased, for example with a p<0.01.<br /> b. It would be worth choosing a few genes called rhythmic in different conditions (mutant or wild-type. LD or DD), and using qPCR to validate the RNAseq results. For example, in Figure 3D, Myh7 RNAseq data are shown, and they do not look convincing. I am surprised this would be called a circadian rhythm. In wild-type, the curve seems arrhythmic to me, with three peaks, and a rather large difference between the first and second ZT0 time point. In the Clock mutants, rhythms seem to have a 12hr period, so they should not be called rhythmic according to the material and methods, which says that only ca 24hr period mRNA rhythms were considered rhythmic. Also, the result section does not say anything about Myh7 rhythms. What do they tell us? Why were they presented at all?

      7) The authors should explain better why only the genes that are both rhythmic in LD and DD are considered to be clock-controlled genes (CCGs). In theory, any gene rhythmic in DD could be a CCG. However, Leach and Reitzel actually found that most genes in DD1 do not cycle the next day (DD2)? This suggests that most "rhythmic" genes might show a transient change in expression due to prolonged obscurity and/or the stress induced by the absence of a light-dark cycle, rather than being clock controlled. Is this why the authors saw genes rhythmic under both LD and DD as actual CCGs? I would suggest verifying that in DD the phase of the oscillation for each CCG is similar to that in LD. If a gene is just responding to obscurity, it might show an elevated expression at the end of the dark period of LD, and then a high level in the first hours of DD. Such an expression pattern would be very unlikely to be controlled by the circadian clock.

      8) Since there are still rhythms in LD in Clock mutants, I wonder whether there is a paralog that could be taking Clock's place, similar to NPAS2 in mammals.

      9) I do not follow the point the authors try to make in lines 268-272. The absence of anticipatory behavior in Drosophila Clk mutants results from disruption of the circadian molecular clock, due to the loss of Clk's circadian function. Which light-dependent function of Clock are the authors referring to, then? Also, following this, it should be kept in mind that clock mutant mice have a weakened oscillator. The effect on entrainment is secondary to the weakening of the oscillator, rather than a direct effect on the light input pathway (weaker oscillators have increased response to environmental inputs). The authors thus need to more clearly explain why they think there is a conservation of circadian and photic clock function.

    1. Reviewer #2 (Public Review):

      Summary: The authors investigate the assembly of the Q-nMT, a stable microtubule structure that is assembled during quiescence. Notably, the authors show that the formation of the Q-nMT cannot be solely explained by changes in the physicochemical properties of quiescent cells. The authors report that Q-nMT assembly occurs in three regulated steps and identify kinesin motor proteins involved in the assembly and disassembly of the structure.

      Strengths: The findings provide new insight into the assembly and possible function of the Q-nMT with respect to the response of haploid budding yeast to glucose starvation.

      Weaknesses: The manuscript would benefit from more precise language and requires additional clarification regarding how claims are supported by the evidence. Clear definitions are also required, for example, "active process" is not defined. Some conclusions are not supported by the results, for example, the claim that the Q-nMT functions as a checkpoint effector that inhibits re-entry into the cell cycle.

    1. Reviewer #2 (Public Review):

      This work describes transcriptome profiling of dissected skin of zebrafish at post-embryonic stages, at a time when adult structures and patterns are forming. The authors have used the state-of-the-art combinatorial indexing RNA-seq approach to generate single cell (nucleus) resolution. The data appears robust and is coherent across the four different genotypes used by the authors.

      The authors present the data in a logical and accessible manner, with appropriate reference to the anatomy. They include helpful images of the biology and schematics to illustrate their interpretations.

      The datasets are then interrogated to define cell and signalling relationships between skin compartments in six diverse contexts. The hypotheses generated from the datasets are then tested experimentally. Overall, the experiments are appropriate and rigorously performed. They ask very interesting questions of interactions in the skin and identify novel and specific mechanisms. They validate these well.

      The authors use their datasets to define lineage relationships in the dermal scales and also in the epidermis. They show that circumferential pre-scale forming cells are precursors of focal scale forming cells while there appeared a more discontinuous relationship between lineages in the epidermis.

      The authors present transcriptome evidence for enamel deposition function in epidermal subdomains. This is convincingly confirmed with an ameloblastin in situ. They further demonstrate distinct expression of SCPP and collagen genes in the SFC regions.

      The authors then demonstrate that Eda and TH signalling to the basal epidermal cells generates FGF and PDGF ligands to signal to surrounding mesenchyme, regulating SFC differentiation and dermal stratification respectively.

      Finally, they exploit RNA-seq data performed in parallel in the bnc2 mutants to identify the hypodermal cells as critical regulators of pigment patterning and define the signalling systems used.

      Whilst these six interactions in the skin are disparate, the stories are unified by use of the sci-RNA-seq data to define interactions. Overall, it's an assembly of work which identifies novel and interesting cell interactions and cross-talk mechanisms.

      The paper provides robust evidence of cell interrelationships in the skin undergoing morphogenesis and will be a welcome dataset for the field.

    2. Reviewer #2 (Public Review):

      This work describes transcriptome profiling of dissected skin of zebrafish at post-embryonic stages, at a time when adult structures and patterns are forming. The authors have used the state-of-the-art combinatorial indexing RNA-seq approach to generate single cell (nucleus) resolution. The data appears robust and is coherent across the four different genotypes used by the authors.

      The authors present the data in a logical and accessible manner, with appropriate reference to the anatomy. They include helpful images of the biology and schematics to illustrate their interpretations.

      The datasets are then interrogated to define cell and signalling relationships between skin compartments in six diverse contexts. The hypotheses generated from the datasets are then tested experimentally. Overall, the experiments are appropriate and rigorously performed. They ask very interesting questions of interactions in the skin and identify novel and specific mechanisms. They validate these well.

      The authors use their datasets to define lineage relationships in the dermal scales and also in the epidermis. They show that circumferential pre-scale forming cells are precursors of focal scale forming cells while there appeared a more discontinuous relationship between lineages in the epidermis.

      The authors present transcriptome evidence for enamel deposition function in epidermal subdomains. This is convincingly confirmed with an ameloblastin in situ. They further demonstrate distinct expression of SCPP and collagen genes in the SFC regions.

      The authors then demonstrate that Eda and TH signalling to the basal epidermal cells generates FGF and PDGF ligands to signal to surrounding mesenchyme, regulating SFC differentiation and dermal stratification respectively.

      Finally, they exploit RNA-seq data performed in parallel in the bnc2 mutants to identify the hypodermal cells as critical regulators of pigment patterning and define the signalling systems used.

      Whilst these six interactions in the skin are disparate, the stories are unified by use of the sci-RNA-seq data to define interactions. Overall, it's an assembly of work which identifies novel and interesting cell interactions and cross-talk mechanisms.

      The paper provides robust evidence of cell interrelationships in the skin undergoing morphogenesis and will be a welcome dataset for the field.

    1. Reviewer #2 (Public Review):

      In this manuscript, Aydemir et al. utilized the large TEDDY study and examined the effect of previously identified tri-SNP in the HLA-DRA gene on the risk of type 1 diabetes (T1D) and celiac disease (CD). They confirmed the protective effect of the tri-SNP haplotype "101" on T1D development. Meanwhile, the same haplotype appeared to be positively associated with risk for CD and the development of CD autoimmunity. The authors further explored the molecular effect of different tri-SNP haplotypes. They proposed that C4A and C4B might be the downstream target.

      Overall, the study is rigorously conducted with proper statistical methods applied. The tri-SNP could be used as an additional risk factor when estimating T1D and celiac disease susceptibility in genetic screening. However, how this locus be incorporated into the current scheme of genetic screening is not discussed and is unlikely to be straightforward.

  2. Aug 2023
    1. Reviewer #2 (Public Review):

      In this study, Aso and Rubin generated new split-GAL4 lines to label Drosophila mushroom body output neurons (MBONs) that previously lacked specific GAL4 drivers. The MBONs represent the output channels for the mushroom body (MB), a computational center in the fly brain. Prior research identified 21 types of typical MBONs whose dendrites exclusively innervate the MB and 14 types of atypical MBONs whose dendrites also innervate brain regions outside the MB. These MBONs transmit information from the MB to other brain areas and form recurrent connections to dopaminergic neurons whose axonal terminals innervate the MB. Investigating the functions of the MBONs is crucial to understanding how the MB processes information and regulates behavior. The authors previously established a collection of split-GAL4 lines for most of the typical MBONs and one atypical MBON. That split-GAL4 collection has been an invaluable tool for researchers studying the MB. This work extends their previous effort by generating additional driver lines labeling the MBON types not covered by the previous split-GAL4 collection. Using these new driver lines, the authors also activated the labeled MBONs using optogenetics and assessed their role in learning, locomotion, and valence coding. The expression patterns of the new split-GAL4 lines and the behavioral analysis presented in this manuscript are generally convincing. I believe that these new lines will be a valuable resource for the fly community.

    1. Reviewer #2 (Public Review):

      Summary:

      This manuscript is part of the Wright lab's ongoing studies that investigate whether the bumblebee B. terrestris can detect the presence of pesticides when feeding. Previously, they showed that B. terrestris cannot detect neonicotinoids and would prefer food containing neonicotinoids (Kessler et al. 2015). However, in that paper, they showed that B. terrestris cannot taste neonicotinoids but did not provide evidence on why B. terrestris prefer food containing neonicotinoids. In the current paper, the authors continue to suggest that B. terrestris cannot taste neonicotinoids as well as another insecticide, sulfoxaflor, based on additional behavioral experiments and electrophysiological experiments focusing on specific GRNs. While the data from these experiments continue to suggest that B. terrestris cannot taste these insecticides using their mouthparts, whether B. terrestris can actually perceive these insecticides, and why this species prefers food containing these compounds is still unknown.

      Strengths:

      The authors provided additional evidence that B. terrestris cannot taste neonicotinoids with their mouthparts.

      Weaknesses:

      There are too many overgeneralizations in the manuscript and parts of it are written in a way that seems to sound combative towards studies from other groups that came to slightly different conclusions from their previous paper.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This main purpose of this investigation was to 1) compare the effects of a single knockout (sKO) of Numb or a double knockout (dKO) of Numb and NumbL on ex-vivo physiological properties of the extensor digitorium longus (EDL) muscle in C57BL/6NCrl mice; and 2) analyze protein complexes isolated from C2C12 myotubes via immunoprecipitation and LC/MS/MS for potential Numb binding partners. The main findings are 1) the muscles from sKO and dKO were significantly weaker with little difference between the sKO and dKO lines, indicating the reduced force is mainly due to the inactivation of the Numb gene; and 2) there were 11 potential Numb binding proteins that were identified and cytoskeletal specific proteins including Septin 7.

      Strengths:<br /> Strait-forward yet elegant design to help determine the important role the Numb has in skeletal muscle.

      Weaknesses: There were a limited number of samples (3-6) that were used for the physiological experiments; however, there was a very large effect size in terms of differences in muscle tension development between the induced KO models and the controls.

    1. Reviewer #2 (Public Review):

      Summary:

      This article takes an expansive look at the potential role of DUX4 in cancer treatment and prognosis, including its correlation with other key biomarkers, the potential for cancer to be resistant to treatment, and risk prediction.

      Strengths:

      The primary strength of this work is the breadth of the analyses. The authors have linked DUX4 to not just one but multiple points in the trajectory of cancer, which increases the face validity of their conclusion that DUX4 is meaningfully related to the course of a cancer as well as the prognosis for a patient.

      Statistically, the authors have taken care to properly validate their findings using appropriate bootstrapping and testing strategies.

      Weaknesses:

      Several weaknesses are noted. First, there is little-to-no description of the underlying sample population. It is only stated that "several large cohorts of patients with different metastatic cancers" were analyzed, and that a cohort of patients with advanced urothelial cancer was used for estimating associations with clinical outcomes. Lacking is information on the sampling mechanism, inclusion/exclusion criteria, treatment modalities, the definition of 'time = 0', the number of events observed, or even the sample size. Knowledge about the underlying study design would help explain some counterintuitive results, e.g. that the hazard of death among patients with Stage IV cancer is half that of those with Stage I cancer (Table 1); presumably this is not because Stage IV is actually protective but rather an artifact of the sampling scheme for these data. Second, the definition of negative versus positive DUX4 expression varies throughout the paper. In Figure 2A and Figure 3A, it is defined as >1 TPM vs. <= 1 TPM; in Figure 3C, it is defined as >1 TPM vs. < 0.5 TPM; in Figure 4A and Figure 5A, it is defined as >1 TPM vs. < 0.25 TPM; in Figure S1C it is partitioned into four groups, with boundaries defined at 0.25 TPM, 1 TPM, and 5 TPM. If categorization is needed, a rationale should be provided (ideally prospectively and not based upon the observed data, so as to avoid the perception of forking paths analyses), and it should be consistently applied. Third and finally, data seem to be occasionally excluded without rationale. For example, as mentioned above, the Cox model presented in Figure 4A seems to exclude all patients with DUX4 TPM between 0.25 and 1. Figure 3C excludes patients with either DUX4 TPM between 0.5 and 1 and/or with TMB in the lowest quartile (although the latter decision was ostensibly to control for TMB confounding, there are more appropriate ways to do so that don't result in loss of data, e.g. a stratified KM plot). Excluding patients based upon a particular region of the covariate space makes interpreting the resulting model awkward.

    1. Reviewer #2 (Public Review):

      Summary: In this manuscript, the authors performed single cell RNA-sequencing of cells from the penises of healthy and diabetes mellitus model (STZ injection-based) mice, identified *Lbh* as a marker of penis pericytes, and report that penis-specific overexpression of *Lbh* is sufficient to rescue erectile function in diabetic animals. In public human single cell RNA-sea datasets, the authors report that *LBH* is similarly specific to pericytes and down regulated in diabetic patients. Additionally, the authors report discovery of CRYAB and VIM1 as protein interacting partners with LBH.

      The authors contributions are of interest to the erectile dysfunction community and their *Lbh* overexpression experiments are especially interesting and well-conducted. However, claims in the manuscript regarding the specificity of *Lbh* as a pericyte marker, the mechanism by which *Lbh* overexpression rescues erectile function, cell-cell interactions impaired by diabetes, and protein-interaction partners require qualification or further evidence to justify.

      Major claims and evidence:

      1. Marker gene specificity and quantification: One of the authors' major contributions is the identification of *Lbh* as a marker of pericytes in their data. The authors present qualitative evidence for this marker gene relationship, but it is unclear from the data presented if *Lbh* is truly a specific marker gene for the pericyte lineage (either based on gene expression or IF presented in Fig. 2D, E). Prior results (see Tabula Muris Consortium, 2018) suggest that *Lbh* is widely expressed in non-pericyte cell types, so the claims presented in the manuscript may be overly broad. Even if *Lbh* is not a globally specific marker, the authors' subsequent intervention experiments argue that it is still an important gene worth studying.<br /> 2. Cell-cell communication and regulon activity changes in the diabetic penis: The authors present cell-cell communication analysis and TF regulon analysis in Fig 3 and report differential activities in healthy and DM mice. These results are certainly interesting, however, no statistical analyses are performed to justify claimed changes in the disease state and no validations are performed. It is therefore challenging to interpret these results, and the relevant claims do not seem well supported.<br /> 3. Rescue of ED by Lbh overexpression: This is a striking and very interesting result that warrants attention. By simple overexpression of the pericyte marker gene Lbh, the authors report rescue of erectile function in diabetic animals. While mechanistic details are lacking, the phenomenon appears to have a large effect size and the experiments appear sophisticated and well conducted. If anything, the authors appear to underplay the magnitude of this result.<br /> 4. Mechanistic claims for rescue of ED by Lbh overexpression: The authors claim that cell type-specific effects on MPCs are responsible for the rescue of erectile function induced by Lbh overexpression. This causal claim is unsupported by the data, which only show that Lbh overexpression influences MPC performance. In vivo, it's likely that Lbh is being over expressed by diverse cell types, any of which could be the causal driver of ED rescue. In fact, the authors report rescue of cell type abundance in endothelial cells and neuronal cells. Therefore, it cannot be concluded that MPC effects alone or in principal are responsible for ED rescue.<br /> 5. Protein interaction data: The authors claim that CRYAB and VIM1 are novel interacting partners of LBH. However, the evidence presented (2 blots in Fig. 6A,B) lack the relevant controls. It is possible that CRYAB and VIM1 are cross-reactive with the anti-LBH antibody or were not washed out completely. The abundance of bands on the Coomassie stain in Fig. 6A suggests that either event is plausible. Therefore, the evidence presented is insufficient to support the claim that CRYAB and VIM1 are protein interacting partners of LBH.

      **Impact**: These data will trigger interest in Lbh as a target gene within the erectile dysfunction community.

    1. Reviewer #2 (Public Review):

      The authors aimed at elucidating the development of high altitude polycythemia which affects mice and men staying in a hypoxic atmosphere at high altitude (hypobaric hypoxia; HH). HH causes increased erythropoietin production which stimulates the production of red blood cells. The authors hypothesize that increased production is only partially responsible for exaggerated red blood cell production, i.e. polycythemia, but that decreased erythrophagocytosis in the spleen contributes to high red blood cells counts.

      The main strength of the study is the use of a mouse model exposed to HH in a hypobaric chamber. However, not all of the reported results are convincing due to some smaller effects which one may doubt to result in the overall increase in red blood cells as claimed by the authors. Moreover, direct proof for reduced erythrophagocytosis is compromised due to a strong spontaneous loss of labelled red blood cells, although effects of labelled E. coli phagocytosis are shown.

      Their discussion addresses some of the unexpected results, such as the reduced expression of HO-1 under hypoxia but due to the above mentioned limitations much of the discussion remains hypothetical.

      In response to the reviewers´comments the authors extensively tried to address the points that were raised. They provided additional data, removed figures from the initial manuscript and referred to ongoing or further work. Nevertheless, not all questions could be answered leaving some hand-waiving and hypothetical explanations for some unexpected results.

    1. Reviewer #2 (Public Review):

      The work presented here by Morgun et al is performed in the context of vaccine development, a field especially active in the context of tuberculosis (TB). The generation of a new vaccine either enhancing or replacing the 100-year-old BCG is urgently needed.

      Most subunit vaccines integrate protein antigens formulated with adjuvants and there are few examples of the performance of subunit vaccines integrating lipid antigens. Considering the hydrophobic and lipid nature of the mycobacterial cell envelope studies, assessing the suitability of mycobacterial lipids in vaccine formulations may contribute to generate new vaccines to tackle the disease.

      The mycobacterial lipid antigens under study are mycolic acids (MA), which are located at the cell wall covalently linked to arabinogalactan. These lipids carry extremely long chain fatty acids of up to 60-90 carbons.

      The group has previously shown that formulating MA into micellar nanocarriers and vaccinating mice intranasally it could activate CD1-restricted T cells. However, this formulation did not allow for the incorporation of protein antigens.

      This work is novel, and it brings new data of high relevance for the TB vaccine field pointing to alternative formulations and antigens and immune mechanisms.

      Authors assay different routes of vaccination but the main results are obtained using non-conventional vaccination routes. Although, it maybe out of the scope of the paper, no protection studies are provided.

    1. Reviewer #2 (Public Review):

      Summary: This paper takes on the important topic of preprocessing of single cell/nuclei RNA-seq prior to testing for differential gene expression. However, the manuscript has a number of critical weaknesses.

      Strengths: This is an important topic and a key dataset for illustration.

      Weaknesses: A major contribution is the use of the authors' own inhouse pipeline for data preparation (scFLOW), but this software is unpublished since 2021 and consequently not yet refereed. It isn't reasonable to take this pipeline as being validated in the field.

      The authors claim that Mathys' analysis didn't use batch correction prior to analysis and claim that such processing is routine in the field, but the only citation they give is to the above-mentioned scFLOW. Batch correction for DEG analysis isn't the field standard, for example, Bryois et al. (2022) PMID: 35915177 doesn't perform batch correction. Whether or not to do such preprocessing is certainly arguable, but the authors need to argue it, not presuppose it.

      The authors spend considerable effort in discounting the pseudoreplication analysis of Mathys. It is well understood that this analysis yields a lot of false positives, but Mathys only used this approach for removing genes, not as a valid test in and of itself. They also worry that the significant findings in Mathys' paper are influenced by the number of cells of each type. I'm sure it is since power is a function of sample size, but is this a bad thing? It seems odd that their approach is not influenced by sample size.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors use previously characterised FRET methods to measure distances between intracellular segments of ASIC and with the membrane. The distances are measured across different conditions and at multiple positions in a very complete study. The picture that emerges is that the N- and C-termini do not associate.

      Strengths:<br /> Good controls, good range of measurements, advanced, well-chosen and carefully performed FRET measurements. The paper is a technical triumph. Particularly, given the weak fluorescence of ANAP, the extent of measurements and the combination with TETAC is noteworthy.

      The distance measurements are largely coherent and favour the interpretation that the N and C terminus are not close together as previously claimed.

      Weaknesses:<br /> One difficulty is that we do not have a positive control for what binding of something to either N- or C-terminus would look like (either in FRET or otherwise).

      One limitation that is not mentioned is the unroofing. The concept of interaction with intracellular domains is being examined. But the authors use unroofing to measure the positions, fully disrupting the cytoplasm. Thus it is not excluded that the unroofing disrupts that interaction. This should be mentioned as a possible (if unlikely) limitation.

    1. Reviewer #2 (Public Review):

      This manuscript by Nagel et al provides a comprehensive examination of the chemical composition of mouse urine (an important source of semiochemicals) across strain and sex, and correlates these differences with functional responses of vomeronasal sensory neurons (an important sensory population for detecting chemical social cues). The strength of the work lies in the careful and comprehensive imaging and chemical analyses, the rigor of quantification of functional responses, and the insight into the relevance of olfactory work on lab-derived vs wild-derived mice.

      With regards to the chemical analysis, the reader should keep in mind that a difference in the concentration of a chemical across strain or sex does not necessarily mean that that chemical is used for chemical communication. In the most extreme case, the animals may be completely insensitive to the chemical. Thus, the fact that the repertoire of proteins and volatiles could potentially allow sex and/or strain discrimination, it is unclear to what degree both are used in different situations.

    1. Reviewer #2 (Public Review):

      The Xerces Blue is an iconic species, now extinct, that is a symbol for invertebrate conservation. Using genomic sequencing of century-old specimens of the Xerces Blue and its closest living relatives, the authors hypothesize about possible genetic indicators of the species' demise. Although the limited range and habitat destruction are the most likely culprits, it is possible that some natural reasons have been brewing to bring this species closer to extinction.

      The importance of this study is in its generality and applicability to any other invertebrate species. The authors find that low effective population size, high inbreeding (for tens of thousands of years), and higher fraction of deleterious alleles characterize the Xerces colonies prior to extinction. These signatures can be captured from comparative genomic analysis of any target species to evaluate its population health.

      It should be noted that it remains unclear if these genomic signatures are indeed predictive of extinction, or populations can bounce back given certain conditions and increase their genetic diversity somehow.

      Methods are detailed and explained well, and the study could be replicated. I think this is a solid piece of work. Interested researchers can apply these methods to their chosen species and eventually, we will assemble datasets to study extinction process in many species to learn some general rules.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, Yuasa et al. aimed to study the spatial resolution of modulations in alpha frequency oscillations (~10Hz) within the human occipital lobe. Specifically, the authors examined the receptive field (RF) tuning properties of alpha oscillations, using retinotopic mapping and invasive electroencephalogram (iEEG) recordings. The authors employ established approaches for population RF mapping, together with a careful approach to isolating and dissociating overlapping, but distinct, activities in the frequency domain. Whereby, the authors dissociate genuine changes in alpha oscillation amplitude from other superimposed changes occurring over a broadband range of the power spectrum. Together, the authors used this approach to test how spatially tuned estimated RFs were when based on alpha range activity, vs. broadband activities (focused on 70-180Hz). Consistent with a large body of work, the authors report clear evidence of spatially precise RFs based on changes in alpha range activity. However, the size of these RFs were far larger than those reliably estimated using broadband range activity at the same recording site. Overall, the work reflects a rigorous approach to a previously examined question, for which improved characterization leads to improved consistency in findings and some advance of prior work.

      Strengths:<br /> Overall, the authors take a careful and well-motivated approach to data analyses. The authors successfully test a clear question with a rigorous approach and provide strong supportive findings. Firstly, well-established methods are used for modeling population RFs. Secondly, the authors employ contemporary methods for dissociating unique changes in alpha power from superimposed and concomitant broadband frequency range changes. This is an important confound in estimating changes in alpha power not employed in prior studies. The authors show this approach produces more consistent and robust findings than standard band-filtering approaches. As noted below, this approach may also account for more subtle differences when compared to prior work studying similar effects.

      Weaknesses:<br /> -Theoretical framing: The authors frame their study as testing between two alternative views on the organization, and putative functions, of occipital alpha oscillations: i) alpha oscillation amplitude reflects broad shifts in arousal state, with large spatial coherence and uniformity across cortex; ii) alpha oscillation amplitude reflects more specific perceptual processes and can be modulated at local spatial scales. However, in the introduction this framing seems mostly focused on comparing some of the first observations of alpha with more contemporary observations. Therefore, I read their introduction to more reflect the progress in studying alpha oscillations from Berger's initial observations to the present. I am not aware of a modern alternative in the literature that posits alpha to lack spatially specific modulations. I also note this framing isn't particularly returned to in the discussion. A second important variable here is the spatial scale of measurement. It follows that EEG based studies will capture changes in alpha activity up to the limits of spatial resolution of the method (i.e. limited in ability to map RFs). This methodological distinction isn't as clearly mentioned in the introduction, but is part of the author's motivation. Finally, as noted below, there are several studies in the literature specifically addressing the authors question, but they are not discussed in the introduction.

      -Prior studies: There are important findings in the literature preceding the author's work that are not sufficiently highlighted or cited. In general terms, the spatio-temporal properties of the EEG/iEEG spectrum are well known (i.e. that changes in high frequency activity are more focal than changes in lower frequencies). Therefore, the observations of spatially larger RFs for alpha activities is highly predicted. Specifically, prior work has examined the impact of using different frequency ranges to estimate RF properties, for example ECoG studies in the macaque by Takura et al. NeuroImage (2016) [PubMed: 26363347], as well as prior ECoG work by the author's team of collaborators (Harvey et al., NeuroImage (2013) [PubMed: 23085107]), as well as more recent findings from other groups (Luo et al., (2022) BioRxiv: https://doi.org/10.1101/2022.08.28.505627). Also, a related literature exists for invasively examining RF mapping in the time-voltage domain, which provides some insight into the author's findings (as this signal will be dominated by low-frequency effects). The authors should provide a more modern framing of our current understanding of the spatial organization of the EEG/iEEG spectrum, including prior studies examining these properties within the context of visual cortex and RF mapping. Finally, I do note that the author's approach to these questions do reflect an important test of prior findings, via an improved approach to RF characterization and iEEG frequency isolation, which suggests some important differences with prior work.

      -Statistical testing: The authors employ many important controls in their processing of data. However, for many results there is only a qualitative description or summary metric. It appears very little statistical testing was performed to establish reported differences. Related to this point, the iEEG data is highly nested, with multiple electrodes (observations) coming from each subject, how was this nesting addressed to avoid bias?

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors proposed that grid cells may be aligned by simpler, 1D attractors, and they showed that the structure and the representational space of an attractor network can be two different topological objects.

      Strengths:<br /> It is very interesting that the toroidal topology of the population activity (the representational space) and the structure of the attractor network do not necessarily to be the same. The authors carried out extensive computational modeling to support such evidence. The results presented by the authors in this study could have an impact in the grid cell field, which will motivate future experimental studies to examine the detailed structure of the grid cell population.

      Weaknesses:<br /> The authors mentioned that "the recurrent collateral structure defines the geometry of the manifold..." and pointed out that this assumption is wrong. I am afraid this claim is too strong. The Gardner Torus paper showed evidence of the 2D CAN exists in the EC as a possible substrate of the grid pattern. Do the authors mean here that even the population activity in the grid cells show the torus structure, it does not necessarily mean that the grid cells form a 2D CAN? I understand that from the computational modeling view, it is doable to find counter-examples (like the 1D attractor network) in which the representational space is a torus but the structure is different. However, from the experimental view, do you expect that the grid cell network is a low-dimensional attractor network? To prove this, is there any evidence from the experimental data?

    1. Reviewer #2 (Public Review):

      Summary:<br /> Mitochondria in synapses are important to support functional needs, such as local protein translation and calcium buffering. Thus, they may be strategically localized to maximize functional efficiency. In this study, the authors examine whether a correlation exists between the positioning of mitochondria and the structure or function of dendritic spines in the visual cortex of a ferret. Unexpectedly, the authors found no correlation between structural measures of synaptic strength to mitochondria positioning, which may indicate that they are not localized only because of the local energy needs. Instead, the authors discover that mitochondria are positioned preferably in spines that display heterogeneous responses, showing that they are localized to support specific functional needs probably distinct from ATP output.

      Strengths:<br /> The thorough analysis provides a yet unprecedented insight into the correlation between synaptic tuning and mitochondrial positioning in the visual cortex in vivo.

      Weaknesses:<br /> The study defined 1 μm and 5 μm as short and extended ranges relative to the synapse and examined the correlation between mitochondria volume and multiple parameters within that defined range. Results showed that mitochondria display preferences towards spines that respond differently to visual stimuli or areas with low local calcium activity. However, it is not known whether this mitochondria preference is a cause or a result of spine heterogeneity. It will be interesting to see the correlation of spine volume relative to mitochondrial positioning in 1µm and 5µm ranges around mitochondria.

      Analysis of this study suggested that mitochondrial volume does not correlate with the structural measure of synaptic strength (e.g. spine volume and post-synaptic density (PSD) area). However, the authors did not examine whether mitochondrial volume correlates to synaptic transmission frequency or plasticity. It may still be possible that mitochondria are localized in positions that exhibit a high frequency of transmission or a high degree of plasticity. Future studies will have to determine the underlying cause of mitochondria positioning preference.

    1. 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".

    1. Reviewer #2 (Public Review):

      The manuscript presents compelling evidence for the role of the zona incerta area of the brain in regulating movement and sensory stimuli in mice. The study uses an appropriate and validated methodology in line with the current state-of-the-art, including optogenetic manipulation and recording of single-unit activity. The authors' claims and conclusions are well-supported by their data, which includes a comprehensive review of previous research on the zona incerta. Overall, the manuscript provides solid evidence for the role of the zona incerta in regulating movement and sensory processing.

      Major strengths and weaknesses of the methods and results.<br /> The zona incerta has many integrative functions that link sensory stimuli with motor responses to guide behavior.<br /> The study explored the activation of zona incerta GABAergic neurons during cued avoidance tasks and found that these neurons activate during goal-directed avoidance movement. Optogenetic manipulation of these neurons affected movement speed and performance during active avoidance tasks.<br /> The findings suggest that the zona incerta area of the brain plays a significant role in regulating movement and responding to salient auditory tones in association with movement in mice. The evidence presented is fundamental and provides a comprehensive review of previous research on the zona incerta and its involvement in various behaviors and sensory processing.

      The article is very well written, with a correct hypothesis and a cutting-edge methodology to achieve the expected objectives. Moreover, they use statistical rigorous approaches in the analysis of the results. Also, analyzes are performed using scripts that automate all aspects of data analysis, ensuring their objectivity. The results are very novel, and provide solid evidence for the role of the zona incerta in regulating movement and sensory processing.

    1. Reviewer #2 (Public Review):

      Previous studies have extensively explored the rules by which patterned inputs from the two eyes are combined in visual cortex. Here the authors explore these rules for un-patterned inputs (luminance flicker) at both the level of cortex, using Steady-State Visual Evoked Potentials (SSVEPs) and at the sub-cortical level using pupillary responses. They find that the pattern of binocular combination differs between cortical and sub-cortical levels with the cortex showing less dichoptic masking and somewhat more binocular facilitation.

      Importantly, the present results with flicker differ markedly from those with gratings (Hou et al., 2020, J Neurosci, Baker and Wade 2017 cerebral cortex, Norcia et al, 2000 Neuroreport, Brown et al., 1999, IOVS. When SSVEP responses are measured under dichoptic conditions where each eye is driven with a unique temporal frequency, in the case of grating stimuli, the magnitude of the response in the fixed contrast eye decreases as a function of contrast in the variable contrast eye. Here the response increases by varying (small) magnitudes. The authors favor a view that cortex and perception pool binocular flicker inputs approximately linearly using cells that are largely monocular. The lack of a decrease below the monocular level when modulation strength increase is taken to indicate that previously observed normalization mechanism in pattern vision does not play a substantial role in the processing of flicker. The authors present of computational model of binocular combination that captures features of the data when fit separately to each data set. Because the model has no frequency dependence and is based on scalar quantities, it cannot make joint predictions for the multiple experimental conditions which one of its limitations.

      A strength of the current work is the use of frequency-tagging of both pupil and EEG responses to measure responses for flicker stimuli at two anatomical levels of processing. Flicker responses are interesting but have been relatively neglected. The tagging approach allows one to access responses driven by each eye, even when the other eye is stimulated which is a great strength. The tagging approach can be applied at both levels of processing at the same time when stimulus frequencies are low, which is an advantage as they can be directly compared. The authors demonstrate the versatility of frequency tagging in a novel experimental design which may inspire other uses, both within the present context and others. A disadvantage of the tagging approach for studying sub-cortical dynamics via pupil responses is that it is restricted to low temporal frequencies given the temporal bandwidth of the pupil. The inclusion of a behavioral measure and a model is also a strength, but there are some limitations in the modeling (see below).

      The authors suggest in the discussion that luminance flicker may preferentially drive cortical mechanisms that are largely monocular and in the results that they are approximately linear in the dichoptic cross condition (no effect of the fixed contrast stimulus in the other eye). By contrast, prior research using dichoptic dual frequency flickering stimuli has found robust intermodulation (IM) components in the VEP response spectrum (Baitch and Levi, 1988, Vision Res; Stevens et al., 1994 J Ped Ophthal Strab; France and Ver Hoeve, 1994, J Ped Ophthal Strab; Suter et al., 1996 Vis Neurosci). The presence of IM is a direct signature of binocular interaction and suggests that at least under some measurement conditions, binocular luminance combination is "essentially" non-linear, where essential implies a point-like non-linearity such as squaring of excitatory inputs. The two views are in striking contrast.

      In this revised manuscript, the addition of Figure 8, which shows more complete response spectra, partially addresses this issue. However, it also raises new questions. Critically, intermodulation (IM) has to be generated at or after a point of binocular combination, as it is a mixture of the two monocular frequencies and the monocular frequencies can only mix after a point of binocular combination.

      In equations 1 and 2 and in the late summation and two-stage models of Meese et al (2006), there are divisive binocular cross-links prior to a summation block. This division is a form of binocular interaction. Do equations 1 and 2 generate IM on their own with parameters used for the overall modeling? Multiplication of two inputs clearly does, as the authors indicate in their toy model. If not, then a different binocular summation rule than the one expressed in equation 3 needs to be considered to produce IM.

      The discussion considers flicker processing as manifest in the EEG to be largely monocular, given the relative lack of binocular facilitation and suppression effects. And yet there is robust IM. These are difficult to reconcile as it stands. The authors suggest that their generic modeling framework can predict IM, but can it predict IM with the parameters used to fit the data, e.g. with very low values of the weight of interocular suppression and no other binocular non-linearity?

      Determining whether IM can be generated by the existing non-linear elements in the model is important because previous work on dichoptic flicker IM has considered a variety of simple models of dichoptic flicker summation and has favored models involving either a non-linear combination of linear monocular inputs (Baitch and Levi, Vis Research, 1988) or a non-linear combination of rectified (non-linear) monocular inputs (Regan and Regan, Canadian J Neurol Sci, 1989). In either case, the last stage of binocular combination is non-linear, rather than linear. The authors' model is different - it has a stage of divisive binocular interaction and this "quasi-monocular" stage feeds a linear binocular combination stage.

      There is a second opportunity to test the proposed model that the authors could take advantage of. In the initial review, two of the reviewers were curious about what is predicted for counter-phase inputs to the two eyes. The authors indicate that the class of models they are using could be extended to cover this case. As it turns out, this experiment has been done for dichoptic full-field flicker (Sherrington, BrJPsychiatr, 1904); van der Tweel and Estevez, Ophthalmologica, 1974; Odom and Chao, IntJNeurosci, 1995; Cavonius, QJExpPsych, 1979; Levi et al., BJO, 1982). More importantly, the predictions of several binocular combination models for anti-phase inter-ocular flicker stimulation have been tested for both the VEP and psychophysics (Odom and Chao, Int J Neurosci). Varying the relative phase of the two eyes inputs from in phase to antiphase, Odom and Chao observed that the 2nd harmonic response went to a minimum at 90 deg of interocular phase. This will happen because a 2nd order nonlinearity in the monocular path will double the phase shift of the second harmonic, putting the two eyes' 2nd harmonic response out of phase when the interocular phase is 90 deg. Summing these inputs thus leads to cancellation at 90 deg, rather than 180 deg of interocular phase. Does the authors' model predict this behavior with typical parameters used in the modeling? In the end, to account for details of both VEP and psychophysical data, Odom and Chao favored a two-path model with one path comprising non-linear monocular inputs being combined linearly and a second path combining linear monocular inputs at a non-linear binocular stage. A similar set of results and models has been developed for inter-ocular presentation of gratings (Zemon et al., PNAS, 1995).

      The Odom/Chao/Zemon VEP and psychophysical data are directly relevant to the authors' work and need to be taken into account in sufficient detail so that we can judge the consistency of the proposed framework with their data and the similarities and differences in the model predictions for dichoptic flicker combination. These models are also relevant to the generation of IM, a concern raised above.

    1. Reviewer #2 (Public Review):

      The manuscript by Dacheux et al. reported homozygous deleterious variants of ZMYND12 in four unrelated men with asthenoteratozoospermia. Based on the immunofluorescence assays in human sperm cells, it was shown that ZMYND12 deficiency altered the localization of DNAH1, DNALI1, WDR66 and TTC29 (four of the known key proteins involved in sperm flagellar formation). Trypanosoma brucei and mouse models were further employed for mechanistic studies, which revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1. Their findings are solid, and this manuscript will be very informative for clinicians and basic researchers in the field of human infertility.

    1. Reviewer #2 (Public Review):

      The authors found that the age-related reduction in the serum CNP concentration was highly correlated with decreased oocyte quality. Treatment with exogenous CNP promoted follicle growth and ovulation in aged mice and enhanced meiotic competency and fertilization ability. The cytoplasmic maturation of aged oocytes was thoroughly improved by CNP treatment. CNP treatment also ameliorated DNA damage and apoptosis caused by ROS accumulation in aged oocytes. CNP reversed the defective phenotypes in aged oocytes by alleviating oxidative damage and suppressing excessive PINK1/Parkin-mediated mitophagy. CNP functioned as a cAMP/PKA pathway modulator to decrease PINK1 stability and inhibit Parkin recruitment. CNP may be used to improve the overall success rates of clinically assisted reproduction in older women.

      The author has modified the text and the level of the article has been improved. Additional experiments will further enhance the credibility of the article.

      1)The control also needs to be pre-cultured as that in CNP treatment.

      2)The mechanism is done 6 days later after CNP treatment. It is hard to know whether it is direct or indirect.

    1. Reviewer #2 (Public Review):

      The manuscript by Genzoni et al. provides evidence that trophic eggs laid by the queen in the ant Pogonomyrmex rugosis have an inhibitory effect on queen development. The authors also compare a number of features of trophic eggs, including protein, DNA, RNA, and miRNA content, to reproductive eggs. To support their argument that trophic eggs have an inhibitory effect on queen development, the authors show that trophic eggs have a lower content of protein, triglycerides, glycogen, and glucose than reproductive eggs, and that their miRNA distributions are different relative to reproductive eggs. Although the finding of an inhibitory influence of trophic eggs on queen development is indeed arresting, the egg cross-fostering experiment that supports this finding can be effectively boiled down to a single figure (Figure 6). The rest of the data are supplementary and correlative in nature (and can be combined), especially the miRNA differences shown between trophic and reproductive eggs. This means that the authors have not yet identified the mechanism through which the inhibitory effect on queen development is occurring. To this reviewer, this finding is more appropriate as a short report and not a research article. A full research article would be warranted if the authors had identified the mechanism underlying the inhibitory effect on queen development. Furthermore, the article is written poorly and lacks much background information necessary for the general reader to properly evaluate the robustness of the conclusions and to appreciate the significance of the findings.

    1. Reviewer #2 (Public Review):

      The manuscript by Nishikawa et al. addresses time-dependent changes in the electron transfer energetics in the photosynthetic reaction center from Blastochloris viridis, whose time-dependent structural changes upon light illumination were recently demonstrated by time-resolved serial femtosecond crystallography (SFX) using X-ray free-electron laser (XFEL) (Dods et al., Nature, 2021). Based on the redox potential Em values of bacteriopheophytin in the electron transfer active branch (BL) by solving the linear Poisson-Boltzmann equation, the authors found that Em(HL) values in the charge-separated 5-ps structure obtained by XFEL are not clearly changed, suggesting that the P+HL- state is not stabilized owing to protein reorganization. Furthermore, chlorin ring deformation upon HL- formation, which was expected from their QM/MM calculation, is not recognized in the 5-ps XFEL structure. Then the authors concluded that the structural changes in the XFEL structures are not related to the actual time course of charge separation. They argued that their calculated changes in Em and chlorin ring deformations using the XEFL structures may reflect the experimental errors rather than the real structural changes; they mentioned this problem is due to the fact that the XFEL structures were obtained at not high resolutions (mostly at 2.8 Å). I consider that their systematic calculations may suggest a useful theoretical interpretation of the XFEL study.

      Comments on latest version:

      The authors have satisfied my concerns. I consider that their present manuscript is more attractive and informative for readers.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors tried to identify new genes involved in melanin metabolism and its spatial distribution in the silkworm Bombyx mori. They identified the gene Bm-mamo as playing a role in caterpillar pigmentation. By functional genetic and in silico approaches, they identified putative target genes of the Bm-mamo protein. They showed that numerous cuticular proteins are regulated by Bm-mamo during larval development.

      Strengths:<br /> -preliminary data about the role of cuticular proteins to pattern the localization of pigments<br /> - timely question<br /> - challenging question because it requires the development of future genetic and cell biology tools at the nanoscale

      Weaknesses:<br /> - statistical sampling limited<br /> - the discussion would gain in being shorter and refocused on a few points, especially the link between cuticular proteins and pigmentation. The article would be better if the last evolutionary-themed section of the discussion is removed.

      A recent paper has been published on the same gene in Bombyx mori (https://www.sciencedirect.com/science/article/abs/pii/S0965174823000760) in August 2023. The authors must discuss and refer to this published paper through the present manuscript.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors report the successful retrieval of mitogenomes from extinct Pleistocene megafauna (woolly Mammoth and woolly rhino) from recent sediment cores from two close Siberian lakes. The cores are too recent to represent real time points of these two extinct species (known to have been extinct for several thousands of years) and therefore, the most plausible interpretation is that permafrost thawing and similar physical processes in the lakes have made surface old ancient DNA, maybe from nearby, deep-buried carcasses.

      Strengths:<br /> The pattern of postmortem damage at the end of the Mammoth DNA reads as well as the length distribution (reported in Figure 1) is expected for authentic ancient DNA extracts (besides the phylogenetic evidence). These results pose a question, in my view, on the general reliability of sedimentary DNA in similar contexts, especially in the absence of direct radiocarbon dating of associated remains and in the absence of an understanding of the local geo-physical dynamics. At the same time, the evidence reported here suggests that, at least in Siberian lakes, the sediments can preserve a rich ancient DNA record that it is worth surveying.

      Weaknesses:<br /> Although admittedly the work can represent two cases of environments with singular thermal conditions and geodynamics, it opens also the possibility of studying more lake sediments for trying to understand if these findings can be generalized.

    1. Reviewer #2 (Public Review):

      Neininger-Castro et al report on their original study entitled "Independent regulation of Z-lines and M-lines during sarcomere assembly in cardiac myocytes revealed by the automatic image analysis software sarcApp", In this study, the research team developed two software, yoU-Net and sarcApp, that provide new binarization and sarcomere quantification methods. The authors further utilized human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) as their model to verify their software by staining multiple sarcomeric components with and without the treatment of Blebbistatin, a known myosin II activity inhibitor. With the treatment of different Blebbistatin concentrations, the morphology of sarcomeric proteins was disturbed. These disrupted sarcomeric structures were further quantified using sarcApp and the quantification data supported the phenotype. The authors further investigated the roles of muscle myosins in sarcomere assembly by knocking down MYH6, MYH7, or MYOM in hiCMs. The knockdown of these genes did not affect Z-line assembly yet the knockdown of MYOM affected M-line assembly. The authors demonstrated that different muscle myosins participate in sarcomere assembly in different manners.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors' main goal was to evaluate how both behavioral responses to odor, and their early sensory representations are modified by repeated exposure to odor, asking whether the process of adaptation is equivalent to reducing the concentration of an odor. They open with behavioral experiments that actually establish that repeated odor presentation increases the likelihood of evoking a behavioral response in their experimental subjects - locusts. They then examine neural activity patterns at the second layer of the olfactory circuit. At the population level, repeated odor exposure reduces total spike counts, but at the level of individual cells there seems to be no consistent guiding principle that describes the adaptation-related changes, and therefore no single mechanism could be identified.

      Both population vector analysis and pattern correlation analysis indicate that odor intensity information is preserved through the adaptation process. They make the closely related point that responses to an odor in the adapted state are distinct from responses to lower concentration of the same odor. These analyses are appropriate, but the point could be strengthened by explicitly using some type of classification analysis to quantify the adaptation effects. e.g. a confusion matrix might show if there is a gradual shift in odor representations, or whether there are trials where representations change abruptly.

      Strengths:<br /> One strength is that the work has both behavioral read-out of odor perception and electrophysiological characterization of the sensory inputs and how both change over repeated stimulus presentations. It is particularly interesting that behavioral responses increase while neuronal responses generally decrease. Although the behavioral effect could occur fully downstream of the sensory responses the authors measure, at least those sensory responses retain the core features needed to drive behavior despite being highly adapted.

      Weaknesses:<br /> Ultimately no clear conceptual framework arises to understand how PN responses change during adaptation. Neither the mechanism (vesicle depletion versus changes in lateral inhibition) nor even a qualitative description of those changes. Perhaps this is because much of the analysis is focused on the entire population response, while perhaps different mechanisms operate on different cells making it difficult to understand things at the single PN level.

      From the x-axis scale in Fig 2e,f it appeared to me that they do not observe many strong PN responses to these stimuli, everything being < 10 spikes/sec. So perhaps a clearer effect would be observed if they managed to find the stronger responding PNs than captured in this dataset.

    1. 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 significance of the findings is moderately impacted due to the following considerations:

      a) Fibrosis in human NAFLD is a significant problem as a predictor of liver related mortality and is associated with type 1 and type 3 collagen. However, the reduction in COL1 in stellate cells did not amount to a reduction in liver fibrosis even in cell specific KO (in Fig 7E, there is no indication of whether Sirius red staining was different between HSC KO and control mice- the authors mention a downward trend in the text). The authors postulate that type 1 COL may not be the more predominant form of fibrosis in the model. This does not seem likely, since the same ob/ob mouse model was used to determine that fibrosis was enhanced with hepatocyte specific MCT-1 KO and decreased with Chol MCT-1KO. Measurements of different types of collagens in their model and the effect of MCT-1 on different types could be more informative. In particular, although collagens are the structural building blocks for hepatic fibrosis, fibrosis can also be controlled by matrix remodeling factors such as Timp1, Serpine 1, PAI-1 and Lox.

      b) The authors use multiple animal models including cell specific KO to conclude that stellate cell MCT-1 inhibition decreases COL-1. However, the mechanisms behind this reduced expression of COL-1 are not discussed or explored, making it descriptive.

      c) Different types of diets are used in this study which could impact lactate availability. Choline deficiency diets are reported to cause weight loss, and importantly have none of the metabolic features of human NASH. Therefore, their utility is doubtful, especially for this study which proposes to investigate if metabolic dysregulation and substrate availability could be a tool for therapy.

      d) Hepatocyte specific MCT-1 KO mice seem to have increased COL-1 production, despite no noticeable difference in hepatocyte steatosis. The reasons for this are not discussed. Fibrosis in NASH is thought to be from stellate cell activation secondary to signals from hepatocellular damage. There is no evidence that there was a difference in either of these parameters in the mouse models used.

      e) The authors report that serum lactate levels did not rise after MCT-1 silencing, but the reasons behind this are unclear. There is insufficient data about lactate production and utilization in this model, which would be useful to interpret data regarding steatosis and fibrosis development. For example, does the MCT-1 KO prevent hepatocyte and stellate cell net import or export of lactate? What is the downstream metabolic consequence in terms of pyruvate, acetylCoA and the NAD/NADH levels. Does the KO have downstream effects on mitochondrial TCA cycling?

      f) MCT-1 protein expression is measured only in the in vitro assay. Similar quantitation through western blot is not shown in the animal models.

    1. Reviewer #2 (Public Review):

      The authors present new data of endocranial surface details from the early Homo specimen KNM-ER 3732 and discuss the evolution of brain surface features that might be related to the evolution of language in the hominin lineage.

      Comments and issues raised by the reviewers have been addressed adequately. I am sure that this contribution will revive discussion about these issues.

    1. Reviewer #2 (Public Review):

      In this work, the authors investigated the pectoralis work loop and the function of the supracoracoideus muscle in the down stroke during slow flight in doves. The aim of this study was to determine how aerodynamic force is generated, using simultaneous high-speed measurements of the wings' kinematics, aerodynamics, and activation and strain of pectoralis muscles during slow flight. The measurements show a reduction in the angle of attack during mid-downstroke, which induces a peak power factor and facilitates the tensioning of the supracoracoideus tendon with pectoralis power, which then can be released in the up-stroke. By combining the data with a muscle mechanics model, the timely tuning of elastic storage in the supracoracoideus tendon was examined and showed an improvement of the pectoralis work loop shape factor. Finally, other bird species were integrated into the model for a comparative investigation.

      The major strength of the methods is the simultaneous application of four high-speed techniques - to quantify kinematics, aerodynamics and muscle activation and strain - as well as the implementation of the time-resolved data into a muscle mechanics model. With a thorough analysis which supports the conclusions convincingly, the authors achieved their goal of reaching an improved understanding of the interplay of the pectoralis and supracoracoideus muscles during slow flight and the resulting energetic benefits.

    1. Reviewer #2 (Public Review):

      Summary:

      The manuscript by Knecht et al entitled "Non-cognate immunity proteins provide broader defenses against interbacterial effectors in microbial communities" aims at characterizing a new type VI secretion system (T6SS) effector immunity pair using genetic and biochemical studies primarily focused on Proteus mirabilis and metagenomic analysis of human-derived data focused on Rothia and Prevotella sequences. The authors provide evidence that RdnE and RdnI of Proteus constitute an E-I pair and that the effector likely degrades nucleic acids. Further, they provide evidence that expression of non-cognate immunity derived from diverse species can provide protection against RdnE intoxication. Overall, this general line of investigation is underdeveloped in the T6SS field and conceptually appropriate for a broad audience journal. The paper is well-written and, aside from a few cases, well-cited. As detailed below however, there are several aspects of this paper where the evidence provided is somewhat insufficient to support the claims. Further, there are now at least two examples in the literature of non-cognate immunity providing protection against intoxication, one of which is not cited here (Bosch et al PMID 37345922 - the other being Ting et al 2018). In general therefore I think that the motivating concept here in this paper of overturning the predominant model of interbacterial effector-immunity cognate interactions is oversold and should be dialed back.

      Strengths:

      One of the major strengths of this paper is the combination of diverse techniques including competition assays, biochemistry, and metagenomics surveys. The metagenomic analysis in particular has great potential for understanding T6SS biology in natural communities. Finally, it is clear that much new biology remains to be discovered in the realm of T6SS effectors and immunity.

      Weaknesses:

      The authors have not formally shown that RdnE is delivered by the T6SS. Is it the case that there are not available genetics tools for gene deletion for the BB2000 strain? If there are genetic tools available, standard assays to demonstrate T6SS-dependency would be to interrogate function via inactivation of the T6SS (e.g. by deleting tssC).

      For swarm cross-phyla competition assays (Figure 4), at what level compared to cognate immunity are the non-cognate immunity proteins being expressed? This is unclear from the methods and Figure 4 legend and should be elaborated upon. Presumably these non-cognate immunity proteins are being overexpressed. Expression level and effector-to-immunity protein stoichiometry likely matters for interpretation of function, both in vitro as well as in relevant settings in nature. It is important to assess if native expression levels of non-cognate cross-phyla immunity (e.g. Rothia and Prevotella) protect similarly as the endogenously produced cognate immunity. This experiment could be performed in several ways, for example by deleting the RdnE-I pair and complementing back the Rothia or Prevotella RdnI at the same chromosomal locus, then performing the swarm assay. Alternatively, if there are inducible expression systems available for Proteus, examination of protection under varying levels of immunity induction could be an alternate way to address this question. Western blot analysis comparing cognate to non-cognate immunity protein levels expressed in Proteus could also be important. If the authors were interested in deriving physical binding constants between E and various cognate and non-cognate I (e.g. through isothermal titration calorimetry) that would be a strong set of data to support the claims made. The co-IP data presented in supplemental Figure 6 are nice but are from E. coli cells overexpressing each protein and do not fully address the question of in vivo (in Proteus) native expression.

      Lines 321-324, the authors infer differences between E and I in terms of read recruitment (greater abundance of I) to indicate the presence of orphan immunity genes in metagenomic samples (Figure 5A-D). It seems equally or perhaps more likely that there is substantial sequence divergence in E compared to the reference sequence. In fact, metagenomes analyzed were required only to have "half of the bases on reference E-I sequence receiving coverage". Variation in coverage again could reflect divergent sequence dipping below 90% identity cutoff. I recommend performing metagenomic assemblies on these samples to assess and curate the E-I sequences present in each sample and then recalculating coverage based on the exact inferred sequences from each sample.

      A description of gene-level read recruitment in the methods section relating to metagenomic analysis is lacking and should be provided.

    1. Reviewer #2 (Public Review):

      The work by Bærentsen et al., entitled "Structural basis for regulation of a tripartite toxin-antitoxin system by dual phosphorylation" deals with the structural aspects of the control of the hipBST TA operon, the role of auto-phosphorylation in the activation and neutralisation of the enzyme and the direct effects of HipS and HipB in neutralisation. This is a follow-up to the Vang Nielsen et al., and Gerdes et al., papers from the same authors on this very unique TA module, that brings forth a thorough and well written dissection of an unusually complex regulatory system.

      This is a much improved manuscript, the paper is more focused and the message is now clear.

    1. Reviewer #2 (Public Review):

      Tian et al. perform a meta-analysis of 113 genome-wide origin profile datasets in humans to assess the reproducibility of experimental techniques and shared genomics features of origins. Techniques to map DNA replication sites have quickly evolved over the last decade, yet little is known about how these methods fare against each other (pros and cons), nor how consistent their maps are. The authors show that high-confidence origins recapitulate several known features of origins (e.g., correspondence with open chromatin, overlap with transcriptional promoters, CTCF binding sites). However, surprisingly, they find little overlap between ORC/MCM binding sites and origin locations.

      Overall, this meta-analysis provides the field with a good assessment of the current state of experimental techniques and their reproducibility, but I am worried about: (a) whether we've learned any new biology from this analysis; (b) how binding sites and origin locations can be so mismatched, in light of numerous studies that suggest otherwise; and (c) some methodological details described below.

      Major comments:

      -- Line 26: "0.27% were reproducibly detected by four techniques" -- what does this mean? Does the fragment need to be detected by ALL FOUR techniques to be deemed reproducible? And what if the technique detected the fragment is only 1 of N experiments conducted; does that count as "detected"? Later in Methods, the authors (line 512) say, "shared origins ... occur in sufficient number of samples" but what does *sufficient* mean? Then on line 522, they use a threshold of "20" samples, which seems arbitrary to me. How are these parameters set, and how robust are the conclusions to these settings? An alternative to setting these (arbitrary) thresholds and discretizing the data is to analyze the data continuously; i.e., associate with each fragment a continuous confidence score.

      -- Line 20: "50,000 origins" vs "7.5M 300bp chromosomal fragments" -- how do these two numbers relate? How many 300bp fragments would be expected given that there are ~50,000 origins? (i.e., how many fragments are there per origin, on average)? This is an important number to report because it gives some sense of how many of these fragments are likely nonsense/noise. The authors might consider eliminating those fragments significantly above the expected number, since their inclusion may muddle biological interpretation.

      -- Line 143: I'm not terribly convinced by the PCA clustering analysis, since the variance explained by the first 2 PCs is only ~25%. A more robust analysis of whether origins cluster by cell type, year etc is to simply compute the distribution of pairwise correlations of origin profiles within the same group (cell type, year) vs the correlation distribution between groups. Relatedly, the authors should explain what an "origin profile" is (line 141). Is the matrix (to which PCA is applied) of size 7.5M x 113, with a "1" in the (i,j) position if the ith fragment was detected in the jth dataset?

      -- It's not clear to me what new biology (genomic features) has been learned from this meta-analysis. All the major genomic features analyzed have already been found to be associated with origin sites. For example, the correspondence with TSS has been reported before:

      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320713/<br /> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547456/

      So what new biology has been discovered from this meta-analysis?

      -- Line 250: The most surprising finding is that there is little overlap between ORC/MCM binding sites and origin locations. The authors speculate that the overlap between ORC1 and ORC2 could be low because they come from different cell types. Equally concerning is the lack of overlap with MCM. If true, these are potentially major discoveries that butts heads with numerous other studies that have suggested otherwise. More needs to be done to convince the reader that such a mis-match is true. Some ideas are below:

      Idea 1) One explanation given is that the ORC1 and ORC2 data come from different cell types. But there must be a dataset where both are mapped in the same cell type. Can the authors check the overlap here? In Fig S4A, I would expect the circles to not only strongly overlap but to also be of roughly the same size, since both ORC's are required in the complex. So something seems off here.

      Idea 2) Another explanation given is that origins fire stochastically. One way to quantify the role of stochasticity is to quantify the overlap of origin locations performed by the same lab, in the same year, in the same experiment, in the same cell type -- i.e., across replicates -- and then compute the overlap of mapped origins. This would quantify how much mis-match is truly due to stochasticity, and how much may be due to other factors.

      Idea 3) A third explanation is that MCMs are loaded further from origin sites in human than in yeast. Is there any evidence of this? How far away does the evidence suggest, and what if this distance is used to define proximity?

      Idea 4) How many individual datasets (i.e., those collected and published together) also demonstrate the feature that ORC/MCM binding locations do not correlate with origins? If there are few, then indeed, the integrative analysis performed here is consistent. But if there are many, then why would individual datasets reveal one thing, but integrative analysis reveal something else?

      Idea 5) What if you were much more restrictive when defining "high-confidence" origins / binding sites. Does the overlap between origins and binding sites go up with increasing restriction?

      Overall, I have the sense that these experimental techniques may be producing a lot of junk. If true, this would be useful for the field to know! But if not, and there are indeed "unexplored mechanisms of origin specification" that would be exciting. But I'm not convinced yet.

      -- It would be nice in the Discussion for the authors to comment about the trade-offs of different techniques; what are their pros and cons, which should be used when, which should be avoided altogether, and why? This would be a valuable prescription for the field.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors provide new evidence for motion-induced sound camouflage and can link the hunting approach to hunting success (detailing the adaptation and inferring a fitness consequence).

      Strengths:<br /> Strong evidence by combining high-resolution accelerometer data with a ground-truthed data set on prey provisioning at nest boxes. A good set of co-variates to control for some of the noise in the data provides some additional insights into owl hunting attempts.

      Weaknesses:<br /> There is a disconnect between the hypotheses tested and the results presented, and insufficient detail is provided on the statistical approach. R2 values of the presented models are very small compared to the significance of the effect presented. Without more detail, it is impossible to assess the strength of the evidence. The authors seem to overcome persisting challenges associated with the validation and calibration of accelerometer data by ground-truthing on-board measures with direct observations in captivity, but here the methods are not described any further and sample sizes (2 owls - how many different loggers were deployed?) might be too small to achieve robust behavioural classifications.

    1. Reviewer #2 (Public Review):

      This is a well-written manuscript about a strong comparative study of diversity of facial movements in three macaque species to test arguments about social complexity influencing communicative complexity.

    1. Reviewer #2 (Public Review):

      The ATPase protein machine cohesin shapes the genome by loop extrusion and holds sister chromatids together by topological entrapment. When executing these functions, cohesin is tightly regulated by multiple cofactors, such as Scc2/Nipbl, Pds5, Wapl, and Eco1/Esco1/2, and it undergoes dynamic conformational changes with ATP binding and hydrolysis. The mechanisms by which cohesin extrudes DNA loops and medicates siter-chromatid cohesion are still not understood. A major reason for the lack of understanding of cohesin dynamics and regulation is the failure to capture the structures of intact cohesin in different nucleotide-bound states and in complex with various regulators. So far only the ATP state cohesin bound to NIPBL and DNA have been experimentally determined.

      In this manuscript, Nasmyth et al. made use of the powerful protein structure prediction tool, AlphaFold2 (AF), to predict the models of tens of cohesin subcomplexes from different species. The results provide important insight into how the Smc3-Scc1 DNA exiting gate is opened, how Pds5 and Wapl maintain the opened gate, how Pds5 and Scc3/SA recruit different cofactors, how Eco1 and Sororin antagonize Wapl, and how Scc2/Nipbl interacts with Scc3/SA. The models are for the most part consistent with published mutations in these proteins that affect cohesin's functions in vitro and in vivo and raise testable hypotheses of cohesin dynamics and regulation. This study also serves as an example of how to use AF to build models of protein complexes that involve the docking of flexible regions to globular domains.

      Major points

      (1)The revised manuscript is still too long and would be difficult for readers to read. While the authors have made some efforts to streamline their presentations and remove excessive speculations and models of minor importance, the changes are not enough.<br /> (2) AF has been accurate in predicting both the fold and sidechain conformations of globular domains. It is less accurate in predicting structural regions with conformational flexibility. Comparisons of predicted and determined structures of large protein complexes have shown considerable differences, particularly with respect to regions lacking tertiary fold. The authors should be more cautious in interpreting some of their models, particularly when the predicted models are inconsistent with determined structures and published biochemical data. For example, human WAPL-C in isolation does not interact with the SA-SCC1 complex while the N-terminal region of WAPL does.<br /> (3) The predicted SA/Scc3-Pds5-Scc1-WaplC quaternary complex is fascinating. Can the authors provide some experimental evidence to support the formation of this quaternary complex or at least the formation of the SA/Scc3-Pds5-WaplC ternary complex? In vitro pulldown or gel filtration can be used to test their predictions. The authors have decided not the test their models experimentally.

    1. Reviewer #2 (Public Review):

      The present study by Ye et al. characterizes some of the major effects of ferroptotic stress on tooth morphogenesis.

      The strengths of this study are its innovative nature and beautiful histology. Mechanistic data are convincing Overall, the study is well done.

    1. Reviewer #2 (Public Review):

      In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei. While these data do not definitively show a role for this pathway in antigenic variation, the data are critical for establishing this pathway as a potential way the parasite could control antigenic variation and thus represent a fundamental discovery.

      The methods used in the study are generally well-controlled. The authors provide evidence that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript. Readers should take into consideration that the epitope tags on RAP1 could alter its function, however.

      There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome which does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be mis-assigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

      Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

      Overall, this is an excellent study which represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

    1. Reviewer #2 (Public Review):

      This paper aims at establishing the role of WRN-interacting protein 1 (WRNIP1) and its UBZ domain (an N-terminal ubiquitin-binding zinc finger domain) on genome instability caused by mild inhibition of DNA synthesis by aphidicolin. The authors used human MRC5 fibroblasts investigated with standard methods in the field. The results clearly showed that WRNIP1 silencing and UBZ-mutation (D37A) increased DNA damage, chromosome aberrations, and transcription-replication conflicts caused by aphidicolin.

      The conclusions of the paper are overall well supported by results, however, aspects of some data analyses would need to be clarified and/or extended.

      1 The methods (immunofluorescence microscopy and dot-blots) to determine R-loop levels can lack sensitivity and specificity. In particular, since the S9.6 antibody can bind to other structures besides heteroduplex, dot-blot analyses only grossly assess R-loop levels in cellular samples of purified nucleic acids, which are constituted by many different types of DNA/RNA structures.

      2 Experimental plan has analyzed the impact of WRNIP1 lack or mutations at steady-state conditions. Thus, the possible role of WRNIP1 at an early step of the mechanism would require some sort of kinetics analysis of the molecular process, therefore not at steady-state conditions. The findings of a co-localization of R-loops and WRNIP1 have been obtained with the S9.6 antibody, which recognizes DNA-RNA heteroduplexes. Since WRNIP1 is known to be recruited at stalled forks and DNA cleavage sites, it is not surprising that WRNIP1 is very close to heteroduplexes, abundant structures at replication forks and cleavage sites. Similar interpretations may also be valid for Rad51/S9.6 co-localization findings.

      3 Determination of DNA damage, chromosome aberration, and co-localization data are reported as means of measurements with appropriate statistics. However, the fold-change values relative to corresponding untreated samples are not reported. In some instances, it seems that WRNIP1 silencing or mutations actually reduce or do not affect aphidicolin effects. That leaves open the interpretation of specific results.

    1. Reviewer #2 (Public Review):

      Summary:

      Spikol et al performed a technical tour de force by combining numerous novel tools and approaches to investigate for the first time the connectivity and motor functions of nucleus incertus subset of neurons genetically defined by the expression of specific markers in the larval zebrafish brain.

      Strengths:

      By using expression of the specific markers relaxin 3 and gsc2, the authors generated novel knock-in transgenic lines enabling them to investigate the connectivity, recruitment and roles of these neurons in locomotion. Their work should enable numerous subsequent studies in zebrafish & inspire new paths of investigations in other animal models.

      Weaknesses:

      More precision is required for the anatomical data and further analysis is needed to describe the recruitment and role in spontaneous exploration of the rln3- and gsc2- expressing neurons.

    1. Reviewer #2 (Public Review):

      Peterson et al., perform a series of behavioral experiments to study the repertoire and variance of Mongolian gerbil vocalizations across social groups (families). A key strength of the study is the use of a behavioral paradigm which allows for long term audio recordings under naturalistic conditions. This experimental set-up results in the identification of additional vocalization types. In combination with state of the art methods for vocalization analysis, the authors demonstrate that the distribution of sound types and the transitions between these sound types across three gerbil families is different. This is a highly compelling finding which suggests that individual families may develop distinct vocal repertoires. One potential limitation of the study lies in the cluster analysis used for identifying distinct vocalization types. The authors use a Gaussian Mixed Model (GMM) trained on variational auto Encoder derived latent representation of vocalizations to classify recorded sounds into clusters. Through the analysis the authors identify 70 distinct clusters and demonstrate a differential usage of these sound clusters across families. While the authors acknowledge the inherent challenges in cluster analysis and provide additional analyses (i.e. maximum mean discrepancy, MMD), additional analysis would increase the strength of the conclusions. In particular, analysis with different cluster sizes would be valuable. An additional limitation of the study is that due to the methodology that is used, the authors can not provide any information about the bioacoustic features that contribute to differences in sound types across families which limits interpretations about how the animals may perceive and react to these sounds in an ethologically relevant manner.

      The conclusions of this paper are well supported by data, but certain parts of the data analysis should be expanded and more fully explained.

      • Can the authors comment on the potential biological significance of the 70 sound clusters? Does each cluster represent a single sound type? How many vocal clusters can be attributed to a single individual? Similarly, can the authors comment on the intra-individual and inter-individual variability of the sound types within and across families?<br /> • As a main conclusion of the paper rests on the different distribution of sound clusters across families, it is important to validate the robustness of these differences across different cluster parameters. Specifically, the authors state that "we selected 70 clusters as the most parsimonious fit". Could the authors provide more details about how this was fit? Specifically, could the authors expand upon what is meant by "prior domain knowledge about the number of vocal types...". If the authors chose a range of cluster values (i.e. 10, 30, 50, 90) does the significance of the results still hold?<br /> • While VAEs are powerful tools for analyzing complex datasets in this case they are restricted to analysis of spectrogram images. Have the authors identified any acoustic differences (i.e. in pitch, frequency, and other sound components) across families?

    1. Reviewer #2 (Public Review):

      In this work the IGluSnFR3 sensor, recently developed by Marvin et al (2023) is mutated position S72, which was previously reported to switch the specificity from Glu to Asp. They made 3 mutations at this position, selected a S72P mutant, then made a second mutation at S27 to generate an Asp-specific version of the sensor. This was then characterized thoroughly and used on some test experiments, where it was shown to detect and allow visualization of aspartate concentration changes over time. It is an incremental advance on the iGluSnFR3 study, where 2 predictable mutations are used to generate a sensor that works on a close analog of Glu, Asp. It is shown to have utility and will be useful in the field of Asp-mediated biological effects.

    1. Reviewer #2 (Public Review):

      Making state-of-the-art (super-resolution) microscopy widely available has been the subject of many publications in recent years as correctly referenced in the manuscript. By advocating the ideas of open-microscopy and trying to replace expensive, scientific-grade components such as lasers, cameras, objectives, and stages with cost-effective alternatives, interested researchers nowadays have a number of different frameworks to choose from. In the iteration of the theme presented here, the authors used the existing modular UC2 framework, which consists of 3D printable building blocks, and combined a cheapish laser, detector and x,y,(z) stage with expensive filters/dichroics and a very expensive high-end objective (>15k Euros). This particular choice raises a first technical question, to which extent a standard NA 1.3 oil immersion objective available for <1k would compare to the chosen NA 1.49 one.

      The choice of using the UC2 framework has the advantage, that the individual building blocks can be 3D printed, although it should be mentioned that the authors used injection-molded blocks that will have a limited availability if not offered commercially by a third party. The strength of the manuscript is the tight integration of the hardware and the software (namely the implementations of imSwitch as a GUI to control data acquisition, OS SMLM algorithms for fast sub-pixel localisation and access to Napari).

      The presented experimental data is convincing, demonstrating (1) extended live cell imaging both using bright-field and fluorescence in the incubator, (2) single-particle tracking of quantum dots, and (3) and STORM measurements in cells stained against tubulin.

      In the following I will raise two aspects that currently limit the clarity and the potential impact of the manuscript.

      First, the manuscript would benefit from further refinement. Elements in Figure 1d/e are not described properly. Figure 2c is not described in the caption. GPI-GFP is not introduced. MMS (moment scaling spectrum) could benefit from a one sentence description of what it actually is. In Figure 6, the size of the STORM and wide-field field of views are vastly different, the distances between the peaks on the tubuili are given in micrometers rather than nanometers. (more in the section on recommendations for the author)

      Second, and this is the main criticism at this point, is that although all the information and data is openly available, it seems very difficult to actually build the setup due to a lack of proper documentation (as of early July 2023).<br /> 1. The bill of materials (https://github.com/openUC2/UC2-STORM-and-Fluorescence#bill-of-material) should provide a link to the commercially available items. Some items are named in German. Maybe split the BoM in commercially available and 3D printable parts (I first missed the option to scroll horizontally).<br /> 2. The links to the XY and Z stage refer to the general overview site of the UC2 project (https://github.com/openUC2/) requiring a deep dive to find the actual information.<br /> 3. Detailed building instructions are unfortunately missing. How to assemble the cubes (pCad files showing exploded views, for example)? Trouble shooting?<br /> 4. Some of the hardware details (e.g. which laser was being used, lenses, etc) should be mentioned in the manuscript (or SI)

      I fully understand that providing such level of detail is very time consuming, but I hope that the authors will be able to address these shortcomings.

    1. Reviewer #2 (Public Review):

      This study compares the activity of neural populations in the primary and non-primary auditory cortex of ferrets while the animals actively behaved or passively listened to a sound discrimination task. Using a variety of methods, the authors convincingly show differential effects of task engagement on population neural activity in primary vs non-primary auditory cortex; notably that in the primary auditory cortex, task-engagement (1) improves discriminability for both task-relevant and non-task relevant dimensions, and (2) improves the alignment between covariability and sound discrimination axes; whereas in the non-primary auditory cortex, task-engagement (1) improves discriminability for only task-relevant dimensions, and (2) does not affect the alignment between covariability and sound discrimination axes. They additionally show that task-engagement changes in gain can account for the selectivity noted in the discriminability of non-primary auditory neurons. They also admirably attempt to isolate task-engagement from arousal fluctuations, by using fluctuations in pupil size as a proxy for physiological arousal. This is a well-carried out study with thoughtful analyses which in large part achieves its aims to evaluate how task-engagement changes neural activity across multiple auditory regions. As with all work, there are several caveats or areas for future study/analysis. First, the sounds used here (tones, and narrow-band noise) are relatively simple sounds; previous work suggests that exactly what activity is observed within each region (e.g., sensory only, decision-related, etc) may depend in part upon what stimuli are used. Therefore, while the current study adds importantly to the literature, future work may consider the use of more varied stimuli. Second, the animals here were engaged in a behavioral task; but apart from an initial calculation of behavioral d', the task performance (and its effect on neural activity) is largely unaddressed.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Through a series of four experiments, Yuan, Wang and Jiang examined pupil size responses to emotion signals in point-light motion stimuli. Experiment 1 examined upright happy, sad and neutral point-light biological motion (BM) walkers. The happy BM induced a significantly larger pupil response than the neutral, whereas the sad BM evoked a significantly smaller pupil size than the neutral BM. Experiment 2 examined inverted BM walkers. Experiment 3 examined BM stimuli with acceleration removed. No significant effects of emotion were found in neither Experiment 2 nor Experiment 3. Experiment 4 examined scrambled BM stimuli, in which local motion features were preserved while the global configuration was disrupted. Interestingly, the scrambled happy and sad BM led to significantly greater pupil size than the scrambled neutral BM at a relatively early time, while no significant difference between the scrambled happy and sad BM was found. Thus, the authors argue that these results suggest multi-level processing of emotions in life motion signals.

      Strengths:<br /> The experiments were carefully designed and well-executed, with point-light stimuli that eliminate many potential confounding effects of low-level visual features such as luminance, contrast, and spatial frequency.

      Weaknesses:<br /> Correlation results with limited sample size should be interpreted with extra caution.

      It would be helpful to add discussions as a context to compare the current results with pupil size reactions to emotion signals in picture stimuli.

      Overall, I think this is a well-written paper with solid experimental results that support the claim of the authors, i.e., the human visual system may process emotional information in biological motion at multiple levels. Given the key role of emotion processing in normal social cognition, the results will be of interest not only to basic scientists who study visual perception, but also to clinical researchers who work with patients of social cognitive disorders. In addition, this paper suggests that examining pupil size responses could be a very useful methodological tool to study brain mechanisms underlying emotion processing.

    1. Reviewer #2 (Public Review):

      This short manuscript by Zhu et al. describes an investigation into the role of gamma protocadherins in synaptic connectivity in the mouse cerebral cortex. First, the authors conduct a single-cell RNA-seq survey of postnatal day 11 mouse cortical neurons, using an adapted 10X Genomics method to capture the 5' sequences that are necessary to identify individual gamma protocadherin isoforms (all 22 transcripts share the same three 3' "constant" exons, so standard 3'-biased methods can't distinguish them). This method adaptation is an advance for examining individual gamma transcripts, and it is helpful to publish the method, the characterization of which is improved in this revised manuscript. The results largely confirm what was known from other approaches, which is that a few of the 19 A and B subtype gamma protocadherins are expressed in an apparently stochastic and combinatorial fashion in each cortical neuron, while the 3 C subtype genes are expressed ubiquitously. Second, using elegant paired electrophysiological recordings, the authors show that in gamma protocadherin cortical slices, the likelihood of two neurons on layers 2/3 being synaptically connected is increased. That suggests that gamma protocadherins generally inhibit synaptic connectivity in the cortex; again, this has been reported previously using morphological assays, but it is important to see it confirmed here with physiology. Finally, the authors use an impressive sequential in utero electroporation method to provide evidence that the degree of isoform matching between two neurons negatively regulates their reciprocal synaptic connectivity. These are difficult experiments to do, and while some caveats remain, the main result is consistent. Strengths include the impressive methodology and improved demonstration of the previously-reported finding that gamma protocadherins work via homophilic matching to put a brake on synapse formation in the cortex. Weaknesses include the writing, which even in the revision fails to completely put the new results in context with prior work, which together has largely shown similar results; a still-incomplete characterization of a new alpha protocadherin KO mouse (a minor point but it should still be addressed); and a lack of demonstration of protein levels in electroporated brains. Because of the unique organization and expression pattern of the gamma protocadherins, it is unlikely that these results will be directly applicable to the broader understanding of the role of cell adhesion molecules in synapse development. However, the methodology, which is now better described, should be applicable more broadly and the improved demonstration of the role of gamma protocadherin's negative role in cortical synaptogenesis is helpful.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, Liang et al. investigate whether an abstract social space is neurally represented by a grid-like code. They trained participants to 'navigate' around a two-dimensional space of social agents characterized by the traits of warmth and competence, then measured neural activity as participants imagined navigating through this space. The primary neural analysis consisted of three procedures: 1) identifying brain regions exhibiting the hexagonal modulation characteristic of a grid-like code, 2) estimating the orientation of each region's grid, and 3) testing whether the strength of the univariate neural signal increases when a participant is navigating in a direction aligned with the grid, compared to a direction that is misaligned with the grid.<br /> From these analyses, the authors find the clearest evidence of a grid-like code in the prefrontal cortex and weaker evidence in the entorhinal cortex.

      Strengths:<br /> The work demonstrates the existence of a grid-like neural code for a socially-relevant task, providing evidence that such coding schemes may be relevant for a variety of two-dimensional task spaces.

      Weaknesses:<br /> In various parts of this manuscript, the authors appear to use a variety of terms to refer to the (ostensibly) same neural regions: prefrontal cortex, frontal pole, ventromedial prefrontal cortex (vmPFC), and orbitofrontal cortex (OFC). It would be useful for the authors to use more consistent terminology to avoid confusing readers.

      Claims about a grid code in the entorhinal cortex are not well-supported by the analyses presented. The whole-brain analysis does not suggest that the entorhinal cortex exhibits hexagonal modulation; the strength of the entorhinal BOLD signal does not track the putative alignment of the grid code there; multivariate analyses do not reveal any evidence of a grid-like representational geometry.

      On a conceptual level, it is not entirely clear how this work advances our understanding of grid-like encoding of two-dimensional abstract spaces, or of social cognition. The study design borrows heavily from Constantinescu et al. 2016, which is itself not an inherent weakness, but the Constantinescu et al. study already suggests that grid codes are likely to underlie two-dimensional spaces, no matter how abstract or arbitrary. If there were a hypothesis that there is something unique about how grid codes operate in the social domain, that would help motivate the search for social grid codes specifically, but no such theory is provided. The authors do note that warmth and competence likely have ecological importance as social traits, but other past studies have used slightly different social dimensions without any apparent loss of generality (e.g., Park et al. 2021). There are some (seemingly) exploratory analyses examining how individual difference measures like social anxiety and avoidance might affect the brain and behavior in this study, but a strong theoretical basis for examining these particular measures is lacking.

      I found it difficult to understand the analyses examining whether behavior (i.e., reaction times) and individual difference measures (i.e., social anxiety and avoidance) can be predicted by the hexagonal modulation strength in some region X, conditional on region X having a similar estimated grid alignment with some other region Y. It is possible that I have misunderstood the authors' logic and/or methodology, but I do not feel comfortable commenting on the correctness or implications of this approach given the information provided in the current version of this manuscript.

      It was puzzling to see passing references to multivariate analyses using representational similarity analysis (RSA) in the main text, given that RSA is only used in analyses presented in the supplementary material.

      References:<br /> Constantinescu, A. O., O'Reilly, J. X., & Behrens, T. E. (2016). Organizing conceptual knowledge in humans with a gridlike code. Science, 352(6292), 1464-1468.

      Park, S. A., Miller, D. S., & Boorman, E. D. (2021). Inferences on a multidimensional social hierarchy use a grid-like code. Nature Neuroscience, 24(9), 1292-1301.

    1. Reviewer #2 (Public Review):

      This manuscript by Xue et al. describes the effects of a long noncoding RNA, lncDACH1, on the localization of Nav channel expression, the magnitude of INa, and arrhythmia susceptibility in the mouse heart. Because lncDACH1 was previously reported to bind and disrupt membrane expression of dystrophin, which in turn is required for proper Nav1.5 localization, much of the findings are inferred through the lens of dystrophin alterations.

      The results report that cardiomyocyte-specific transgenic overexpression of lncDACH1 reduces INa in isolated cardiomyocytes; measurements in whole heart show a corresponding reduction in conduction velocity and enhanced susceptibility to arrhythmia. The effect on INa was confirmed in isolated WT mouse cardiomyocytes infected with a lncDACH1 adenoviral construct. Importantly, reducing lncDACH1 expression via either a cardiomyocyte-specific knockout or using shRNA had the opposite effect: INa was increased in isolated cells, as was conduction velocity in heart. Experiments were also conducted with a fragment of lnDACH1 identified by its conservation with other mammalian species. Overexpression of this fragment resulted in reduced INa and greater proarrhythmic behavior. Alteration of expression was confirmed by qPCR.

      The mechanism by which lnDACH1 exerts its effects on INa was explored by measuring protein levels from cell fractions and immunofluorescence localization in cells. In general, overexpression was reported to reduce Nav1.5 and dystrophin levels and knockout or knockdown increased them.

    1. Reviewer #2 (Public Review):

      This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

      Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox (and should be justifed/caveats mentioned in the main text), however, this does not affect the main finding of the study.

      Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

    1. Reviewer #2 (Public Review):

      The manuscript by Kaneko set out to understand the mechanisms underlying cell proliferation in hepatocytes lacking Shp2 signals. To do this, the authors focused on CD133 as the proliferating clusters of cells in the Shp2 knockout (SKO) livers are CD133 expressing. After excluding the contribution of progenitors that are CD133 to this cell population, the authors focused on the intrinsic regulation of CD133 by Met/Shp2 regulated Ras/Erk parthway and showed upregulation of CD133 to be a compensatory signal to overcome loss of Ras/Erk signal and suggested Wnt10a in the regulation of CD133 signal. The study then focused on the observed filament localization of CD133 in the CD133+ cluster of cells. The study went on to identify the CD133+ vesicles that contain primarily mRNA vs. microRNA like other EVs. Specifically, the authors identified several mRNA species that encode IEGs, indicating a potential role for these CD133+ vesicles in cell proliferation signal transmission to neighboring cells via delivery of the IEG mRNAs as cargos. Finally, they showed that the induction of CD133 (and by derivative, the CD133+ vesicles) are necessary for maintaining cell proliferation in the cell cluster with high proliferation capacities in the SKO livers; and in intestinal crypt organoids treated with Met inhibitors to block Ras/ERk signal. In the revised manuscript, the authors more definitively identified the CD133+ vesicles. The authors also provided additional experimental evidence demonstrating the role of these CD133+ vesicles in cell-cell communication. The functional significance of CD133 on this cell-cell communication was further demonstrated with genetic knockout studies.

    1. Reviewer #2 (Public Review):

      Summary:

      The preprint by Pang, Deluca, et al. investigates the molecular events occurring during germline stem cell (GSC) differentiation into an oocyte. The study highlights several critical observations:

      1. Gene Expression and Chromatin State: GSCs exhibit an open chromatin state and express a large number of genes. However, during differentiation, the number of genes expressed decreases.<br /> 2. Gene Clustering and Chromatin Domains: Genes promoting GSC fate are found in clusters close to centric heterochromatin domains.<br /> 3. Epigenetic Marks: The transition from GSC fate to oocyte/nurse cell fate is marked by an increase in H3K27me3 and H3K9me3 on regions, including centric heterochromatin.<br /> 4. Metabolic Rewiring: Genes related to metabolism undergo changes during this fate transition, indicating metabolic rewiring.

      Strengths:<br /> The conclusions are strongly supported by a substantial amount of data. Multiple complementary methods are employed, such as increased H3K9me3 heterochromatin and reporter assays, to validate the increase of H3K9me3 during meiosis.

      The wealth of data presented will be valuable to the scientific community, providing further insights into critical molecular events during GSC differentiation.

      The study uncovers new biology, notably the proximity of stem cell genes to centric heterochromatin and its regulation.

      Key observations include the low H3K9me3 levels on transposons in GSCs, which warrant further investigation.

      Weaknesses:<br /> To make the paper more accessible to a broader audience, the authors can use fewer jargon terms. In particular, the abbreviations used for staging can be confusing.

      Some sections in the results contain extensive discussion that may be better suited for the discussion section. For example, see page 9.

    1. Reviewer #2 (Public Review):

      Please note that I am not a structural biologist and cannot critically evaluate the details of figures 1 to 3; my review focuses on the cell biology experiments in figures 4 and 5.

      Paine and colleagues investigated structural requirements for the interaction between the ESCRT-III subunit IST1 and the protease CAPN7. This is a continuation of previous work by the same group (Wenzel et al., eLife 2022), which showed that Capn7 is recruited to the midbody by Ist1 and that Capn7 promotes both normal abscission and NoCut abscission checkpoint function. In this article, the structural determinants of the Ist1-Capn7 interaction are characterised in more detail, focusing on the structure of Capn7 MIT domains and their binding to Ist1. Notably, point mutations in Capn7 MIT domains known to mediate binding to Ist1 and midbody recruitment are shown here to be required for abscission functions, as expected from the authors' previous paper. Furthermore, the report shows that a Capn7 point mutant lacking proteolytic activity behaves as a loss-of-function in abscission assays, despite showing normal midbody localisation. These are important results that will help in future studies to understand how the Capn7 protease regulates abscission mechanistically.

      The report is clearly written and the results support the main conclusions. Some technical limitations and alternative interpretations of the data should be discussed in the text, as outlined below.

      1. It is not always clearly stated how the results presented in this report relate to those in the Wenzel paper. For example, the finding that Ist1 recruits Capn7 to midbodies (p. 6 and figure 4) was first shown in the Wenzel paper. The novelty here is not that Capn7 MIT mutants fail to localise to midbodies, but that they phenocopy the previously described knockdown of Capn7, failing to support normal abscission and NoCut function (fig. 5). This supports and extends the findings of Wenzel et al. It is important to make this explicit and explain the conceptual advances shown here more clearly.<br /> 2. The NoCut checkpoint can be triggered by chromatin bridges, DNA replication stress, and nuclear basket defects, but only basket defects are tested here. Therefore, it is not clear if NoCut is still functional in Capn7-defective cells after replication stress and/or with chromatin bridges. Ideally, this should be tested experimentally, or alternatively discussed in the text, especially since the molecular details of how NoCut is engaged under different conditions remain unclear. For example, "abscission checkpoint bodies" proposed to control abscission timing form in response to nuclear basket defects and aphidicolin treatment, but not in the presence of chromatin bridges (Strohacker et al., eLife 2021).<br /> 3. The current data suggest that Capn7 is a regulator of abscission timing, but in my opinion do not quite establish this, for two main reasons. First, abscission timing is not directly measured in this study. Time-lapse imaging would be required to rule out alternative interpretations of the data in figure 5. For example, a delay in an earlier cell cycle stage could in principle lead to a decrease in the overall fraction of midbody-stage cells. Second, the absence of the midbody is not necessarily a marker of complete abscission. Indeed, midbody disassembly is associated with the completion of abscission in unchallenged HeLa cells, but not in cells with chromatin bridges (Steigemann et al, Cell 2009). Midbodies remain a useful marker for pre-abscission cells, but the absence of midbodies should not be immediately interpreted as completion of abscission without further assays. Formally, a direct measurement of abscission timing would require imaging of the plasma membrane, for example using time-lapse phase-contrast microscopy (Fremont et al., 2016 Nat Comm). These limitations should be mentioned in the text.<br /> 4. IST1 plays a role in nuclear envelope sealing by recruiting the co-factor Spastin (Vietri et al., Nature 2015), a known IST1 co-factor also confirmed in the previous interactome screen (Wenzel et al. 2022). CAPN7 could have a role in maintaining nuclear integrity upon the KD of Nup153 and Nup50 (Mackay et al. 2010) instead of/in addition to its proposed role in delaying abscission as part of the NoCut checkpoint at the midbody. I don't think the authors can differentiate between these two possibilities, and it would be interesting to consider their possible implications on how the "NoCut" checkpoint is triggered.<br /> 5. Figure 5 should include images of representative cells, highlighting midbody-positive and multinucleated cells. Without images, it is not possible to evaluate the quality of these data.

    1. Reviewer #2 (Public Review):

      The cortical hem is one of the main signaling centers in the vertebrate forebrain, regulating neurogenesis of the medial pallium and the generation of Cajal-Retzius neurons. The authors examine how this signaling center is formed and functions. Previously, transcription factors playing instructive roles in the development of the cortical hem have been identified, but a master regulator had not been found so far. The authors build on their previous work studying the transcription factor Lmx1a which is one of the earliest and most specific cortical hem markers.

      By combining loss- and gain-of-function studies, RNA sequencing, histology, and analysis of downstream factors, the authors rigorously show Lmx1a is required for the expression of signaling molecules in the hem, the proliferation and functionality of dentate gyrus neurons, the cell cycle exit and differentiation (and also migration) of cajal-retzius cells and this by activating different downstream regulators.

      They use golden standard experiments in the field such as BrdU-Ki67 cell-cycle exit measurements, RNA sequencing, and patch clamping; combined with state-of-the-art techniques such as RNAscope and laser capture microdissection. These convincingly show that Lmx1a regulates the proliferation of dentate gyrus progenitor cells and a malformation of the transhilar scaffold. The authors also claim a migration deficit for dentate gyrus progenitors, but they do not consider apoptosis or show direct evidence for migration abnormalities.<br /> In the hem, the authors report normal proliferation and apoptosis in the Lmx1a mutants, but aberrant cell-cycle-exit, from which the authors conclude a problem in differentiation. However, this could be a cell cycle progression problem too (stuck in a certain cell cycle phase?), as the RNAseq data suggest. The authors should acknowledge this possibility.

      The RNAseq dataset provides candidate downstream regulators of the observed phenotypes and the authors test the functionality of Wnt3a, Tbr2, and Cdkn1a, showing they are involved in distinct processes.<br /> Strikingly, Wnt3a is not significantly downregulated in the RNAseq data in the Lmx1a mutant, but quantification of in situ hybridization signal (which is less robust) did reveal a significant difference. Is this a splice variant issue? A timing issue or specificity of the RNAscope probe? The authors should look into this more carefully.

      To study the role of Cdkn1a, the authors performed rescue experiments using in utero electroporation, which is a standard in the field. However, they argued before that "CR cell migration and DG morphogenesis are complex processes that require precise expression levels of key genes" when studying downstream factors Wnt3a and Tbr2. Why is this no longer an issue studying Cdkn1a?<br /> To study cell-cycle exit in this model, the authors quantified GFP and Ki67. Since electroporation not only targets the progenitor cells (see e.g. Govindan et al. 2018, Nature protocols), the authors should confirm these results with a BrdU/Ki67 quantification as in previous experiments, or confirm electroporation only targeted progenitor cells in their model.

      Lastly, the authors ectopically expressed Lmx1a and convincingly show its ability to generate a hem-like structure. Could the authors elaborate on the necessity for a medial signature? Can the hem be ectopically induced in the lateral pallium?

    1. Reviewer #2 (Public Review):

      In the past few years, single-cell transcriptomics analysis has uncovered cellular states associated with disease in experimental models and humans, revealing previously unrecognized disease-associated macrophage states. In particular, a macrophage state characterized by high expression of SPP1 (encoding osteopontin), and by a specific gene expression signature including the expression of TREM2, has been observed in various pathologies and given various names depending on the context e.g. TREM2hi macrophages, lipid-associated macrophages (LAM), disease-associated microglia (DAM), Scar-associated macrophages (SAM), etc... However, a focused investigation and comparison of SPP1+ macrophages across disease contexts were lacking. Here, the authors aimed to systematically analyze SPP1+ macrophages in the context of tissue fibrosis, and integrated single-cell RNA-seq data of >200,000 human macrophages in 6 organs in health and tissue fibrosis.

      Beyond confirming the presence of SPP1+ macrophages with a conserved gene expression module (TREM2, CD9, GPNMB, etc...) across tissues and their association with fibrosis, the authors identified a previously unknown cell subset within SPP1+ macrophages, that was enriched for the expression of genes involved in remodeling of the extracellular matrix, which they termed SPP1+ matrisome-associated macrophages (SPP1+MAM+). The authors further used computational tools to compare these SPP1+MAM+ macrophages to previously described SPP1+ macrophage states (LAM, DAM, SAM), investigate the differentiation and activation trajectory of SPP1+MAM+ macrophages, and identify potential transcriptional regulators involved in their differentiation. Finally, the authors show that SPP1+MAM+ macrophages are associated with ageing in both humans and mice.

      Overall, the conclusions of the authors are well supported by the data. The authors made excellent use of available computational tools, and the figures are clear and informative. The methods are well-described and appropriately used. In particular, the authors made a nice effort in explaining and justifying some key decisions in their scRNA-seq data analysis workflow, including a data-driven approach to decisions in the clustering analysis.

      The author's findings are of broad interest to the fields of tissue inflammation, fibrosis, macrophage biology, and immunology, and their report constitutes a valuable resource, and a basis for further investigations of macrophage differentiation mechanisms in tissue fibrosis, and how macrophages could be targeted to alleviate pathological tissue fibrosis.

    1. Reviewer #2 (Public Review):

      In this study, the authors pursue a line of inquiry related to the impacts of cholesterol depletion on macrophage gene expression. The authors find that depletion of cholesterol with either statins or methyl-cyclodextrin induces robust gene expression changes, including changes to JMJD3 expression, an epigenetic regulator.

      The authors then seek to dissect the mechanistic determinants of the regulation of JMJD3 in macrophages converging on a metabolic regulation hypothesis that requires mitochondrial activity.

      A strength of the paper is the use of multiple macrophage cell models and multiple tools for perturbation to improve the rigor of their conclusions. A weakness of the paper is that it relies heavily on chemical approaches without ever using genetic tools to confirm that their conclusions can be supported using an alternative approach, and when perturbing metabolic pathways as described here, it is difficult to understand how the entire cell state has changed. In fact, the unique focus on JMJD3 without utilizing a control set of genes to show that the impacts of these metabolic perturbations are specific to JMJD3 makes it hard to understand if this is a truly specific pathway for JMJD3 or a general cellular health change.

      The authors make an interesting claim in the early part of their manuscript about the potential for statins to regulate the epigenome which they show; however, in the present presentation, it is unclear if this is related to the JMJD3 effect or a separate form of regulation.

      This work has the potential to contribute to an improved understanding of the impact of statins on immune function.

    1. Reviewer #2 (Public Review):

      For most organs including lung produced by blastocyst complementation, certain cells including the blood vessels are still derived from host tissues, making them unfit for transplantation. To address this issue, Miura et al. explored the origin and the program of whole lung epithelium and mesenchyme, and identified the crucial Foxa2 lineage for lung organogenesis by using lineage tracing mice and human iPSC derived lung differentiation. They found that Foxa2 lineage cells contribute to both lung epithelium and mesenchyme formation, which suggest targeting Fox2 lineage cells could create an empty developmental niche for blastocyst complementation in mice. They further deplete Fgfr2 gene in Foxa2 lineage cells to induce the lung agenesis phenotype in mice, and donor mouse iPSCs injected into Fgfr2 mutant blastocysts occupied the empty niche and formed the missing lung.

      Strengths:

      To fill our knowledge gap of the origin of all lung cell types, especially pulmonary mesenchyme and endothelium, the authors investigated the lineage hierarchy of specified lung precursors in gastrulating mesendoderm. Using mouse lineage trancing and human iPSC derived lung differentiation, they clarified the msendoderm gene Expression pattern and progression, and compared the contributions of Pdgfra and Foxa2 lineage cells during lung development. They further demonstrate that the defective Foxa2 lineage in critically important for efficient lung complementation, which provide insight for next generation lung transplant therapies.

      Weakness:

      1. Several lineage tracing experiment lack rigorous quantification, the authors using "partially labels" or "labels a part of" in the text to describe their finding and conclusion, which make the evidence less solid.

      2. The ideal lung for transplant should be functional for gas exchange, the lung complementation was only analyzed at E17.5 and E14.5, these two stages were too early to determine the function of the lungs generated via CBC.

      3. Immune cells contribute large proportion in the lung, and are critical for lung transplant, the chimerism analysis of immune cells is missing in this study.