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    1. Reviewer #2 (Public Review):

      The data generated for this paper provides an important resource for the neuroscience community. The locus coeruleus (LC) is the known seed of noradrenergic cells in the brain. Due to its location and size, it remains scarcely profiled in humans. Despite the physically minute structure containing these cells, its impact is wide-reaching due to the known neuromodulatory function of norepinephrine (NE) in processes like attention and mood. As such, profiling NE cells has important implications for most neurological and neuropsychiatric disorders. This paper generates transcriptomic profiles that are not only cell-specific but which also maintain their spatial context, providing the field with a map for the cells within the region.


      Using spatial transcriptomics in a morphologically distinct region is a very attractive way to generate a map. Overlaying macroscopic information, i.e. a region with greater pigmentation, with its corresponding molecular profile in an unbiased manner is an extremely powerful way to understand the specific cellular and molecular composition of that brain structure.

      The technologies were used with an astute awareness of their limitations, as such, multiple technologies were leveraged to paint a more complete and resolved picture of the cellular composition of the region. For example, the lack of resolution in the spatial transcriptomic platform was compensated by complementary snRNA-seq and single molecule FISH.

      This work has been made publicly available and accessible through a user-friendly application such that any interested researcher can investigate the level of expression of their gene of interest within this region.

      Two important implications from this work are 1) the potential that the gene regulatory profiles of these cells are only partially conserved across species, humans, and rodents, and 2) that there may be other neuromodulatory cell types within the region that were otherwise not previously localized to the LC


      Given that the markers used to identify cells are not as specific as they need to be to definitively qualify the desired cell type, the results may be over-interpreted. Specifically, TH is the primary marker used to qualify cells as noradrenergic, however, TH catalyzes the synthesis of L-DOPA, a precursor to dopamine, which in turn is a precursor for epinephrine and norepinephrine suggesting some of the cells in the region may be dopaminergic and not NE cells. Indeed, there are publications to support the presence of dopaminergic cells in the LC (see Kempadoo et al. 2016, Takeuchi et al., 2016, Devoto et al. 2005). This discrepancy is further highlighted by the apparent lack of overlap per given Visium spots with TH, SCL6A2, or DBH. While the single-nucleus FISH confirms that some of the cells in the region are noradrenergic, others very possibly represent a different catecholamine. As such it is suggested that the nomenclature for the cells be reconsidered.

      The authors are unable to successfully implement unsupervised clustering with the spatial data, this greatly reduces the impact of the spatial technology as it implies that the transcriptomic data generated in the study did not have enough resolution to identify individual cell types.

      The sample contribution to the results is highly unbalanced, which consequently, may result in ungeneralizable findings in terms of regional cellular composition, limiting the usefulness of the publicly available data.

      This study aimed to deeply profile the LC in humans and provide a resource to the community. The combination of data types (snRNA-seq, SRT, smFISH) does in fact represent this resource for the community. However, due to the limitations, of which, some were described in the manuscript, we should be cautious in the use of the data for secondary analysis. For example, some of the cellular annotations may lack precision, the cellular composition also may not reflect the general population, and the presence of unexpected cell types may represent the accidental inclusion of adjacent regions, in this case, serotonergic cells from the Raphe nucleus.

      Nonetheless having a well-developed app to query and visualize these data will be an enormous asset to the community especially given the lack of information regarding the region in general.

    1. Reviewer #2 (Public Review):


      The authors set out to discover a developmental pathway leading to functionally diverse mTEC subsets. They show that Ccl21 is expressed early during thymus ontogeny in the medullary area. Fate-mapping gives evidence for the Ccl21 positive history of Aire positive mTECs as well as of thymic tuft cells and postnatally of a certain percentage of cTECs. Therefore, the differentiation potential of Ccl21+ TECs is tested in reaggregate thymus experiments - using embryonic or postnatal Ccl21+ TECs. From these experiments, the authors conclude that at least embryonic mTECs in large part pass through a Ccl21 positive stage prior to differentiation towards an Aire expressing or tuft cell stage.

      The authors are using Ccl21a as a marker for a bipotent progenitor that is detectable in the embryonic thymus and is still present at the adult stage mainly giving rise to mTECs. The choice of this marker gene is very interesting since Ccl21 expression can directly be linked to an important aspect in thymus biology: the expression of Ccl21 by cells in the thymic medulla allows trafficking of T cells into the medulla in order to undergo T cell selection.

      Making use of the Ccl21 detection, the authors can nicely show that cells actively expressing Ccl21 are localized throughout the medulla at an embryonic stage but also in adult thymus tissue. This suggests, that this progenitor is not accumulating at a specific area inside the medulla. This is a new finding.

      Moreover, the finding that a Ccl21+ progenitor population plays a functional role in thymocyte trafficking towards the medulla has not been described. Thus, Ccl21 expression may be used to localize a late bipotent progenitor in the thymic lobes.<br /> In addition, in Fig.8, the authors provide evidence that these progenitor cells have the potential to self-maintain as well as to differentiate in reaggregate experiments at E17 (not at 4 weeks of age). The first point is of great interest and importance since these cells in theory can be of therapeutic use.

      Overall assessment:

      The authors highlight a developmental pathway starting from a Ccl21-expressing TEC progenitor that contributes to a functionally diverse mTEC repertoire. This is a welcome addition to current knowledge of TEC differentiation.

    1. Reviewer #2 (Public Review):


      This manuscript describes the study protocol, structure and logic of the PAVE strategy. The PAVE study is a multicentric study to evaluate a novel cervical screen-triage-treat strategy for resource-limited settings as part of a global strategy to reduce cervical cancer burden. The PAVE strategy involves: 1) screening with self-sampled HPV testing; 2) triage of HPV-positive participants with a combination of extended genotyping and visual evaluation of the cervix assisted by deep-learning-based automated visual evaluation (AVE); and 3) treatment with thermal ablation or excision (Large Loop Excision of the Transformation Zone). The PAVE study has two phases: efficacy (2023-2024) and effectiveness (planned to begin in 2024-2025). The efficacy phase aims to refine and validate the screen-triage portion of the protocol. The effectiveness phase will examine few implementation of the PAVE strategy into clinical practice. In following phases implementation will further explored.

      Strengths and weaknesses

      The Pave Study develops and evaluates a novel strategy that combines HPV self-collection -that has been proven effective to increase screening coverage in different settings-, with genotyping and Automated Visual Evaluation as triage. The proposed strategy combined three key innovations to improve an important step in the cervical cancer care continuum. If the strategy is effective it will contribute to enhance cervical cancer prevention in low resource settings.

      As authors mentioned, despite the existence of effective preventive technologies (e.g., HPV vaccine and HPV test) translation of the HPV prevention methods has not yet occurred in many Low-Middle-Income Countries. So, in this context, new screen-triage-treat strategies are needed and if PAVE strategy were effective, it could be a landmark for cervical cancer prevention.

      The PAVE Study is a solid and important study that is aimed to be carried out in nine countries and recruit tens thousands of women. It is a study with a large and diverse sample that can provide useful information for the development of this new screen-triage-treat strategy. Another strength is the fact that the PAVE project is integrated into the screening activities placed in the selected countries that will allow to evaluate efficacy and effectiveness in real-word context.

      The manuscript does not present results because its aim is to describe the study protocol, structure and logic of the PAVE strategy.

      Phase 1 aims to evaluate efficacy of the strategy. Methods are well described and are consistent with the study aims.

      Phase 2 aims to evaluate the implementation of the PAVE strategy in clinical practice. The inclusion of implementation evaluation in this type of studies is an important milestone in the field of cervical cancer prevention. It has been shown that many strategies that have proven to be effective in controlled studies face barriers when they are implemented in real life. In that sense, results of phase 2 are key to ensure the future implementation of the strategy.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This research involves conducting experiments to determine the role of Fmnl2 during oocyte meiosis I.

      Strengths:<br /> Identifying the role of Fmnl2 during oocyte meiosis I is significant.

      Weaknesses:<br /> The quantitative analysis and the used approach to perturb FMNL2 function are currently incomplete and would benefit from more confirmatory approaches and rigorous analysis.

      1- Most of the results are expected. The new finding here is that FMNL2 regulates cytoplasmic F-actin in mouse oocytes, which is also expected given the role of FMNL2 in other cell types. Given that FMNL2 regulates cytoplasmic F-actin, it is very expected to see all the observed phenotypes. It is already established that F-actin is required for spindle migration to the oocyte cortex, extruding a small polar body and normal organelle distribution and functions.

      2-The authors used Fmnl2 cRNA to rescue the effect of siRNA-mediated knockdown of Fmnl2. It is not clear how this works. It is expected that the siRNA will also target the exogenous cRNA construct (which should have the same sequence as endogenous Fmnl2) especially when both of them were injected at the same time. Is this construct mutated to be resistant to the siRNA?

      3-The authors used only one approach to knockdown FMNL2 which is by siRNA. Using an additional approach to inhibit FMNL2 would be beneficial to confirm that the effect of siRNA-mediated knockdown of FMNL2 is specific.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors solved the crystal structure of CDV H-protein head domain at 3,2 A resolution to better understand the detailed mechanism of membrane fusion triggering. The structure clearly showed that the orientation of the H monomers in the homodimer was similar to that of measles virus H and different from other paramyxoviruses. The authors used the available co-crystal strictures of the closely related measles virus H structures with the SLAM and Nectin4 receptors to map the receptor binding site on CDV H. The authors also confirmed which N-linked sites were glycosylated in the CDV H protein and showed that both wildtype and vaccine strains of CDV H have the same glycosylation pattern. The authors documented that the glycans cover a vast majority of the H surface while leaving the receptor binding site exposed, which may in part explain the long-term success of measles virus and CDV vaccines. Finally, the authors used HS-AFM to visualize the real-time dynamic characteristics of CDV-H under physiological conditions. This analysis indicated that homodimers may dissociate into monomers, which has implications for the model of fusion triggering.

      The structural data and analysis were thorough and well-presented. However, the HS-AFM data, while very exciting, was not presented in a manner that could be easily grasped by readers of this manuscript. I have some suggestions for improvement.

      1) The authors claim their structure is very similar to the recently published croy-EM structure of CDV H. Can the authors provide us with a quantitative assessment of this statement?

      2) The results for the HS-AFM are difficult to follow and it is not clear how the authors came to their conclusions. Can the authors better explain this data and justify their conclusions based on it?

      3) The fusion triggering model in Figure 8 is ambiguous as to when H-F interactions are occurring and when they may be disrupted. The authors should clarify this point in their model.

    1. Reviewer #2 (Public Review):

      This study provides the proteomic and phosphoproteomics data for our understanding of the molecular alterations in adipose tissue and skeletal muscle from women with PCOS. This work is useful for understanding of the characteristics of PCOS, as it may provide potential targets and strategies for the future treatment of PCOS. While the manuscript presents interesting findings on omics and phenotypic research, the lack of in-depth mechanistic exploration limits its potential impact.

      The study primarily presents findings from omics and phenotypic research, but fails to provide a thorough investigation into the underlying mechanisms driving the observed results. Without a thorough elucidation of the mechanistic underpinnings, the significance and novelty of the study are compromised.

    1. Reviewer #2 (Public Review):

      In the study by Hreich et al, the potency of P2RX7-specific positive modulator HEI3090, developed by the authors, for the treatment of Idiopathic pulmonary fibrosis (IPF) was investigated. Recently, the authors have shown that HEI3090 can protect against lung cancer by stimulating dendritic cell P2RX7, resulting in IL-18 production that stimulates IFN-γ production by T and NK cells (DOI: 10.1038/s41467-021-20912-2). Interestingly, HEI3090 increases IL-18 levels only in the presence of high eATP. Since the treatment options for IPF are limited, new therapeutic strategies and targets are needed. The authors first show that P2RX7/IL-18/IFNG axis is downregulated in patients with IPF. Next, they used a bleomycin-induced lung fibrosis mouse model to show that the use of a positive modulator of P2RX7 leads to the activation of the P2RX7/IL-18 axis in immune cells that limits lung fibrosis onset or progression. Mechanistically, treatment with HEI3090 enhanced IL-18-dependent IFN-γ production by lung T cells leading to a decreased production of IL-17 and TGFβ, major drivers of IPF. The major novelty is the use of the small molecule HEI3090 to stimulate the immune system to limit lung fibrosis progression by targeting the P2RX7, which could be potentially combined with current therapies available. Overall, the study was well performed and the manuscript is clear. However, there is need for more details on the description and interpretation of the adoptive transfer experiments, as well as the statistical analyses and number of replicate independent experiments.

    1. Reviewer #2 (Public Review):


      The authors identified miR-199b-5p as a potential OA target gene using serum exosomal small RNA-seq from human healthy and OA patients. Their RNA-seq results were further compared with publicly available datasets to validate their finding of miR-199b-5p. In vitro chondrocyte culture with miR-199b-5p mimic/inhibitor and in vivo animal models were used to evaluate the function of miR-199b-5p in OA. The possible genes that were potentially regulated by miR-199b-5p were also predicted (i.e., Fzd6 and Gcnt2) and then validated by using Luciferase assays.


      1. Strong in vivo animal models including pain tests.<br /> 2. Validates the binding of miR-199b-5p with Fzd6 and binding of miR-199b-5p with Gcnt2.


      1. The authors may overinterpret their results. The current work shows the possible bindings between miR-199b-5p and Fzd6 as well as bindings between miR-199b-5p and Gcnt2. However, whether miR-199b-5p truly functions through Fzd6 and/or Gcnt2 requires genetic knockdown of Fzd6 and Gcnt2 in the presence of miR-199b-5p.<br /> 2. In vitro chondrocyte experiments were conducted in a 2D manner, which led to chondrocyte de-differentiation and thus may not represent the chondrocyte response to the treatments.<br /> 3. There is a lack of description for bioinformatic analysis.<br /> 4. There are several errors in figure labeling.

    1. Reviewer #2 (Public Review):


      In this study, Christin Krause et al mapped the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, and identified miR-182-5p and its target genes LRP6 as potential drivers of dysregulated glucose tolerance and fatty acid metabolism in obese T2-diabetics.


      This study contains some interesting findings and is valuable for the understanding of the key regulatory role of miRNAs in the pathogenesis of T2D.


      The authors didn't systemically investigate the function of miR-182 in T2DM or NAFLD.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this paper, Chamness and colleagues make a pioneering effort to map epistatic interactions among mutations in a membrane protein. They introduce thousands of mutations to the mouse GnRH Receptor (GnRHR), either under wild-type background or two mutant backgrounds, representing mutations that destabilize GnRHR by distinct mechanisms. The first mutant background is W107A, destabilizing the tertiary fold, and the second, V276T, perturbing the efficiency of cotranslational insertion of TM6 to the membrane, which is essential for proper folding. They then measure the surface expression of these three mutant libraries, using it as a proxy for protein stability, since misfolded proteins do not typically make it to the plasma membrane. The resulting dataset is then used to shed light on how diverse mutations interact epistatically with the two genetic background mutations. Their main conclusion is that epistatic interactions vary depending on the degree of destabilization and the mechanism through which they perturb the protein. The mutation V276T forms primarily negative (aggravating) epistatic interactions with many mutations, as is common to destabilizing mutations in soluble proteins. Surprisingly, W107A forms many positive (alleviating) epistatic interactions with other mutations. They further show that the locations of secondary mutations correlate with the types of epistatic interactions they form with the above two mutants.

      Strengths:<br /> Such a high throughput study for epistasis in membrane proteins is pioneering, and the results are indeed illuminating. Examples of interesting findings are that: (1) No single mutation can dramatically rescue the destabilization introduced by W107A. (2) Epistasis with a secondary mutation is strongly influenced by the degree of destabilization introduced by the primary mutation. (3) Misfolding caused by mis-insertion tends to be aggravated by further mutations. The discussion of how protein folding energetics affects epistasis (Fig. 7) makes a lot of sense and lays out an interesting biophysical framework for the findings.

      Weaknesses:<br /> The major weakness comes from the potential limitations in the measurements of surface expression of severely misfolded mutants. This point is discussed quite fairly in the paper, in statements like "the W107A variant already exhibits marginal surface immunostaining" and many others. It seems that only about 5% of the W107A makes it to the plasma membrane compared to wild-type (Figures 2 and 3). This might be a low starting point from which to accurately measure the effects of secondary mutations.

      Still, the authors claim that measurements of W107A double mutants "still contain cellular subpopulations with surface immunostaining intensities that are well above or below that of the W107A single mutant, which suggests that this fluorescence signal is sensitive enough to detect subtle differences in the PME of these variants". I was not entirely convinced that this was true. Firstly, I think it would be important to test how much noise these measurements have and how much surface immunostaining the W107A mutant displays above the background of cells that do not express the protein at all. But more importantly, it is not clear if under this regimen surface expression still reports on stability/protein fitness. It is unknown if the W107A retains any function or folding at all. For example, it is possible that the low amount of surface protein represents misfolded receptors that escaped the ER quality control. The differential clustering of epistatic mutations (Fig. 6) provides some interesting insights as to the rules that dictate epistasis, but these too are dominated by the magnitude of destabilization caused by one of the mutations. In this case, the secondary mutations that had the most interesting epistasis were exceedingly destabilizing. With this in mind, it is hard to interpret the results that emerge regarding the epistatic interactions of W107A. Furthermore, the most significant positive epistasis is observed when W107A is combined with additional mutations that almost completely abolish surface expression. It is likely that either mutation destabilizes the protein beyond repair. Therefore, what we can learn from the fact that such mutations have positive epistasis is not clear to me. Based on this, I am not sure that another mutation that disrupts the tertiary folding more mildly would not yield different results.

      With that said, I believe that the results regarding the epistasis of V276T with other mutations are strong and very interesting on their own.

      Additionally, the study draws general conclusions from the characterization of only two mutations, W107A and V276T. At this point, it is hard to know if other mutations that perturb insertion or tertiary folding would behave similarly. This should be emphasized in the text.

      Some statistical aspects of the study could be improved:

      1. It would be nice to see the level of reproducibility of the biological replicates in a plot, such as scatter or similar, with correlation values that give a sense of the noise level of the measurements. This should be done before filtering out the inconsistent data.

      2. The statements "Variants bearing mutations within the C- terminal region (ICL3-TMD6-ECL3-TMD7) fare consistently worse in the V276T background relative to WT (Fig. 4 B & E)." and "In contrast, mutations that are 210 better tolerated in the context of W107A mGnRHR are located 211 throughout the structure but are particularly abundant among residues 212 in the middle of the primary structure that form TMD4, ICL2, and ECL2 213 (Fig. 4 C & F)." are both hard to judge. Inspecting Figures 4B and C does not immediately show these trends, and importantly, a solid statistical test is missing here. In Figures 4E and F the locations of the different loops and TMs are not indicated on the structure, making these statements hard to judge.

      3. The following statement lacks a statistical test: "Notably, these 98 variants are enriched with TMD variants (65% TMD) relative to the overall set of 251 variants (45% TMD)." Is this enrichment significant? Further in the same paragraph, the claim that "In contrast to the sparse epistasis that is generally observed between mutations within soluble proteins, these findings suggest a relatively large proportion of random mutations form epistatic interactions in the context of unstable mGnRHR variants". Needs to be backed by relevant data and statistics, or at least a reference.

    1. Reviewer #2 (Public Review):

      Members of the EphB family of tyrosine kinase receptors are involved in a multitude of diverse cellular functions, ranging from the control of axon growth to angiogenesis and synaptic plasticity. In order to provide these diverse functions, it is expected that these receptors interact in a cell-type specific manner with a diverse variety of downstream signalling molecules.

      The authors have used proteomics approaches to characterise some of these molecules in further detail. This molecule, myc-binding protein 2 (MYCBP2) is also known as highwire, has been identified in the context of establishment of neural connectivity. Another molecule coming up on this screen was identified as FBXO45.

      The authors use classical methods of co-IP to show a kinase-independent binding of MYCBP2 to EphB2. They further showed that FBXO45 within a ternary complex increased the stability of the EphB2/MYCBP2 complex.

      To define the interacting domains, they used clearly designed swapping experiments to show that the extracellular and transmembrane domains are necessary and sufficient for the formation of the ternary complex.

      Using a cellular contraction assay, the authors showed the necessity of MYCBP2 in mediating the cytoskeletal response of EphB2 forward signalling. Furthermore, they used the technically challenging stripe assay of alternating lanes of ephrinB-Fc and Fc to show that also in this migration-based essay MYCBP2 is required for EphB mediated differential migration pattern.

      MYCBP2 in addition is necessary to stabilize EphB2, that is in the absence of MYCBP2, EphB2 is degraded in the lysosomal pathway.

      Interestingly, the third protein in this complex, Fbxo45, was further characterized by overexpression of the domain of MYCBP2, known to interact with Fbxo45. Here the authors showed that this approach led to the disruption of the EphB2 / MYCBP2 complex, and also abolished the ephrinB mediated activation of EphB2 receptors and their differential outgrowth on ephrinB2-Fc / Fc stripes.

      Finally, the authors demonstrated an in vivo function of this complex using another model system, C elegans where they were able to show a genetic interaction.

      Data show in a nice set of experiments a novel level of EphB2 forward signalling where a ternary complex of this receptor with multifunctional MYCBP2 and Fbxo45 controls the activity of EphB2, allowing a further complex regulation of this important receptors. Additionally, the authors challenge pre-existing concepts of the function of MYCBP2 which might open up novel ways to think about this protein.<br /> Of interest is this work also in terms of development of the retinotectal projection in zebrafish where MYCBP2/highwire plays a crucial role, and thus might lead to a better understanding of patterning along the DV axis, for which it is known that EphB family members are crucial.

      Overall, the experiments are classical experiments of co-immunoprecipitations, swapping experiments, collapse assays, and stripe assays which all are well carried out and are convincing.

    1. Reviewer #2 (Public Review):

      In this work, Jarc et al. describe a method to decouple the mechanisms supporting progenitor self-renewal and expansion from feed-forward mechanisms promoting their differentiation.

      The authors aimed at expanding pancreatic progenitor (PP) cells, strictly characterized as PDX1+/SOX9+/NKX6.1+ cells, for several rounds. This required finding the best cell culture conditions that allow sustaining PP cell proliferation along cell passages while avoiding their further differentiation. They achieve this by comparing the transcriptome of PP cells that can be expanded for several passages against the transcriptome of unexpanded (just differentiated) PP cells.

      The optimized culture conditions enabled the selection of PDX1+/SOX9+/NKX6.1+ PP cells and their consistent, 2000-fold, expansion over ten passages and 40-45 days. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. These optimized culture conditions consisted in substituting the Vitamin A containing B27 supplement with a B27 formulation devoid of vitamin A (to avoid retinoic acid (RA) signaling from an autocrine feed-forward loop), substituting A38-01 with the ALK5 II inhibitor (ALK5i II) that targets primarily ALK5, supplementation of medium with FGF18 (in addition to FGF2) and the canonical Wnt inhibitor IWR-1, and cell culture on vitronectin-N (VTN-N) as a substrate instead of Matrigel.

      The strength of this work relies on a clever approach to identify cell culture modifications that allow expansion of PP cells (once differentiated) while maintaining, if not reinforcing, PP cell identity. Along the work, it is emphasized that PP cell identity is associated to the co-expression of PDX1, SOX9 and NKX6.1. The optimized protocol is unique (among the other datasets used in the comparison shown here) at inducing a strong upregulation of GP2, a unique marker of human fetal pancreas progenitors. Importantly GP2+ enriched hPS cell-derived PP cells are more efficiently differentiating into pancreatic endocrine cells (Aghazadeh et al., 2022; Ameri et al., 2017).

      The unlimited expansion of PP cells reported here would allow scaling-up the generation of beta cells, for the cell therapy of diabetes, by eliminating a source of variability derived from the number of differentiation procedures to be carried out when starting at the hPS cell stage each time. The approach presented here would allow selection of the most optimally differentiated PP cell population for subsequent expansion and storage. Among other conditions optimized, the authors report a role for Vitamin A in activating retinoic acid signaling in an autocrine feed-forward loop, and the supplementation with FGF18 to reinforce FGF2 signaling.

      This is a relevant topic in the field of research, and some of the cell culture conditions reported here for PP expansion might have important implications in cell therapy approaches. Thus, the approach and results presented in this study could be of interest for researchers working in the field of in vitro pancreatic beta cell differentiation from hPSCs. Table S1 and Table S4 are clearly detailed and extremely instrumental to this aim.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Transposable elements are known to have a strong potential to generate diversity and impact gene regulation, and they are thought to play an important role in plant adaptation to changing environments. Nevertheless, very few studies have performed genome-wide analyses to understand the global effect of selection on TEs in natural populations. Horvath et al. used available whole-genome re-sequencing data from a representative panel of B. distachyon accessions to detect TE insertion polymorphisms (TIPs) and estimate their time of origin. Using a thorough combination of population genomics approaches, the authors demonstrate that only a small amount of the TE polymorphisms are targeted by positive selection or potentially involved in adaptation. By comparing the age-adjusted population frequencies of TE polymorphisms and neutral SNPs, the authors found that retrotransposons are affected by purifying selection independently of their distance to genes. Finally, using forward simulations they were able to quantify the strength of selection acting on TE polymorphisms, finding that retrotransposons are mainly under moderate purifying selection, with only a minority of the insertions evolving neutrally.

      Strengths:<br /> Horvath et al., use a convincing set of strategies, and their conclusions are well supported by the data. I think that incorporating polymorphism's age into the analysis of purifying selection is an interesting way to reduce the possible bias introduced by the fact that SNPs and TEs polymorphisms do not occur at the same pace. The fact that TE polymorphisms far from genes are also under purifying selection is an interesting result that reinforces the idea that the trans-regulatory effect of TE insertions might not be a rare phenomenon, a matter that may be demonstrated in future studies.

      Weaknesses:<br /> TEs from different classes and orders strongly differ in multiple features such as size, the potential impact of close genes upon insertion, insertion/elimination ratio (ie, MITE/TIR excision, solo-LTR formation), or insertion preference. Given such diversity, it is expected that their survival rates on the genome and the strength of selection acting on them could be different. The authors differentiate DNA transposons and retrotransposons in some of the analyses, the specificities of the most abundant plant TE types (ie, LTR/Gypsy, LTR/Copia, MITE DNA transposons) are not considered.

      The authors used a short-read-based approach to detect TIPs and TAPs. It is known that detecting TE polymorphisms is challenging and can lead to false negatives, depending on the method used and the sequencing coverage. The methodology used here (TEPID) has been previously applied to other species, but it is unclear if the sensitivity of the TIP/TAP caller is equivalent to that of the SNP caller and how these potential differences may affect the results.

    1. Reviewer #2 (Public Review):

      The Kinesin superfamily motors mediate the transport of a wide variety of cargos which are crucial for cells to develop into unique shapes and polarities. Kinesin-3 subfamily motors are among the most conserved and critical classes of kinesin motors which were shown to be self-inhibited in a monomeric state and dimerize to activate motility along microtubules. Recent studies have shown that different members of this family are uniquely activated by to undergo transition from monomers to dimers.

      Niwa and colleagues study two well-described members of the kinesin-3 superfamily, unc104 and KLP6, to uncover the mechanism of monomer to dimer transition upon activation. Their studies reveal that although both Unc104 and KLP6 are both self-inhibited monomers, their propensities for forming dimers are quite different. The authors relate this difference to a region in the molecules called CC2 which has a higher propensity for forming homodimers. Unc104 readily forms homodimers if its self-inhibited state is disabled while KLP6 does not.

      The work suggests that although mechanisms for self-inhibited monomeric states are similar, variations in the kinesin-3 dimerization may present a unique forms of kinesin-3 motor regulation with implications on the forms of motility functions carried out by these unique kinesin-3 motors.

    1. Reviewer #2 (Public Review):

      Hersperger et al. investigated the importance of Drosophila immune cells, called hemocytes, in the response to oxidative stress in adult flies. They found that hemocytes are essential in this response, and using state-of-the-art single-cell transcriptomics, they identified expression changes at the level of individual hemocytes. This allowed them to cluster hemocytes into subgroups with different responses, which certainly represents very valuable work. One of the clusters appears to respond directly to oxidative stress and shows a very specific expression response that could be related to the observed systemic metabolic changes and energy mobilization.

      Using hemocyte-specific genetic manipulation, the authors convincingly show that the DNA damage response in hemocytes regulates JNK activity and subsequent expression of the JAK/STAT ligand Upd3. Silencing of the DNA damage response or excessive activation of JNK and Upd3 leads to increased susceptibility to oxidative stress. This nicely demonstrates the importance of tight control of JNK-Upd3 signaling in hemocytes during oxidative stress. The treatment the authors used is quite harsh, and in such a situation it is simply better not to use upd3 signaling, but it is still worth bearing in mind that upd3 signaling may have a protective role under milder stresses, but Upd3 could require very tight control - this could be an interesting objective for future studies.

      The authors demonstrate that hemocytes play an important role in energy mobilization during oxidative stress, suggesting that control of energy mobilization by hemocytes is essential for the response. They further postulate that "hemocyte-derived upd3 is most likely released by the activated plasmatocyte cluster C6 during oxidative stress in vivo and is subsequently controlling energy mobilization and subsequent tissue wasting upon oxidative stress." It is important to note here that the association of upd3 with the observed changes in energy metabolism has not been tested, and the subsequent tissue wasting allegedly caused by excessive upd3 as a cause of death remains an open question.

    1. Reviewer #2 (Public Review):

      In this study, the authors tried to gauge the effect of human activity on three species, (1) the Hooded grow, an urban exploiter, (2) the Rose ring parakeet, an invasive, alien species that has adapted to exploit human resources, and (3) the Graceful prinia, an urban adapter, which is relatively shy of humans. A goal of the study was to increase awareness of the importance of urban parks.

      Strengths:<br /> Strengths of the study include the fact that it was conducted at 17 different sites, including parks, roads and residential areas, and included three species with different habitat preferences. Each species produced relatively loud and repeatable vocalizations. To avoid the effect of seasonal changes, sounds were sampled within a 10 day period of the lockdown as well as post-lockdown. The analysis included a comparison of the number of sound files, binary values indicating emission of a common syllable, and also the total number of syllables emitted as a measurement of bird activity. Ambient temperatures and sound levels of human activity were also recorded. All of these factors speak to the comprehensive approach and analysis adopted in this study. The results are based on a rigorous statistical analysis, ruling out the effects of various extraneous parameters.

      Weaknesses:<br /> Most significant changes may occur near the ambient noise levels and this could lead to a different conclusion, but the authors authors acknowledge this possibility and clarify that they only analyzed vocalizations with high signal-to-noise signals to avoid ambiguity. In the revised version, they also replaced the previous ambient noise parameter with an estimate of ambient noise under 1kHz, assuming that it reflects most anthropogenic noise (not restricted to human speech). This seems reasonable and this new model gave very similar results to the previous one.

      In interpreting the data, the authors mention the effect of human activity on bird vocalizations in the context of inter-species predator-prey interactions; however, the presence of humans could also modify intraspecies interactions by acting as triggers for communication of warning and alarm, and/or food calls (as may sometimes be the case) to conspecifics. The behavioral significance of the syllables used to monitor animal activity could be informative in this context; however, the authors acknowledge this possibility in the Discussion. Most importantly, the authors acknowledge the possibility of the above-noted bias, and the potential of a transient nature of the observed effects.

      Conclusion:<br /> In general, the authors achieved their aim of illustrating the complexity of the affect of human activity on animal behavior notwithstanding the caveats noted above. Their study also makes it clear that estimating such affects is not simple given the dynamics of animal behavior. For example, seasonality, temperature changes, animal migration and movement, as well as interspecies interactions, such as those related to predator-prey behavior, and inter/intra-species competition in other respects can all play into site-specific changes in the vocal activity of a particular species.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Caflisch and coworkers investigate the methyltransferase activity of the complex of methyltransferase-like proteins 3 and 14 (METTL3-14). To obtain a high-resolution description of the complete catalytic cycle they have carefully designed a combination of experiments and simulations. Starting from the identification of bisubstrate analogues (BAs) as binders to stabilise a putative transition state of the reaction, they have determined multiple crystal structures and validated relevant interactions by mutagenesis and enzymatic assays.

      Using the resolved structure and classical MD simulations they obtained a kinetic picture of the binding and release of the substrates. Of note, they accumulated very good statistics on these processes using 16 simulation replicates over a time scale of 500 ns. To compare the time scale of the release of the products with that of the catalytic step they performed state-of-the-art QM/MM free energy calculations (testing multiple levels of theory) and obtained a free energy barrier that indicates how the release of the product is slower than the catalytic step.

      Strengths:<br /> All the work proceeds through clear hypothesis testing based on a combination of literature and new results. Eventually, this allows them to present in Figure 10 a detailed step-by-step description of the catalytic cycle. The work is very well crafted and executed.

      Weaknesses:<br /> To fulfill its potential of guiding similar studies for other systems as well as to allow researchers to dig into their vast work, the authors should share the results of their simulations (trajectories, key structures, input files, protocols, and analysis) using repositories like Zenodo, the plumed-nest, figshare or alike.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this manuscript, the authors reveal that GIF/MT-3 regulates zinc homeostasis depending on the cellular redox status. The manuscript technically sounds, and their data concretely suggest that the recombinant MTs, not only GIF/MT-3 but also canonical MTs such as MT-1 and MT-2, contain sulfane sulfur atoms for the Zn-binding. The scenario proposed by the authors seems to be reasonable to explain the Zn homeostasis by the cellular redox balance.

      Strengths:<br /> The data presented in the manuscript solidly reveal that recombinant GIF/MT-3 contains sulfane sulfur.

      Weaknesses:<br /> It is still unclear whether native MTs, in particular, induced MTs in vivo contain sulfane sulfur or not.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Bates TA. et al. studied the biochemical characteristics of ESAT-6, a major virulence factor of Mycobacterium tuberculosis (Mtb), as part of the heterodimer with CFP10, a molecular chaperon of ESAT-6, as in homodimer and in homotetramer using recombinant ESAT-6 and CFP10 expressed in E. coli by applying several biochemical assays including Biolayer Interferometry (BLI) assay. The main findings show that ESAT-6 forms a tight interaction with CFP10 as a heterodimer at neutral pH, and ESAT-6 forms homodimer and even tetramer-based larger molecular aggregates at acidic pH. Although the discussion of the potential problems associated with the contamination of ESAT-6 preparations with ASB-14 during the LPS removal step is interesting, this research does not test the potential impact of residual ASB-14 contaminant on the biochemical behavior ESAT-6-CFP10 heterodimer and ESAT-6 homodimer or tetramer and their hemolytic activity in comparison with the ones without ASB-14. The main strength of this study is the generation of ESAT-6 specific nanobodies and the demonstration of its anti-tuberculosis efficiency in THP-1 cell lines infected with Mtb strains with reporter genes.

      Strengths:<br /> Generation and demonstration of the anti-ESAT-6 nanobodies against tuberculosis infection in a cell line based Mtb infection model.

      Weaknesses:<br /> Although the biochemistry studies provide quantitative data about the interactions of ESAT-6 with its molecular chaperon CFP10 and the interaction of ESAT-6 homodimer and tetramers, the novel information from these studies is minimal.

    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.


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

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

      Weaknesses: (1) While this has the advantage of a minimal model (model with minimal factors incorporated), it is a disadvantage for predicting in vivo organization; one might need to incorporate the action of many other proteins (for example, PRC, HP1, etc) and several other histone modifications to predict in vivo organization. (2) While this forward model produces features of relevant contact maps, one would need to tune some of the intra-chromatin interaction parameters to obtain an accurate contact map and radius of gyration.

    1. Reviewer #2 (Public Review):

      Strengths:<br /> The authors have conducted a large, well-designed experiment to test the response to eCO2. Overall, the experimental design is sound and appropriate for the questions about how a change in CO2 affects the ionome of Arabidopsis. Most of the conclusions in this area are well supported by the data that the authors present.

      Weakness:<br /> While the authors have done good experiments, it is a big stretch from Arabidopsis grown in an arbitrary concentration of CO2 to relevance to human and animal nutrition in future climates. Arabidopsis is a great model plant, but its leaves are not generally eaten by humans or animals.

      The authors don't justify their choice of a CO2 concentration. Given the importance of the parameter for the experiment, the rationale for selecting 900 ppm as elevated CO2 compared to any other concentration should be addressed. And CO2 is just one of the variables that plants will have to contend with in future climates, other variables will also affect elemental concentrations.

      Given these concerns, I think the emphasis on biofortification for future climates is unwarranted for this study.

      Additionally, I have trouble with these conclusions:

      -Abstract "Finally, we demonstrate that manipulating the function of one of these genes can mitigate the negative effect of elevated CO2 on the plant mineral composition. "<br /> -Discussion "Consistent with these results, we show that manipulating TIP2;2 expressions with a knock-out mutant can modulate the Zn loss observed under high CO2."

      The authors have not included the data to support this conclusion as stated. They have shown that this mutant increases the Zn content of the leaves when compared to WT but have not demonstrated that this response is different than in ambient CO2. This is an important distinction: one way to ameliorate the reduction of nutrients due to eCO2 is to try to identify genes that are involved in the mechanism of eCO2-induced reduction. Another way is to increase the concentration of nutrients so that the eCO2-induced reduction is not as important (i.e. a 10% reduction in Zn due to eCO2 is not as important if you have increased the baseline Zn concentration by 20%). The authors identified tip2 as a target from the GWAS on difference, but their validation experiment only looks at eCO2.

    1. Reviewer #2 (Public Review):

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

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

    1. Reviewer #2 (Public Review):


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


      The manuscript is clearly written and follows a series of logical steps toward connecting multiple matrisome genes and putative AIS effectors in a new framework of pathomechanism. The multidisciplinary nature of the work makes it a strong body of work wherein multiple models offer multiple lines of supportive data.


      This manuscript remains an important multidisciplinary study of the genetic and functional basis of adolescent idiopathic scoliosis (AIS). To the benefit of the overall manuscript quality, the reviewers have addressed most concerns to satisfaction. I have a few remaining suggestions:

      1. Regarding the genetic association of the common COL11A1 variant rs3753841, p.Pro1335Leu, please soften this statement to indicate that the variant could be a "risk locus" rather than "causal" in the following sentence on page 7-8: "These observations suggested that rs3753841 itself could be causal, although our methods would not detect deep intronic variants that could contribute to the overall association signal."

      2. Include the list of three rare missense variants mentioned in the response to reviewers as a supplementary table. Please also include methods for the SKATO rare variant burden analysis.

      3. Thank you for addressing the question of whether p.Pro1335Leu is a loss of function, gain of function, or dominant negative variant. The rationale in the response to reviewers was helpful, so please include this line of reasoning, and that there remains uncertainty, in the Discussion of the main text of the manuscript.

    1. Reviewer #2 (Public Review):


      In this article, the authors employed modified CRISPR screens ["guide-only (GO)-CRISPR"] in the attempt to identify the genes which may mediate cancer cell dormancy in the high grade serous ovarian cancer (HGSOC) spheroid culture models. Using this approach, they observed that abrogation of several of the components of the netrin (e.g., DCC, UNC5Hs) and MAPK pathways compromise the survival of non-proliferative ovarian cancer cells. This strategy was complemented by the RNAseq approach which revealed that a number of the components of the netrin pathway are upregulated in non-proliferative ovarian cancer cells and that their overexpression is lost upon disruption of DYRK1A kinase that has been previously demonstrated to play a major role in survival of these cells. Perampalam et al. then employed a battery of cell biology approaches to support the model whereby the Netrin signaling governs the MEK-ERK axis to support survival of non-proliferative ovarian cancer cells. Moreover, the authors show that overexpression of Netrins 1 and 3 bolsters dissemination of ovarian cancer cells in the xenograft mouse model, while also providing evidence that high levels of the aforementioned factors are associated with poor prognosis of HGSOC patients.


      Overall it was thought that this study is of potentially broad interest inasmuch as it provides previously unappreciated insights into the potential molecular underpinnings of cancer cell dormancy, which has been associated with therapy resistance, disease dissemination, and relapse as well as poor prognosis. Notwithstanding the potential limitations of cellular models in mimicking cancer cell dormancy, it was thought that the authors provided sufficient support for their model that netrin signaling drives survival of non-proliferating ovarian cancer cells and their dissemination. Collectively, it was thought that these findings hold a promise to significantly contribute to the understanding of the molecular mechanisms of cancer cell dormancy and in the long term may provide a molecular basis to address this emerging major issue in the clinical practice.


      Several issues were observed regarding methodology and data interpretation. The major concerns were related to the reliability of modelling cancer cell dormancy. To this end, it was relatively hard to appreciate how the employed spheroid model allows to distinguish between dormant and e.g., quiescent or even senescent cells. This was in contrast to solid evidence that netrin signaling stimulates abdominal dissemination of ovarian cancer cells in the mouse xenograft and their survival in organoid culture. Moreover, the role of ERK in mediating the effects of netrin signaling in the context of the survival of non-proliferative ovarian cancer cells was found to be somewhat underdeveloped.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study seeks to advance our knowledge of how vitamin D may be protective in allergic airway disease in both adult and neonatal mouse models. The rationale and starting point are important human clinical, genetic/bioinformatic data, with a proposed role for vitamin D regulation of 2 human chromosomal loci (Chr17q12-21.1 and Chr17q21.2) linked to the risk of immune-mediated/inflammatory disease. The authors have made significant contributions to this work specifically in airway disease/asthma. They link these data to propose a role for vitamin D in regulating IL-2 in Th2 cells implicating genes associated with these loci in this process.

      Strengths:<br /> Here the authors draw together evidence form. multiple lines of investigation to propose that amongst murine CD4+ T cell populations, Th2 cells express high levels of VDR, and that vitamin D regulates many of the genes on the chromosomal loci identified to be of interest, in these cells. The bottom line is the proposal that vitamin D, via Ikfz3/Aiolos, suppresses IL-2 signalling and reduces IL-2 signalling in Th2 cells. This is a novel concept and whilst the availability of IL-2 and the control of IL-2 signalling is generally thought to play a role in the capacity of vitamin D to modulate both effector and especially regulatory T cell populations, this study provides new data.

      Weaknesses:<br /> Overall, this is a highly complicated paper with numerous strands of investigation, methodologies etc. It is not "easy" reading to follow the logic between each series of experiments and also frequently fine detail of many of the experimental systems used (too numerous to list), which will likely frustrate immunologists interested in this. There is already extensive scientific literature on many aspects of the work presented, much of which is not acknowledged and largely ignored. For example, reports on the effects of vitamin D on Th2 cells are highly contradictory, especially in vitro, even though most studies agree that in vivo effects are largely protective. Similarly other reports on adult and neonatal models of vitamin D and modulation of allergic airway disease are not referenced. In summary, the data presentation is unwieldy, with numerous supplementary additions, that makes the data difficult to evaluate and the central message lost. Whilst there are novel data of interest to the vitamin D and wider community, this manuscript would benefit from editing to make it much more readily accessible to the reader.

      Wider impact: Strategies to target the IL-2 pathway have long been considered and there is a wealth of knowledge here in autoimmune disease, transplantation, GvHD etc - with some great messages pertinent to the current study. This includes the use of IL-2, including low dose IL-2 to boost Treg but not effector T cell populations, to engineered molecules to target IL-2/IL-2R.

    1. Reviewer #2 (Public Review):


      This manuscript from Liu et al. examines the role of Fat and Dachsous, two transmembrane proto-cadherins that function both in planar cell polarity and in tissue growth control mediated by the Hippo pathway. The authors developed a new method for measuring growth of the wing imaginal disc during late larval development and then used this approach to examine the effects of disruption of Fat/Dachsous function on disc growth. The authors show that during mid to late third instar the wing imaginal disc normally grows in a linear rather than exponential fashion and that this occurs due to slowing of the mitotic cell cycle as the disc grows during this period. Consistent with their known role in regulating Hippo pathway activity, this slowing of growth is disrupted by loss of Fat/Dachsous function. The authors also observed a previously unreported gradient of Fat protein across the wing blade. However, graded expression of Fat or Dachsous is not necessary for proper growth regulation in the late third instar because ectopic Dachsous expression, which affects gradients of both Dachsous and Fat, has no growth phenotype.


      Although the role of the Hippo pathway in growth control has been extensively studied, our understanding of how the pathway controls growth during normal development remains relatively weak. This work addresses this question by examining normal growth of the wing imaginal disc during part of its development in the larva and characterizing the effects of Fat/Dachsous manipulation on that growth. The authors developed tools for measuring wing growth by measuring wing volume, an approach that could be useful in future studies of tissue growth.


      1) Although the approach used to measure volume is new to this study, the basic finding that imaginal disc growth slows at the mid-third instar stage has been known for some time from studies that counted disc cell number during larval development (Fain and Stevens, 1982; Graves and Schubiger, 1982). Although these studies did not directly measure disc volume, because cell size in the disc is not known to change during larval development, cell number is an accurate measure of tissue volume. However, it is worth noting that the approach used here does potentially allow for differential growth of different regions of the disc.

      2) Related to point 1, a main conclusion of this study, that cell cycle length scales with growth of the wing, is based on a developmentally limited analysis that is restricted to the mid-third instar larval stage and later (early third instar begins at 72 hr - the authors' analysis started at 84 hr). The previous studies cited above made measurements from the beginning of the 3rd instar and combined them with previous histological analyses of cell numbers starting at the beginning of the 2nd instar. Interestingly, both studies found that cell number increases exponentially from the start of the 2nd instar until mid-third instar, and only after that point does the cell cycle slow resulting in the linear growth reported here. The current study states that growth is linear due to scaling of cell cycle with disc size as though this is a general principle, but from the earlier studies, this is not the case earlier in disc development and instead applies only to the last day of larval life.

      3) The analysis of the roles of Fat and Dachsous presented here has weaknesses that should be addressed. It is very curious that the authors found that depletion of Fat by RNAi in the wing blade had essentially no effect on growth while depletion of Dachsous did, given that the loss of function overgrowth phenotype of null mutations in fat is more severe than that of null mutations in dachsous (Matakatsu and Blair, 2006). An obvious possibility is that the Fat RNAi transgene employed in these experiments is not very efficient. The authors tried to address this by doubling the dose of the transgene, but it is not clear to me that this approach is known to be effective. The authors should test other RNAi transgenes and additionally include an analysis of growth of discs from animals homozygous for null alleles, which as they note survive to the late larval stages.

      4) It is surprising that the authors detect a gradient of Fat expression that has not been seen previously given that this protein has been extensively studied. It is also surprising that they find that expression of Nubbin Gal4 is graded across the wing blade given that previous studies indicate that it is uniform (ie. Martín et al. 2004). These two surprising findings raise the possibility that the quantification of fluorescence could be inaccurate. The curvature of the wing blade makes it a challenging tissue to image, particularly for quantitative measurements.

      5) Overall, in my view the impact of these findings is limited. The focus on growth solely at the end of larval development, when there are a number of potentially confounding variables (for example hormonal cues), makes the generality of the findings reported here difficult to judge. Additionally, the functional analysis of Fat/Dachsous function in this process is limited - for example does disruption of other Hippo pathway components have a similar effect?

    1. Reviewer #2 (Public Review):

      In the main text, the authors apply their metrics to a data set that was published by Mira et al. in 2015. The data consist of growth rate measurements for a combinatorially complete set of 16 genetic variants of the antibiotic resistance enzyme beta-lactamase across 10 drugs and drug combinations at 3 different drug concentrations, comprising a total of 30 different environmental conditions. In my previous report I had asked the authors to specify why they selected only 7 out of 30 environments for their analysis, with only one concentration for drug, but a clear explanation is still lacking. In the Data section of Material and Methods, the authors describe their criterion for data selection as follows: "we focus our analyses on drug treatments that had a significant negative effect on the growth of wildtype/TEM-1 strains". However, in Figure 2 it is seen that, even for the selected data sets, not all points are significant compared to wild type (grey points). So what criterion was actually applied?

      In effect, for each chosen drug or drug combination, the authors choose the data set corresponding to the highest drug concentration. As a consequence, they cannot assess to what extent their metrics depend on drug concentration. This is a major concern, since Mira et al. concluded in their study that the differences between growth rate landscapes measured at different concentrations were comparable to the differences between drugs. I argued before that, if the new metrics display a significant dependence on drug concentration, this would considerably limit their usefulness. The authors challenge this, saying in their rebuttal that "no, that drug concentration would<br /> be a major actor in the value of the metrics does not limit the utility of the metric. It is simply another variable that one can consider when computing the metrics." While this is true in principle, I don't think any practicing scientist would disagree with the statement that the existence of additional confounding factors (in particular if they are unknown) reduces the usefulness<br /> of a quantitative metric.

      As a consequence of the small number of variant-drug-combinations that are used, the conclusions that the authors draw from their analysis are mostly tentative. For example, on line 123 the authors write that the observation that<br /> the treatment of highest drug applicability is a combination of two drugs "fits intuition". In the Discussion this statement is partly retracted with reference to the piperacillin/tazobactam-combination which has low drug applicability. Being based on only a handful of data points, both observations are essentially anecdotal and it is unclear what the reader is supposed to learn.

      To assess the environment-dependent epistasis among the genetic mutations comprising the variants under study, the authors decompose the data of Mira et al. into epistatic interactions of different orders. This part of the analysis is incomplete in two ways. First, in their study, Mira et al. pointed out that a fairly large fraction of the fitness differences between variants that they measured were not statistically significant. This information has been removed in the depiction of the Mira et al. fitness landscapes in Figure 1 of the present manuscript, and it does not seem to be reflected in the results of the interaction analysis in Figure 4. Second, the interpretation of the coefficients obtained from the epistatic decomposition depends strongly on the formalism that is being used. In a note added on page 15 of the revised manuscript, the authors write that they have used the LASSO regression for their analysis and refer the reader to a previous publication (Guerrero et al. 2019) which however (as far as I could see) also does not fully explain how the method works. To give an example of the difficulty of interpreting the data in Figure 4 without further information: The substitution C (G238S) is well known to have a strong positive effective in cefotaxime, but the corresponding coefficient is essentially zero. So whatever the LASSO regression does, it cannot simply measure the effect on growth.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Gaikwad et al. investigated the role of eIF2A in translational response to stress in yeast. For this purpose, the authors conducted ribosome profiling under SM treatment in an eIF2A-depleted strain. Data analysis revealed that eIF2A did not influence translation from mRNAs bearing uORFs or cellular IRESes, in the stress condition, broadly. The authors found that only a small number of mRNAs were supported by eIF2A. The data should be helpful for researchers in the field.

      Major points:<br /> 1. The weakness of this work is the lack of clarification on the function of eIF2A in general. The novelty of this study was limited.

      2. Related to this, it would be worth investigating common features in mRNAs selectively regulated (surveyed in Figure 3A). Also, it would be worth analyzing the effect of eIF2A deletion on elongation (ribosome occupancy on each codon and/or global ribosome footprint distribution along CDS) and termination/recycling (footprint reads on stop codon and on 3′ UTR).

      3. Regarding Figure 3D, the reporters were designed to include promoter and 5′ UTR of the target genes. Thus, it should be worth noting that reporter design was based on the assumption that eIF2A-dependency in translation regulation was not dependent on 3′ UTR or CDS region. The reason why the effects on ribosome profiling-supported mRNAs could not be recapitulated in reporter assay may originate from this design. This should be also discussed.

      4. Related to the point above, the authors claimed that eIF2A affects "possibly only one" (HKR1) mRNA. However, this was due to the reporter assay which is technically variable and could not allow some of the constructs to pass the authors' threshold. Alternative wording for this point should be considered.

      5. For Figure 3D, it would be worth considering testing the #-marked genes (in Figure 3C) in this set up.

      6. In box plots, the authors should provide the statistical tests, at least where the authors explained in the main text.

    1. Reviewer #2 (Public Review):


      The authors describe the discovery of a filovirus neutralizing antibody, AF03, by phage display, and its subsequent improvements to include NPC2 that resulted in a greater breadth of neutralization. Overall, the manuscript would benefit from considerable grammatical review, which would improve the communication of each point to the reader. The authors do not convincingly map the AF03 epitope, nor do they provide any strong support for their assumption that AF03 targets the NPC1 binding site. However, the authors do show that AF03 competes for MR78 binding to its epitope, and provides good support for the internalization of AF03-NL as the mechanism for improved breadth over the original AF03 antibody.


      This study shows convincing binding to Marburgvirus GP and neutralization of Marburg viruses by AF03, as well as convincing neutralization of Ebolaviruses by AF03-NL. While there are no distinct populations of PE-stained cells shown by FACS in Figure 5A, the cell staining data in Figure 5C are compelling to a non-expert in endosomal staining like me. The control experiments in Figure 7 are compelling showing neutralization by AF03-NL but not AF03 or NPC2 alone or in combination. Altogether these data support the internalisation and stabilisation mechanism that is proposed for the gain in neutralization breadth observed for Ebolaviruses by AF03-NL over AF03 alone.


      Overall, this reviewer is of the opinion that this paper is constructed haphazardly. For instance, the neutralization of mutant pseudoviruses is shown in Figure 2 before the concept of pseudovirus neutralization by AF03 is introduced in Figure 3. Similarly, the control experiments for AF03+NPC2 are described in Figure 7 after the data for breadth of neutralization are shown in Figure 6. GP quality controls are shown in Figure 2 after GP ELISAs / BLI experiments are done in Figure 1. This is disorienting for the reader.

      Figure 1: The visualisation of AF03 modelling and docking endeavours is extremely difficult to interpret. Firstly, there is no effort to orient the non-specialist reader with respect to the Marburgvirus GP model. Secondly, from the figures presented it is impossible to tell if the Fv docks perfectly onto the GP surface, or if there are violent clashes between the deeply penetrating AF03 CDRs and GP. This information would be better presented on a white background, perhaps showing GP in surface view from multiple angles and slices. The authors attempt to label potential interactions, but these are impossible to read, and labels should be added separately to appropriately oriented zoomed-in views.

      Figure 2: The neutralization of mutant pseudoviruses cannot be properly assessed using bar graphs. These data should be plotted as neutralization curves as they were done for the wild-type neutralization data in Figure 3. The authors conclude that Q128 & N129 are contact residues, but the neutralization data for this mutant appear odd as the lowest two concentrations of AF03 show higher neutralization than the second highest AF03 concentration. Neutralization of T204/Q205/T206 (green), Y218 (orange), K222 (blue), or C226 (purple) appears to be better than neutralization of the wild-type MARV. The authors do not discuss this oddity. What are the IC50's? The omission of antibody concentrations on the x-axis and missing IC50 values give a sense of obscuring the data, and the manuscript would benefit from greater transparency, and be much easier to interpret if these were included. I am intrigued that the Q128S/N129S mutant is reported as having little effect on the neutralization of MR78. The bar graph appears to show some effect (difficult to interpret without neutralization curves and IC50 data), and indeed PDB:5UQY seems to suggest that these amino acids form a central component of the MR78 epitope (Q128 forms potential hydrogen bonds with CDRH1 Y35 and CDRL3 Y91, while N129 packs against the MR78 CDRH3 and potentially makes additional polar contact with the backbone). Lastly, since neutralization was tested in both HEK293T cells and Huh7 cells in Figure 3, the authors should clarify which cells were used for neutralization in Figure 2.

      Figure 3: The first two images in Figure 3C showing bioluminescent intensity from pseudovirus-injected mice pretreated with either 10mg/kg or 3mg/kg AF03 are identical images. This is apparent from the location, shape, and intensity of the bioluminescence, as well as the identical foot placement of each mouse in these two panels. Currently, this figure is incomplete and should be corrected to show the different mice treated with either 10mg/kg or 3mg/kg of AF03.

      Figure 4 would benefit from a control experiment without antibodies comparing infection with GP-cleaved and GP-uncleaved pseudoviruses. The paragraph describing these data was also difficult to read and would benefit from additional grammatical review.

      Figure 5: The authors should clarify in the methods section that the "mock" experiment included the PE anti-human IgG Fc antibody. Without this clarification, the lack of a distinct negative population in the FACS data could be interpreted as non-specific staining with PE. If the PE antibody was added at an equivalent concentration to all panels, what does the directionality of the arrowheads in Figure 5A (labelled PE) and 5B (labelled pHrodo Red) indicate?

      Figure 6B: These data would benefit from the inclusion of IC50, transparency of antibody concentrations used, and consistency in the direction of antibody concentrations (increasing to the right or left of the x-axis) when compared to Figure 2.

    1. Reviewer #2 (Public Review):


      Mandal et al. use WASP-deficient T cells to study the role of WASP in T cell signaling and activation and tying WASP to mechanosensing in T cells. Using both CD8 and CD4 T cells from WASP-deficient animals, the authors show defects in T cell signaling and function as well as defects in mechanosensing in activated CD8 T cells.


      Confirming findings from many previous studies, Mandal et al. demonstrate that WASP-deficiency in T cells leads to defective T cell function (Figs 1, 2, 3, and 4). Fig 3 shows direct effects of mechanical stress on CD8 T cell signaling in the absence of WASP.


      The title does not reflect the data presented as the only data demonstrating a role for WASP in mechanosensing in this manuscript doesn't directly connect WASP mechanosensing with tumors (Fig 3). The results shown in Fig 1 using an actin inhibitor doesn't directly connect WASP with mechanosensing. Fig 4 uses WASP-deficient animals in a tumor model, but doesn't demonstrate any role for mechanosensing in the WASP-deficient animals. The title should reflect the lack of data connecting WASP in mechanosensing to a tumor context.

      One major oversight is the absence of discussion of a previous publication demonstrating a direct role of WASP in mechanosensing to the actin cytoskeleton in dendritic cells and naive CD4 and CD8 T cells (Gaertner et al. Dev Cell 2022). There should be a discussion of how the findings in Gaertner et al. shed light on the results from this manuscript.

      The use of Myca to disrupt the actin cytoskeleton as a "modulator of stiffness" is problematic. While one of the potential effects of disrupting the actin cytoskeleton is changing stiffness, as shown in Figure 1, many other functions are simultaneously disturbed also. The use of B16 tumor cells is simply for antigen presentation, and not in a tumor context, so generalized statements about "stiffness" or "softness" and "tumor cells" in reference to Figure 1 should be changed to account for these alternative explanations.

      Fig S2 shows Myca treatment of BMDCs leads to decreased functionality of OTII CD4s. Interpretation in the manuscript claims "This indicates that leaching of Myca from treated cells does not cause inhibition of bystander cells". This would not be my interpretation of the data. An alternative interpretation is that if Myca is remaining in the media, then effects on APCS (either BMDCs or B16s) could lead to decreased CD4 or CD8 T cell activation and thus be responsible for effects seen in Fig 1. This possibility should be considered.

      Fig 4 claims that high rigidity leads to downstream effects of WASP-/- T cell function. But there is no demonstration of the role of mechanosensing in Figure 4. To make this claim, the authors would need to compare high and low rigidity conditions.

      Fig 4 also shows that WASP-/- showed higher tumor growth in an implanted tumor model. For 4F, since WASP is deficient in all hematopoietic cells, the finding in 4G may not be due to T cells. In 4H-J, because implantation of tumors occurs within 1 day of lymphodepletion and assessing tumor growth prior to reconstitution of the hematopoietic compartment, there should be control experiments shown to demonstrate that other hematopoietic cell types that remain are not function and thus do not participate in the differences seen in tumor growth. Also, statistical tests need to be done to show the significance of the differences between groups in Fig 4I and 4J (also 4G).

    1. Reviewer #2 (Public Review):


      The 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.


      Straight-forward yet elegant design to help determine the important role the Numb has in skeletal muscle.


      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):


      In this manuscript, the authors developed an open-top two-photon light sheet microscopy (OT-TP-LSM) that enables high-throughput and high-depth investigation of 3D cell structures. The data presented here shows that OT-T-LSM could be a complementary technique to traditional imaging workflows of human cancer cells.


      High-speed and high-depth imaging of human cells in an open-top configuration is the main strength of the presented study. An extended depth of field of 180 µm in 0.9 µm thickness was achieved together with an acquisition of 0.24 mm2/s. This was confirmed by 3D visualization of human cancer cells in the skin, pancreas, and prostate.


      The complementary aspect of the presented technique in human pathological samples is not convincingly presented. The traditional hematoxylin and eosin (H&E) staining is a well-established and widely used technique to detect human cancer cells. What would be the benefit of 3D cell visualization in an OT-TP-LSM microscope for cancer detection in addition to H&E staining?

    1. Reviewer #2 (Public Review):

      The authors provide convincing evidence that optogenetic stimulation of ChR2-expressing motor neurons implanted in muscles effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1 mouse model of ALS, and concluded that this method can be applied to selectively control the function of implicated muscles, which was supported by convincing data presented in the paper.

    1. Reviewer #2 (Public Review):


      The authors use single-cell "multi-comics" to study clonal heterogeneity in chronic myeloid leukemia (CML) and its impact on treatment response and resistance. Their main results suggest 1) Cell compartments and gene expression signatures both shared in CML cells (versus normal), yet 2) some heterogeneity of multiomic mapping correlated with ELN treatment response; 3) further definition of s unique combination of CD26 and CD35 surface markers associated with gene expression defined BCR::ABL1+ LSCs and BCR::ABL1- HSCs. The manuscript is well-written, and the method and figures are clear and informative. The results fit the expanding view of cancer and its therapy as a complex Darwinian exercise of clonal heterogeneity and the selective pressures of treatments.


      Cutting-edge technology by one of the expert groups of single-cell 'comics.


      Very small sample sizes, without a validation set.<br /> The obvious main problem with the study is that an enormous amount of results and conjecture arise from a very small data set: only nine cases for the treatment response section (three in each of the ELN categories), only two normal marrows, and only two patient cases for the division kinetic studies. Thus, it is very difficult to know the "noise" in the system - the stability of clusters and gene expression and the normal variation one might expect, versus patterns that may be reproducibly study artifact, effects of gene expression from freezing-thawing, time on the bench, antibody labeling, etc. This is not so much a criticism as a statement of reality: these elegant experiments are difficult, time-consuming, and very expensive. Thus in the Discussion, it would be helpful for the authors to just frankly lay out these limitations for the reader to consider. Also in the Discussion, it would be interesting for the authors to consider what's next: what type of validation would be needed to make these studies translatable to the clinic? Is there a clever way to use these data to design a faster/cheaper assay?

    1. Reviewer #2 (Public Review):


      This manuscript provides important new findings regarding the connection between inflammation and metabolism. It also identifies a new type of post-translational modification and its connection to protein stability. This finding is expected to be generalizable to other protein targets. In vitro evidence is solid. In vivo evidence needs some additional controls.


      A new connection between inflammation and metabolism.

      A novel type of PTM was identified.

      Findings would be of broad interest and the mechanisms are likely generalizable to related control systems.

      In vitro data are well-supported.

      The authors successfully demonstrated that treatment with 4-octyl Itaconate (4-OI), a prodrug form of itaconate, reduces neutral lipid accumulation in the AML12 cell line and primary hepatocytes. They show that 4-OI promotes fatty acid beta-oxidation through increased stability of CPT1a protein, the rate-limiting step in this process.


      Some conclusions involving the Irg1 knockout mice require important controls and clarifications to be fully convincing and some controls are missing.

    1. Reviewer #2 (Public Review):


      The goal of untargeted metabolomics is to identify differences between metabolomes of different biological samples. Untargeted metabolomics identifies features with specific mass-to-charge ratio (m/z) and retention time (RT). Matching those to specific metabolites based on the model compounds from databases is laborious and not always possible, which is why methods for comparing samples on the level of unmatched features are crucial.

      The main purpose of the GromovMatcher method presented here is to merge and compare untargeted metabolomes from different experiments. These larger datasets could then be used to advance biological analyses, for example, for the identification of metabolic disease markers. The main problem that complicates merging different experiments is m/z and RT vary slightly for the same feature (metabolite).

      The main idea behind the GromovMatcher is built on the assumption that if two features match between two datasets (that feature i from dataset 1 matches feature j from dataset 2, and feature k from dataset 1 matches feature l from dataset 2), then the correlations or distances between the two features within each of the datasets (i and k, and j and l) will be similar. The authors then use the Gromov-Wasserstein method to find the best matches matrix from these data.

      The variation in m/z between the same features in different experiments is a user-defined value and it is initially set to 0.01 ppm. There is no clear limit for RT deviations, so the method estimates a non-linear deviation (drift) of RT between two studies. GromovMatcher estimates the drift between the two studies and then discards the matching pairs where the drift would deviate significantly from the estimate. It learns the drift from a weighted spline regression.

      The authors validate the performance of their GromovMatcher method by a validation experiment using a dataset of cord blood. They use 20 different splits and compare the GromovMatcher (both its GM and GMT iterations, whereby the GMT version uses the deviation from estimated RT drift to filter the matching matrix) with two other matching methods: M2S and metabCombiner.

      The second validation was done using a (scaled and centered) dataset of metabolics from cancer datasets from the EPIC cohort that was manually matched by an expert. This dataset was also used to show that using automatic methods can identify more features that are associated with a particular group of samples than what was found by manual matching. Specifically, the authors identify additional features connected to alcohol consumption.


      I see the main strength of this work in its combination of all levels of information (m/z, RT, and higher-order information on correlations between features) and using each of the types of information in a way that is appropriate for the measure. The most innovative aspect is using the Gromov-Wasserstein method to match the features based on distance matrices.

      The authors of the paper identify two main shortcomings with previously established methods that attempt to match features from different experiments: a) all other methods require fine-tuning of user-defined parameters, and, more importantly, b) do not consider correlations between features. The main strength of the GromovMatcher is that it incorporates the information on distances between the features (in addition to also using m/z and RT).


      The first, minor, weakness I could identify is that there seem not to be plenty of manually curated datasets that could be used for validation. The second is also emphasized by the authors in the discussion. Namely, the method as it is set up now can be directly used only to compare two datasets.

    1. Reviewer #2 (Public Review):


      The authors of this study have sought to better understand the timing and location of the attachment of the lpp lipoprotein to the peptidoglycan in E. coli, and to determine whether YafK is the hydrolase that cleaves lpp from the peptidoglycan.


      The method is relatively straightforward. The authors are able to draw some clear conclusions from their results, that lpp molecules get cleaved from the peptidoglycan and then re-attached, and that YafK is important for that cleavage.


      However, the authors make a few other conclusions from their data which are harder to understand the logic of, or to feel confident in based on the existing data. They claim that their 5-time point kinetic data indicates that new lpp is not substantially added to lipidII before it is added to the peptidoglycan, and that instead lpp is attached primarily to old peptidoglycan. I believe that this conclusion comes from the comparison of Fig.s 3A and 3C, where it appears that new lpp is added to old peptidoglycan a few minutes before new lpp is added to new peptidoglycan. However, the very small difference in the timing of this result, the minimal number of time points and the complete lack of any presentation of calculated error in any of the data make this conclusion very tenuous. In addition, the authors conclude that lpp is not significantly attached to septal peptidoglycan. The logic behind this conclusion appears to be based on the same data, but the authors do not provide a quantitative model to support this idea.

      This work will have a moderate impact on the field of research in which the connections between the OM and peptidoglycan are being studied in E. coli. Since lpp is not widely conserved in gram negatives, the impact across species is not clear. The authors do not discuss the impact of their work in depth.

    1. Reviewer #2 (Public Review):

      Summary: CD8+ QFL T cells recognize a peptide, FYAEATPML (FL9), presented on Erap1-deficient cells. QFL T cells are present at a high frequency in the spleen of naïve mice. They express an antigen-experienced phenotype, and about 80% express an invariant TCRα chain Vα3.2Jα21.

      Here, Guan and coll. report that QFL T cells are present not only in the spleen but also in the intestinal epithelium, where they display several phenotypic and functional peculiarities. The establishment of spleen and gut Vα3.2+ QFL T cells is TAP-dependent, and their phenotype is regulated by the presence/absence of Qa-1b and Erap1. Maintenance of gut Vα3.2+ QFL T cells depends on the gut microbiota and is associated with colonization by Pediococcus pentosaceus.


      This article contains in-depth studies of a peculiar and interesting subset of unconventional CD8 T cells, based partly on generating two novel TCR-transgenic models.

      The authors discovered a clear relation between the gut microbiome and the maintenance of gut QFL T cells. One notable observation is that monocolonization of the gut with Pediococcus pentosaceus is sufficient to sustain gut QFL T cells.


      In the absence of immunopeptidomic analyses, the presence or absence of the FL9 peptide on various cell types is inferred based on indirect evidence. Hence, whether the FL9 peptide is presented by some cells that express Qa-1b but not Erap1 remains unknown.

      Analyses of the homology between the FL9 and bacterial peptides were limited to two amino acid residues (P4 and P6). This limitation is mitigated in part by the justifications provided by the authors in the revised preprint.

      The potential function of QFL T cells remains elusive. The present article should provide an incentive for further functional studies.

    1. Reviewer #2 (Public Review):


      The paper examines the role L-cysteine metabolism plays in the biology of Mycobacterium tuberculosis. The authors have preliminary data showing that Mycobacterium tuberculosis has two unique pathways to synthesize cysteine. The data showing new compounds that act synergistically with INH is very interesting.


      RNAseq data is interesting and important.


      The paper would be strengthened if the authors were to add further detail to their genetic manipulations.

      The authors provide evidence that they have successfully made a cysK2 mutant by recombineering. This data looks promising, but I do not see evidence for the cysM deletion. It is also important to state what sort of complementation was done (multicopy plasmid, integration proficient vector, or repair of the deletion). Since these mutants are the basis for most of the additional studies, these details are essential. It is important to include complementation in mouse studies as unexpected loss of PDIM could have occurred.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this paper, the authors have obtained an analytical expression that provides intuition about regimes of interfacial resistance that depend on droplet size. Additionally, through simulations, the authors provide microscopic insight into the arrangement of sticky and non-sticky functional groups at the interface. The authors introduce bouncing dynamics for rationalizing quantity recovery timescales.

      I found several sections that felt incomplete or needed revision and additional data to support the central claim and make the paper self-contained and coherent.

      First, the analytical theory operates with diffusion coefficients for dilute and dense phases. For the dilute phase, this is fine. For the dense phase, I have doubts that dynamics can be described as diffusive. Most likely, dynamics is highly subdiffusive due to crowded, entangled, and viscoelastic environments of densely packed interactive biomolecules. Some explanation and justification are in order here.

      The second major issue is that I did not find a clean comparison of simulations with the derived analytical expression. Simulations test various microscopic properties on the value of k, which is important. But how do we know that it is the same quantity that appears in the expressions? Also, how can we be sure that analytical expressions can guide simulations and experiments as claimed? The authors should provide sound evidence of the predictive aspect of their derived expressions.

      Are the plots in Figure 4 coming from experiment, theory, and simulation? I could not find any information either in the text or in the caption.

    1. Reviewer #2 (Public Review):

      Summary and Major Findings/Strengths:

      Across diverse hosts, microbiota can influence viral infection and transmission. C. elegans is naturally infected by the Orsay virus, which infects intestinal cells and is transmitted via the fecal-oral route. Previous work has demonstrated that host immune defense pathways, such as antiviral RNAi and the intracellular pathogen response (IPR), can influence host susceptibility to virus infection. However, little is known about how bacteria modulate viral transmission and host susceptibility.

      In this study, the authors investigate how diverse bacterial species influence Orsay virus transmission and host susceptibility in C. elegans. When C. elegans is grown in the presence of two Ochrobactrum species, the authors find that animals exhibit increased viral transmission, as measured by the increased proportion of newly infected worms (relative to growth on E. coli OP50). The presence of the two Ochrobactrum species also resulted in increased host susceptibility to the virus, which is reflected by the increased fraction of infected animals following exposure to the exogenous Orsay virus. In contrast, the presence of Pseudomonas lurida MYb11, as well as Pseudomonas PA01 or PA14, attenuates viral transmission and host susceptibility relative to E. coli OP50. For growth in the presence of P. aeruginosa PA01 and PA14, the attenuated transmission and susceptibility are suppressed by mutations in regulators of quorum sensing and the gacA two-component system. The authors also identify six virulence genes in P. aeruginosa PA14 that modulate host susceptibility to virus and viral transmission, albeit to a lesser extent. Based on the findings in P. aeruginosa, the authors further demonstrate that deletion of the gacA ortholog in P. lurida results in loss of the attenuation of viral transmission and host susceptibility.

      Taken together, these findings provide important insights into the species-specific effects that bacteria can have on viral infection in C. elegans. The authors also describe a role for Pseudomonas quorum sensing and virulence genes in influencing viral transmission and host susceptibility.

      Major weaknesses:

      The manuscript has several issues that need to be addressed, such as insufficient rigor of the experiments performed and questions about the reproducibility of the data presented in some places. In addition, confounding variables complicate the interpretations that can be made from the authors' findings and weaken some of the conclusions that are stated in the manuscript.

      1. The authors sometimes use pals-5p::GFP expression to indicate infection, however, this is not necessarily an accurate measure of the infection rate. Specifically, in Figures 4-6, the authors should include measurements of viral RNA, either by FISH staining or qRT-PCR, to support the claims related to differences in infection rate.

      2. In several instances, the experimental setup and presentation of data lack sufficient rigor. For example, Fig 1D and Fig 2B only display data from one experimental replicate. The authors should include information from all 3 experimental replicates for more transparency. In Fig 3B, the authors should include a control that demonstrates how RNA1 levels change in the presence of E. coli OP50 for comparison with the results showing replication in the presence of PA14. In order to support the claim that "P. aeruginosa and P. lurida MYb11 do not eliminate Orsay virus infection", the authors should also measure RNA1 fold change in the presence of PA01 and P. lurida in the context of exogenous Orsay virus. Additionally, the authors should standardize the amount of bacteria added to the plate and specify how this was done in the Methods, as differing concentrations of bacteria could be the reason for species-specific effects on infection.

      3. The authors should be more careful about conclusions that are made from experiments involving PA14, which is a P. aeruginosa strain (isolated from humans), that can rapidly kill C. elegans. To eliminate confounding factors that are introduced by the pathogenicity of PA14, the authors should address how PA14 affects the health of the worms in their assays. For example, the authors should perform bead-feeding assays to demonstrate that feeding rates are unaffected when worms are grown in the presence of PA14. Because Orsay virus infection occurs through feeding, a decrease in C. elegans feeding rates can influence the outcome of viral infection. The authors should also address whether or not the presence of PA14 affects the stability of viral particles because that could be another trivial reason for the attenuation of viral infection that occurs in the presence of PA14.

    1. Ashby's law of requisite variety may also be at play for overloading our system 1 heuristic abilities with respect to misinformation (particularly in high velocity social media settings). Switching context from system 1 to system 2 on a constant basis to fact check everything in our (new digital) immediate environment can be very mentally and emotionally taxing. This can result in both mental exhaustion as well as anxiety.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This work analyses the historical spread and evolution, termed 'population dynamics', of a human bacterial pathogen, Neisseria gonorrhoeae, the cause of the sexually transmitted infection, gonorrhoea. N. gonorrhoeae is classified as a high priority pathogen by the World Health Organisation, due to infections numbering in the tens of millions annually, with high levels of antibiotic resistance and no vaccine available, meaning treating and preventing infections is becoming increasingly more difficult. To implement interventions effectively, important resistant lineages and their transmission routes must be identified on a national and international level.

      In this work, Osnes et al. use genomic data, coupled with geographic, temporal and demographic metadata, to analyse the global population dynamics N. gonorrhoeae using 9,732 genomes. The study also includes a granular analysis of transmission between and within four regions of different sizes with high levels of data coverage: USA, Europe, Norway, and Victoria state in Australia.<br /> The authors built a phylogenetic tree including all genomes using a novel computationally efficient method for removing genome regions resulting from recombination, which would otherwise result in incorrect branch lengths and tree topology. Using the tree, the authors show that the effective population size of N. gonorrhoeae, describing population size and diversity, decreased in the period from 2010 to present day, and was not entirely an artefact of sampling bias. The authors then stratified the tree based on isolates that contained alleles that are associated with resistance to antibiotics commonly used to treat gonorrhoea. The authors found resistance was associated with particular lineages, of which most, but not all, underwent shrinking in effective population size in the last decade.<br /> Using the tree, the authors then inferred likely importation, exportation, and local transmission events, finding notable differences in the contribution of imports to local incidence between locations, as well as the likelihood of exportation. As inference of these events relies on sampling density, the authors used a novel method for identifying whether sampling was representative of the population diversity of a given location. Using this approach, they found that the densely sampled regions, Norway and Victoria, were likely representative of the local N. gonorrhoeae population diversity, whilst the larger, less densely sampled regions, Europe and USA, were not. Finally, they investigated the contribution of specific transmission networks to the spread gonorrhoea, finding that the frequency of males within a transmission network may play a role in the rate of N. gonorrhoeae transmission in Norway, but not Victoria.<br /> This work introduces several novel approaches to the analysis of pathogen population dynamics, and highlights notable differences in N. gonorrhoeae transmission between and within distinct geographic locations.

      Strengths:<br /> • The authors have collated a large global collection of N. gonorrhoeae genomes with associated metadata, and in some cases generated assemblies themselves. A dataset of this size and detail is a valuable asset to the public health community, enabling analysis of both national and international population dynamics.<br /> • The stratification of the phylogenetic tree by antimicrobial resistance gene alleles enables the study of how antibiotic usage has shaped global and regional N. gonorrhoeae populations. Analysis of changes in the effective population size of clades harbouring resistance alleles is particularly impactful, as this can be used to show how changes in treatment patterns affect the growth or decline of drug-resistant pathogen populations. This analysis also enables the determination of the frequency of multiple resistance alleles being present in single isolates, important for determining the scale of multidrug resistance within the N. gonorrhoeae global population.<br /> • The use of ancestral trait reconstruction to quantify importation, exportation and local transmission is an important contribution to public health efforts tackling N. gonorrhoeae spread. Understanding the differences in transmission networks within and between different geographic locations provides public health researchers with crucial information to model and implement effective targeted interventions on regional and international scales.

      Weaknesses:<br /> • The method used to generate the phylogenetic tree and mask regions of recombination is likely flawed. The authors repeatedly down-sampled the whole population to 500 genomes, using Gubbins to identify regions that have recombined and therefore would not follow the clonal history of the N. gonorrhoeae population. This small sample size will result in the same ancient internal nodes being sampled repeatedly, whilst more recent internal nodes will not. Therefore, more recent recombination events would not be identified by this method and were therefore likely included in the whole genome alignment used to build the tree. Furthermore, Gubbins was designed to identify recombination between closely related genomes, not across a whole species, where the background mutation rate will be too high to differentiate between recombined regions and the clonal frame. Both of these factors will mean that the amount of the genome predicted to have recombined will likely be underestimated, resulting in inflated branch lengths and incorrect tree topology. This effect is potentially the cause of the observed drop in N. gonorrhoeae effective population size between 2010-present day in Figure 2, which does not align with gonorrhoea incidence, and the elevated estimated mutation rate of 7.41x10-6 substitutions per site per year, which is higher than previous estimates based on N. gonorrhoeae global populations. The result of underestimation of recombined regions will be two-fold. Inclusion of recombined regions in the alignment will result in inflated branch lengths, which will impact all estimates of effective population size in the study. Furthermore, tree topology may be incorrect, which will impact ancestral trait reconstruction and result in incorrect inference of import, export and local transmission events in Figures 3, 4 and 5. Additionally, the clade-specific resistance gene analyses will be affected in Figure 2, as certain isolates may be incorrectly included or excluded within stratified clades. Therefore, the conclusions made about the changes in effective population size for the global population, and individual clades, as well as the differences in transmission dynamics between locations, are likely to be incorrect.<br /> • The method used to identify sampling bias, shown in Figure 4, is a novel and interesting take on the problem. However, it is not clear whether the effect being measured is the presence of sampling bias or an artefact of differences in N. gonorrhoeae diversity between locations. The results in Figure 4 do align with what is known about the population datasets; the data from Norway and Victoria is more comprehensive than that of the USA and Europe due to the difference in size of the respective human populations, meaning the likelihood of sampling bias will be lower in the smaller population. However, with increased human population size, we would also expect a greater amount of pathogen diversity, due to increased within-region transmission and greater numbers of importation events. Supporting this, we see in Figure 3 that the transmission lineages in the USA and Europe are estimated to have emerged earlier than Norway or Victoria, indicative of a greater amount of standing population diversity. Therefore, the reason why convergence is observed when up-sampling from smaller populations may be because a vast majority of isolates will sit within a small part of the tree, whilst from a larger, more diverse population, isolates will be placed all across the tree and so convergence will never be observed. In effect, it is unknown whether increasing the sample size of the USA and Europe to be truly representative of their respective N. gonorrhoeae populations would ever result in convergence between the two methods of up-sampling. Testing this method using simulations could be used to determine whether it is sensitive to sampling bias, or population diversity.<br /> • In Figure 5, a significant difference in transmission lineage size was only found between male-dominated and mixed lineages in Norway and not Victoria. Therefore, the conclusion that sex distribution within transmission networks affects the size of transmission lineages is not supported by the data, and could also be due to geographical and other demographic differences between the datasets which were not accounted for.

    1. Reviewer #2 (Public Review):

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

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

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

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

      Some additional, specific comments are as follows:

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

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

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

    1. Reviewer #2 (Public Review):

      Li et al present a method to extract "behaviorally relevant" signals from neural activity. The method is meant to solve a problem which likely has high utility for neuroscience researchers. There are numerous existing methods to achieve this goal some of which the authors compare their method to, though there are notable omissions. However, I do believe that d-VAE is a promising approach that has its own advantages.

      That being said, there are issues with the paper as-is. This could have been a straightforward "methods" paper describing their approach and validating it on different ground truth and experimental datasets. Instead, the authors focus on the neuroscientific results and their implications for brain mechanisms. Unfortunately, while the underlying method seems sound and performs well relative to the assessed competition, the scientific results and presentation they put forward were not sufficiently strong to support these claims, especially given the small amount of data (recordings of one monkey per task, with considerable variability between them).

      Specific comments<br /> - Is the apparently increased complexity of encoding vs decoding so unexpected given the entropy, sparseness, and high dimensionality of neural signals (the "encoding") compared to the smoothness and low dimensionality of typical behavioural signals (the "decoding") recorded in neuroscience experiments? This is the title of the paper so it seems to be the main result on which the authors expect readers to focus.

      - I take issue with the premise that signals in the brain are "irrelevant" simply because they do not correlate with a fixed temporal lag with a particular behavioural feature hand-chosen by the experimenter. As an example, the presence of a reward signal in motor cortex [1] after the movement is likely to be of little use from the perspective of predicting kinematics from time-bin to time-bin using a fixed model across trials (the apparent definition of "relevant" for behaviour here), but an entire sub-field of neuroscience is dedicated to understanding the impact of these reward-related signals on future behaviour. Is there method sophisticated enough to see the behavioural "relevance" of this brief, transient, post-movement signal? This may just be an issue of semantics, and perhaps I read too much into the choice of words here. Perhaps the authors truly treat "irrelevant" and "without a fixed temporal correlation" as synonymous phrases and the issue is easily resolved with a clarifying parenthetical the first time the word "irrelevant" is used. But I remain troubled by some claims in the paper which lead me to believe that they read more deeply into the "irrelevancy" of these components.

      - The authors claim the "irrelevant" responses underpin an unprecedented neuronal redundancy and reveal that movement behaviors are distributed in a higher-dimensional neural space than previously thought." Perhaps I just missed the logic, but I fail to see the evidence for this. The neural space is a fixed dimensionality based on the number of neurons. A more sparse and nonlinear distribution across this set of neurons may mean that linear methods such as PCA are not effective ways to approximate the dimensionality. But ultimately the behaviourally relevant signals seem quite low-dimensional in this paper even if they show some nonlinearity may help.

      - Relatedly, I would like to note that the exercise of arbitrarily dividing a continuous distribution of a statistic (the "R2") based on an arbitrary threshold is a conceptually flawed exercise. The authors read too much into the fact that neurons which have a low R2 w.r.t. PDs have behavioural information w.r.t. other methods. To this reviewer, it speaks more about the irrelevance, so to speak, of the preferred direction metric than anything fundamental about the brain.

      - there is an apparent logical fallacy that begins in the abstract and persists in the paper: "Surprisingly, when incorporating often-ignored neural dimensions, behavioral information can be decoded linearly as accurately as nonlinear decoding, suggesting linear readout is performed in motor cortex." Don't get me wrong: the equivalency of linear and nonlinear decoding approaches on this dataset is interesting, and useful for neuroscientists in a practical sense. However, the paper expends much effort trying to make fundamental scientific claims that do not feel very strongly supported. This reviewer fails to see what we can learn about a set of neurons in the brain which are presumed to "read out" from motor cortex. These neurons will not have access to the data analyzed here. That a linear model can be conceived by an experimenter does not imply that the brain must use a linear model. The claim may be true, and it may well be that a linear readout is implemented in the brain. Other work [2,3] has shown that linear readouts of nonlinear neural activity patterns can explain some behavioural features. The claim in this paper, however, is not given enough

      - I am afraid I may be missing something, as I did not understand the fano factor analysis of Figure 3. In a sense the behaviourally relevant signals must have lower FF given they are in effect tied to the temporally smooth (and consistent on average across trials) behavioural covariates. The point of the original Churchland paper was to show that producing a behaviour squelches the variance; naturally these must appear in the behaviourally relevant components. A control distribution or reference of some type would possibly help here.

      - The authors compare the method to LFADS. While this is a reasonable benchmark as a prominent method in the field, LFADS does not attempt to solve the same problem as d-VAE. A better and much more fair comparison would be TNDM [4], an extension of LFADS which is designed to identify behaviourally relevant dimensions.

      [1] https://doi.org/10.1371/journal.pone.0160851<br /> [2] https://doi.org/10.1101/2022.03.31.486635<br /> [3] https://doi.org/10.1038/s41593-017-0028-6<br /> [4] Hurwitz et al, Targeted Neural Dynamical Modeling, NeurIPS 2021.

    1. Reviewer #2 (Public Review):

      In this manuscript, the authors propose a computational method based on deep convolutional neural networks (CNNs) to automatically detect cell divisions in two-dimensional fluorescence microscopy timelapse images. Three deep learning models are proposed to detect the timing of division, predict the division axis, and enhance cell boundary images to segment cells before and after division. Using this computational pipeline, the authors analyze the dynamics of cell divisions in the epithelium of the Drosophila pupal wing and find that a wound first induces a reduction in the frequency of division followed by a synchronised burst of cell divisions about 100 minutes after its induction.

      In general, novelty over previous work does not seem particularly important. From a methodological point of view, the models are based on generic architectures of convolutional neural networks, with minimal changes, and on ideas already explored in general. The authors seem to have missed much (most?) of the literature on the specific topic of detecting mitotic events in 2D timelapse images, which has been published in more specialized journals or Proceedings. (TPMAI, CCVPR etc., see references below). Even though the image modality or biological structure may be different (non-fluorescent images sometimes), I don't believe it makes a big difference. How the authors' approach compares to this previously published work is not discussed, which prevents me from objectively assessing the true contribution of this article from a methodological perspective.

      On the contrary, some competing works have proposed methods based on newer - and generally more efficient - architectures specifically designed to model temporal sequences (Phan 2018, Kitrungrotsakul 2019, 2021, Mao 2019, Shi 2020). These natural candidates (recurrent networks, long-short-term memory (LSTM), gated recurrent units (GRU), or even more recently transformers), coupled to CNNs are not even mentioned in the manuscript, although they have proved their generic superiority for inference tasks involving time series (Major point 2). Even though the original idea/trick of exploiting the different channels of RGB images to address the temporal aspect might seem smart in the first place - as it reduces the task of changing/testing a new architecture to a minimum - I guess that CNNs trained this way may not generalize very well to videos where the temporal resolution is changed slightly (Major point 1). This could be quite problematic as each new dataset acquired with a different temporal resolution or temperature may require manual relabeling and retraining of the network. In this perspective, recent alternatives (Phan 2018, Gilad 2019) have proposed unsupervised approaches, which could largely reduce the need for manual labeling of datasets.

      Regarding the other convolutional neural networks described in the manuscript:

      1) the one proposed to predict the orientation of mitosis performs a regression task, predicting a probability for the division angle. The architecture, which must be different from a simple Unet, is not detailed anywhere, so the way it was designed is difficult to assess. It is unclear if it also performs mitosis detection, or if it is instead used to infer orientation once the timing and location of the division have been inferred by the previous network.

      2) the one proposed to improve the quality of cell boundary images before segmentation is nothing new, it has now become a classic step in segmentation, see for example Wolny et al. eLife 2020.

      As a side note, I found it a bit frustrating to realise that all the analysis was done in 2D while the original images are 3D z-stacks, so a lot of the 3D information had to be compressed and has not been used. A novelty, in my opinion, could have resided in the generalisation to 3D of the deep-learning approaches previously proposed in that context, which are exclusively 2D, in particular, to predict the orientation of the division.

      Concerning the biological application of the proposed methods, I found the results interesting, showing the potential of such a method to automatise mitosis quantification for a particular biological question of interest, here wound healing. However, the deep learning methods/applications that are put forward as the central point of the manuscript are not particularly original.

      Major point 1: generalisation potential of the proposed method.

      The neural network model proposed for mitosis detection relies on a 2D convolutional neural network (CNN), more specifically on the Unet architecture, which has become widespread for the analysis of biology and medical images. The strategy proposed here exploits the fact that the input of such an architecture is natively composed of several channels (originally 3 to handle the 3 RGB channels, which is actually a holdover from computer vision, since most medical/biological images are gray images with a single channel), to directly feed the network with 3 successive images of a timelapse at a time. This idea is, in itself, interesting because no modification of the original architecture had to be carried out. The latest 10-channel model (U-NetCellDivision10), which includes more channels for better performance, required minimal modification to the original U-Net architecture but also simultaneous imaging of cadherin in addition to histone markers, which may not be a generic solution.

      Since CNN-based methods accept only fixed-size vectors (fixed image size and fixed channel number) as input (and output), the length or time resolution of the extracted sequences should not vary from one experience to another. As such, the method proposed here may lack generalization capabilities, as it would have to be retrained for each experiment with a slightly different temporal resolution. The paper should have compared results with slightly different temporal resolutions to assess its inference robustness toward fluctuations in division speed.

      Another approach (not discussed) consists in directly convolving several temporal frames using a 3D CNN (2D+time) instead of a 2D, in order to detect a temporal event. Such an idea shares some similarities with the proposed approach, although in this previous work (Ji et al. TPAMI 2012 and for split detection Nie et al. CCVPR 2016) convolution is performed spatio-temporally, which may present advantages. How does the authors' method compare to such an (also very simple) approach?

      Major point 2: innovatory nature of the proposed method.

      The authors' idea of exploiting existing channels in the input vector to feed successive frames is interesting, but the natural choice in deep learning for manipulating time series is to use recurrent networks or their newer and more stable variants (LSTM, GRU, attention networks, or transformers). Several papers exploiting such approaches have been proposed for the mitotic division detection task, but they are not mentioned or discussed in this manuscript: Phan et al. 2018, Mao et al. 2019, Kitrungrotaskul et al. 2019, She et al 2020.

      An obvious advantage of an LSTM architecture combined with CNN is that it is able to address variable length inputs, therefore time sequences of different lengths, whereas a CNN alone can only be fed with an input of fixed size.

      Another advantage of some of these approaches is that they rely on unsupervised learning, which can avoid the tedious relabeling of data (Phan et al. 2018, Gilad et al. 2019).

      References :<br /> Ji, S., Xu, W., Yang, M., & Yu, K. (2012). 3D convolutional neural networks for human action recognition. IEEE transactions on pattern analysis and machine intelligence, 35(1), 221-231. >6000 citations

      Nie, W. Z., Li, W. H., Liu, A. A., Hao, T., & Su, Y. T. (2016). 3D convolutional networks-based mitotic event detection in time-lapse phase contrast microscopy image sequences of stem cell populations. In Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition Workshops (pp. 55-62).

      Phan, H. T. H., Kumar, A., Feng, D., Fulham, M., & Kim, J. (2018). Unsupervised two-path neural network for cell event detection and classification using spatiotemporal patterns. IEEE Transactions on Medical Imaging, 38(6), 1477-1487.

      Gilad, T., Reyes, J., Chen, J. Y., Lahav, G., & Riklin Raviv, T. (2019). Fully unsupervised symmetry-based mitosis detection in time-lapse cell microscopy. Bioinformatics, 35(15), 2644-2653.

      Mao, Y., Han, L., & Yin, Z. (2019). Cell mitosis event analysis in phase contrast microscopy images using deep learning. Medical image analysis, 57, 32-43.

      Kitrungrotsakul, T., Han, X. H., Iwamoto, Y., Takemoto, S., Yokota, H., Ipponjima, S., ... & Chen, Y. W. (2019). A cascade of 2.5 D CNN and bidirectional CLSTM network for mitotic cell detection in 4D microscopy image. IEEE/ACM transactions on computational biology and bioinformatics, 18(2), 396-404.

      Shi, J., Xin, Y., Xu, B., Lu, M., & Cong, J. (2020, November). A Deep Framework for Cell Mitosis Detection in Microscopy Images. In 2020 16th International Conference on Computational Intelligence and Security (CIS) (pp. 100-103). IEEE.

      Wolny, A., Cerrone, L., Vijayan, A., Tofanelli, R., Barro, A. V., Louveaux, M., ... & Kreshuk, A. (2020). Accurate and versatile 3D segmentation of plant tissues at cellular resolution. Elife, 9, e57613.

    1. Reviewer #2 (Public Review):

      This manuscript by Port and colleagues describes rigorous experiments that provide a wealth of virologic, respiratory physiology, and particle aerodynamic data pertaining to aerosol transmission of SARS-CoV-2 between infected Syrian hamsters. The data is particularly significant because infection is compared between alpha and delta variants, and because viral load is assessed via numerous assays (gRNA, sgRNA, TCID) and in tissues as well as the ambient environment of the cage. The paper will be of interest to a broad range of scientists including infectious diseases physicians, virologists, immunologists and potentially epidemiologists. The strength of evidence is relatively high but limited by unclear presentation in certain parts of the paper.

      Important conclusions are that infectious virus is only detectable in air samples during a narrow window of time relative to tissue samples, that airway constriction increases dynamically over time during infection limiting production of fine aerosol droplets, that variants do not appear to exclude one another during simultaneous exposures and that exposures to virus via the aerosol route lead to lower viral loads relative to direct inoculation suggesting an exposure dose response relationship.

      While the paper is valuable, I found certain elements of the data presentation to be unclear and overly complex.

    1. Reviewer #2 (Public Review):

      Tian et al. performed 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.

      -- I understand better the inclusion/exclusion logic for the samples. But I'm still not sure about the fragments. As the authors wrote, there is both noise and stochasticity; the former is not important but the latter is essential to include. How can these two be differentiated, and what may be the expected overlap as a function of different stochasticity rates?

      -- Many of 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/

      -- 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.

      The key missing dataset is ORC1 and ORC2 CHiP-seq from the same cell type. This shouldn't be too expensive to perform, and I hope someone performs this test soon. Without this, I remain on the fence about how much existing datasets are "junk" vs how much the prevailing hypothesis about replication needs to be revisited. Nonetheless, the authors do perform a nice analysis showing that existing techniques should be carefully used and interpreted.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this study, Mu Qiao employs a bilinear modeling approach, commonly utilized in recommendation systems, to explore the intricate neural connections between different pre- and post-synaptic neuronal types. This approach involves projecting single-cell transcriptomic datasets of pre- and post-synaptic neuronal types into a latent space through transformation matrices. Subsequently, the cross-correlation between these projected latent spaces is employed to estimate neuronal connectivity. To facilitate the model training, connectomic data is used to estimate the ground-truth connectivity map. This work introduces a promising model for the exploration of neuronal connectivity and its associated molecular determinants. However, it is important to note that the current model has only been tested with Bipolar Cell and Retinal Ganglion Cell data, and its applicability in more general neuronal connectivity scenarios remains to be demonstrated.

      Strengths:<br /> This study introduces a succinct yet promising computational model for investigating connections between neuronal types. The model, while straightforward, effectively integrates single-cell transcriptomic and connectomic data to produce a reasonably accurate connectivity map, particularly within the context of retinal connectivity. Furthermore, it successfully recapitulates connectivity patterns and helps uncover the genetic factors that underlie these connections.

      Weaknesses:<br /> 1. The study lacks experimental validation of the model's prediction results.<br /> 2. The model's applicability in other neuronal connectivity settings has not been thoroughly explored.<br /> 3. The proposed method relies on the availability of neuronal connectomic data for model training, which may be limited or absent in certain brain connectivity settings.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors investigate the effect of oscillatory activity on the chaotic dynamics of high-dimensional networks. The network oscillations are internally generated by synaptic delays which are known to produce oscillations. The authors demonstrate that the intensity of the chaos and the dimension of the chaotic attractor picks at a delay value. A similar effect is found when an external input drives the network. In this case, these quantities pick at the network's resonant frequency. This shows that the intensity of the chaotic dynamics can be boosted by internally or externally generated oscillations.

      Strengths:<br /> The paper is technically solid. They introduce a novel method to perform calculations of the Lyapunov spectrum in networks with delays, which have infinite dimensions, effectively transforming it into a network of finite dimensions. The conclusions of the paper are supported by strong analytical calculations and novel and intensive numerical methods.

      Weaknesses:<br /> The main weakness is that is difficult to find the relevance of the paper's findings to neuroscience. It is not clear to me that measures such as the rate of production of entropy of a chaotic attractor in spiking networks, its dimension, and its Lyapunov spectra are experimentally relevant. Moreover, the authors make little to no attempt to provide interpretations for these quantities nor put their work in a broader context in the field of systems neuroscience. The paper also is written in an overly technical way with sometimes the use of technical jargon which might be difficult to follow for a non-expert in mean field theories and statistical physics.

    1. Reviewer #2 (Public Review):

      This study by Adelus et al. profiled the transcriptome and chromatin accessibility in cultured human aortic endothelial cells (ECs) at single-cell resolution. They also stimulated these cells with EC-activating agents, such as IL1b, TGFB2, or si-EGR, to knock down this master transcription factor in ECs. The results show a subpopulation, EC3, with the highest plasticity and sensitivity to perturbations. The authors also reviewed and meta-analyzed three independent publicly available scRNA-seq datasets, identifying two distinct EC subpopulations. Additionally, they aligned CAD-related SNPs with open chromatin regions in EC subpopulations. This study provides fundamental evidence to enrich our understanding of vascular ECs and highlights potential subpopulations that may contribute to health and diseases. The work exhibits the potential impact in the field. While the manuscript is comprehensive, there are some concerns that should be addressed.

      1. My major concern is whether EC4 is derived from ECs. It seems that EC4 showed a lesser reaction to those perturbations and had lower expression levels of EC marker genes. Did the authors evaluate the purity of their isolated HAECs? Please discuss the potential cell lineage mapping of EC4.

      2. Although all the donors are de-identified, is there any information about the severity of their vascular impairment, particularly in the case of patient 5, who exhibits the unique EC5?

      3. The meta-analysis of the published datasets is comprehensive. The identified EC heterogeneity corresponds to their in vitro data. I am wondering, in terms of transcriptome, is there any similarity between endo1 and EC1/EC2, and also endo2 and EC3/EC4?

      4. The in vitro data indicates that EC3 shows the highest plasticity and sensitivity to perturbations, which may act as the major subtype of ECs responding to risk factors. It's very interesting that CAD-related SNPs do not seem to be enriched in EC3. Please discuss this discrepancy.

      5. The last sentence in the legend of Figure 1 seems incomplete: 'Module scores are generated for each cell barcode with Seurat function AddModuleScore().'

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors present a theoretical study of the length dynamics of bundles of actin filaments. They first show a "balance point model" in which the bundle is described as an effective polymer. The corresponding assembly and disassembly rates can depend on bundle length. This model generates a steady-state bundle-length distribution with a variance that is proportional to the average bundle length. Numerical simulations confirm this analytic result. The authors then present an analysis of previously published length distributions of actin bundles in various contexts and argue that these distributions have variances that depend quadratically with the average length. They then consider a bundle of N-independent filaments that each grow in an unregulated way. Defining the bundle length to be that of the longest filament, the resulting length distribution has a variance that scales quadratically with the average bundle length.

      Strengths:<br /> The manuscript is very well written, and the computations are nicely presented. The work gives fundamental insights into the length distribution of filamentous actin structures. The universal dependence of the variance on the mean length is of particular interest. It will be interesting to see in the future, how many universality classes there are, and which features of a growth process determine to which class it belongs.

      Weaknesses:<br /> 1) You present the data in Fig. 3 as arguments against the balance point model. Although I agree that the data is compatible with your description of a bundle of filaments, I think that the range of mean lengths you can explore is too limited to conclusively argue against the balance point model. In most cases, your data extend over half an order of magnitude only. Could you provide a measure to quantify how much your model of independent filaments fits better than the balance point model?

      2) Concerning your bundled-filament model, why do you consider the polymerizing ends to be all aligned? Similarly to the opposite end, fluctuations should be present. Furthermore, it is not clear to me, where the presence of crosslinking proteins enters your description. Finally, linked to my first remark on this model, why is the longest filament determining the length of the bundle in all the biological examples you cite? I am thinking in particular about the actin cables in yeast.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The replication of information-coding polymers and the emergence of catalytic ribozymes pose significant challenges, both experimentally and theoretically, in the study of the RNA world hypothesis. In this context, Tkachenko et al. put forth a novel hypothesis regarding a replication oligomer system based on a cleavage ribozyme. They initially highlighted that the breakage of oligomers could contribute to self-replication, provided that these fragments function as primers for subsequent replications. Next, they proposed a self-replicating system of oligomers founded on a hammerhead structure that catalyzes cleavage. By a simple dynamical model, they demonstrated that such a system is self-sustainable in certain parameter regimes. Furthermore, they delved into discussions regarding the potential emergence of such a system and the evolution toward further optimized ribozymes.

      Strengths:<br /> Although the cleavage (hammerhead) ribozyme has been discussed in the context of the origins of life, the authors are the first to discuss how they could be selected using a mathematical model as far as I know. The idea is simple: ribozyme activity creates fragments by breakage of an oligomer, which works as a primer for the ribozyme itself, resulting in a positive feedback system (i.e., autocatalytic sets in a broader sense). This potentially enables us to resolve at the same time problems on the (i) supply of new primers (but note that there is a major concern on this as described in the 'weakness'), and (ii) the sustaining of the cleavage ribozyme.

      Weaknesses:<br /> The major weakness of their theory is that the ends of the new primers, formed through the breakage/cleavage of polymers, must be chemically active (as the authors have already emphasized in the last paragraph of their discussion) to enable further elongation. Reactivating the ends of preexisting oligomers without enzymes, to the best of our current knowledge, could be a challenging task. Although their model heavily relies on this aspect, the authors do not elaborate on it.

      Another weakness is in the setup of their discussion on evolutionary dynamics. While they claim that their model is robust against replication errors, their approach to evolutionary dynamics appears unconventional, and it remains unclear under what conditions their assumptions are founded. They treat a whole set of oligos as a subject of evolution, rather than each individual oligo. This may necessitate more complex assumptions, such as the encapsulation of sets of oligos inside a protocell, to be adequately rationalized. Thus, it remains uncertain whether the system is indeed robust against replication errors in a more natural context. For example, if a mutant oligo, denoted as b', arises due to an error in the replication of oligo b, and if b' has lower catalytic activity but replicates more rapidly than b, it may ultimately come to dominate the system.

    1. Reviewer #2 (Public Review):

      Overall: This paper describes new material of Acanthomeridion serratum that the authors claim supports its synonymy with Acanthomeridion anacanthus. The material is important and the description is acceptable after some modification. In addition, the paper offers thoughts and some exploration of the possibility of multiple origins of the dorsal facial suture among artiopods, at least once within Trilobita and also among other non-trilobite artiopods. Although this possibility is real and apparently correct, the suggestions presented in this paper are both surprising and, in my opinion, unlikely to be true because the potential homologies proposed with regard to Acanthomeridion and trilobite-free cheeks are unconventional and poorly supported.

      What to do? I can see two possibilities. One, which I recommend, is to concentrate on improving the descriptive part of the paper and omit discussion and phylogenetic analysis of dorsal facial suture distribution, leaving that for more comprehensive consideration elsewhere. The other is to seek to improve both simultaneously. That may be possible but will require extensive effort.

      Major concerns

      Concern 1 - Ventral sclerites as free cheek homolog, marginal sutures, and the trilobite doublure

      Firstly, a couple of observations that bear on the arguments presented - the eyes of A. serratum are almost marginal and it is not clear whether a) there is a circumocular suture in this animal and b) if there was, whether it merged with the marginal suture. These observations are important because this animal is not one in which an impressive dorsal facial suture has been demonstrated - with eyes that near marginal it simply cannot do so. Accordingly, the key argument of this paper is not quite what one would expect. That expectation would be that a non-trilobite artiopod, such as A. serratum, shows a clear dorsal facial suture. But that is not the case, at least with A. serratum, because of its marginal eyes. Rather, the argument made is that the ventral doublure of A. serratum is the homolog of the dorsal free cheeks of trilobites. This opens up a series of issues.

      The paper's chief claim in this regard is that the "teardrop" shaped ventral, lateral cephalic plates in Acanthomeridion serratum are potential homologs of the "free cheeks" of those trilobites with a dorsal facial suture. There is no mention of the possibility that these ventral plates in A. serratum could be homologs of the lateral cephalic doublure of olenelloid trilobites, which is bound by an operative marginal suture or, in those trilobites with a dorsal facial suture, that it is a homolog of only the doublure portions of the free cheeks and not with their dorsal components.

      The introduction to the paper does not inform the reader that all olenelloids had a marginal suture - a circumcephalic suture that was operative in their molting and that this is quite different from the situation in, say, "Cedaria" woosteri in which the only operative cephalic exoskeletal suture was circumocular. The conservative position would be that the olenelloid marginal suture is the homolog of the marginal suture in A. serratum: the ventral plates thus being homolog of the trilobite cephalic doublure, not only potential homolog to the entire or dorsal only part of the free cheeks of trilobites with a dorsal facial suture. As the authors of this paper decline to discuss the doublure of trilobites (there is a sole mention of the word in the MS, in a figure caption) and do not mention the olenelloid marginal suture, they give the reader no opportunity to assess support for this alternative.

      At times the paper reads as if the authors are suggesting that olenelloids, which had a marginal cephalic suture broadly akin to that in Limulus, actually lacked a suture that permitted anterior egression during molting. The authors are right to stress the origin of the dorsal cephalic suture in more derived trilobites as a character seemingly of taxonomic significance but lines such as 56 and 67 may be taken by the non-specialist to imply that olenelloids lacked a forward egression-permiting suture. There is a notable difference between not knowing whether sutures existed (a condition apparently quite common among soft-bodied artiopods) and the well-known marginal suture of olenelloids, but as the MS currently reads most readers will not understand this because it remains unexplained in the MS.

      With that in mind, it is also worth further stressing that the primary function of the dorsal sutures in those which have them is essentially similar to the olenelloid/limulid marginal suture mentioned above. It is notable that the course of this suture migrated dorsally up from the margin onto the dorsal shield and merged with the circumocular suture, but this innovation does not seem to have had an impact on its primary function - to permit molting by forward egression. Other trilobites completely surrendered the ability to molt by forward egression, and there are even examples of this occurring ontogenetically within species, suggesting a significant intraspecific shift in suture functionality and molting pattern. The authors mention some of this when questioning the unique origin of the dorsal facial suture of trilobites, although I don't understand their argument: why should the history of subsequent evolutionary modification of a character bear on whether its origin was unique in the group?

      The bottom line here is that for the ventral plates of A. serratum to be strict homologs of only the dorsal portion of the dorsal free cheeks, there would be no homolog of the trilobite doublure in A. serratum. The conventional view, in contrast, would be that the ventral plates are a homolog of the ventral doublure in all trilobites and ventral plates in artiopods. I do not think that this paper provides a convincing basis for preferring their interpretation, nor do I feel that it does an adequate job of explaining issues that are central to the subject.

      Concern 2. Varieties of dorsal sutures and the coexistence of dorsal and marginal sutures

      The authors do not clarify or discuss connections between the circumocular sutures (a form of dorsal suture that separates the visual surface from the rest of the dorsal shield) and the marginal suture that facilitates forward egression upon molting. Both structures can exist independently in the same animal - in olenelloids for example. Olenelloids had both a suture that facilitated forward egression in molting (their marginal suture) and a dorsal suture (their circumocular suture). The condition in trilobites with a dorsal facial suture is that these two independent sutures merged - the formerly marginal suture migrating up the dorsal pleural surface to become confluent with the circumocular suture. (There are also interesting examples of the expansion of the circumocular suture across the pleural fixigena.) The form of the dorsal facial suture has long figured in attempts at higher-level trilobite taxonomy, with a number of character states that commonly relate to the proximity of the eye to the margin of the cephalic shield. The form of the dorsal facial suture that they illustrate in Xanderella, which is barely a strip crossing the dorsal pleural surface linking marginal and circumocular suture, is comparable to that in the trilobites Loganopeltoides and Entomapsis but that is a rare condition in that clade as a whole. The paper would benefit from a clear discussion of these issues at the beginning - the dorsal facial suture that they are referring to is a merged circumcephalic suture and circumocular suture - it is not simply the presence of a molt-related suture on the dorsal side of the cephalon.

      Concern 3. Phylogenetics<br /> While I appreciate that the phylogenetic database is a little modified from those of other recent authors, still I was surprised not to find a character matrix in the supplementary information (unless it was included in some way I overlooked), which I would consider a basic requirement of any paper presenting phylogenetic trees - after all, there's no a space limit. It is not possible for a reviewer to understand the details of their arguments without seeing the character states and the matrix of state assignments.

      The section "phylogenetic analyses" provides a description of how tree topology changes depending on whether sutures are considered homologous or not using the now standard application of both parsimony and maximum likelihood approaches but, considering that the broader implications of this paper rest of the phylogenetic interpretation, I also found the absence of detailed discussion of the meaning and implications of these trees to be surprising, because I anticipated that this was the main reason for conducting these analysis. The trees are presented and briefly described but not considered in detail. I am troubled by "Circles indicate presence of cephalic ecdysial sutures" because it seems that in "independent origin of sutures" trilobites are considered to have two origins (brown color dot) of cephalic ecdysial sutures - this may be further evidence that the team does not appreciate that olenelloids have cephalic ecdysial sutures, as the basal condition in all trilobites. Perhaps I'm misunderstanding their views, but from what's presented it's not possible to know that. Similarly, in the "sutures homologous" analyses why would there be two independent green dots for both Acanthomeridion and Trilobita, rather than at the base of the clade containing them both, as cephalic ecdysial sutures are basal to both of them? Here again, we appear to see evidence that the team considers dorsal facial sutures and cephalic ecdysial sutures to be synonymous - which is incorrect.

      This point aside, and at a minimum, that team needs to do a more thorough job of characterizing and considering the variety of conditions of dorsal sutures among artiopods, their relationships to the marginal suture and to the circumocular suture, the number, and form of their branches, etc.

    1. Reviewer #2 (Public Review):

      In this work the authors describe the shape and interconnectedness of intracellular structures of malaria blood stage parasites by taking advantage of expansion microscopy. Compared to previous microscopy work with these parasites, the strength of this paper lies in the increased resolution and the fact that the NHE ester highlights protein densities. Together with the BodipyC membrane staining, this results in data that is somewhere in between EM and standard fluorescence microscopy: it has higher resolution than standard fluorescence microscopy and provides some points of reference of different cellular structures due to the NHE ester/BodipyC.

      This study makes many interesting and useful observations and although it is somewhat "old school descriptory" in its presentation, researchers working in many different areas will find something of interest here. This ranges from mitosis, to organisation and distribution of major cellular structures, endocytosis and invasion, overall providing a rich and interesting resource. The results section is long but by taking the space to explain everything in detail, it has the advantage that it clearly transpires how things were done and on how many cells a conclusion is based on. Further the authors often also included a brief interpretation of their findings with a very open assessment what it does and what it does not show, highlighting interesting questions left by the data.

      Overall this is a very nice and useful paper that will be of interest to many, particularly those working on nuclear division, cytokinesis, endocytosis or invasion in malaria parasites. The spatiotemporal arrangement and interconnection of subcellular structures will also give a framework for specific functional studies.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The manuscript by Liu et al. reports a task that is designed to examine the extent to which "past" and "future" information is encoded in working memory that combines a retro cue with rules that indicate the location of an upcoming test probe. An analysis of microsaccades on a fine temporal scale shows the extent to which shifts of attention track the location of the location of the encoded item (past) and the location of the future item (test probe). The location of the encoded grating of the test probe was always on orthogonal axes (horizontal, vertical) so that biases in microsaccades could be used to track shifts of attention to one or the other axis (or mixtures of the two). The overall goal here was then to (1) create a methodology that could tease apart memory for the past and future, respectively, (2) to look at the time-course attention to past/future, and (3) to test the extent to which microsaccades might jointly encode past and future memoranda. Finally, some remarks are made about the plausibility of various accounts of working memory encoding/maintenance based on the examination of these time courses.

      Strengths:<br /> This research has several notable strengths. It has a clear statement of its aims, is lucidly presented, and uses a clever experimental design that neatly orthogonalizes "past" and "future" as operationalized by the authors. Figure 1b-d shows fairly clearly that saccade directions have an early peak (around 300ms) for the past and a "ramping" up of saccades moving in the forward direction. This seems to be a nice demonstration the method can measure shifts of attention at a fine temporal resolution and differentiate past from future-oriented saccades due to the orthogonal cue approach. The second analysis shown in Figure 2, reveals a dependency in saccade direction such that saccades toward the probe future were more likely also to be toward the encoded location than away from the encoded direction. This suggests saccades are jointly biased by both locations "in memory".

      Weaknesses:<br /> 1. The "central contribution" (as the authors characterize it) is that "the brain simultaneously retains the copy of both past and future-relevant locations in working memory, and (re)activates each during mnemonic selection", and that: "... while it is not surprising that the future location is considered, it is far less trivial that both past and future attributes would be retained and (re)activated together. This is our central contribution." However, to succeed at the task, participants must retain the content (grating orientation, past) and probe location (future) in working memory during the delay period. It is true that the location of the grating is functionally irrelevant once the cue is shown, but if we assume that features of a visual object are bound in memory, it is not surprising that location information of the encoded object would bias processing as indicated by microsaccades. Here the authors claim that joint representation of past and future is "far less trivial", this needs to be evaluaed from the standpoint of prior empirical data on memory decay in such circumstances, or some reference to the time-course of the "unbinding" of features in an encoded object.

      2. The authors refer to "future" and "past" information in working memory and this makes sense at a surface level. However, once the retrocue is revealed, the "rule" is retrieved from long-term memory, and the feature (e.g. right/left, top/bottom) is maintained in memory like any other item representation. Consider the classic test of digit span. The digits are presented and then recalled. Are the digits of the past or future? The authors might say that one cannot know, because past and future are perfectly confounded. An alternative view is that some information in working memory is relevant and some is irrelevant. In the digit span task, all the digits are relevant. Relevant information is relevant precisely because it is thought be necessary in the future. Irrelevant information is irrelevant precisely because it is not thought to be needed in the immediate future. In the current study, the orientation of the grating is relevant, but its location is irrelevant; and the location of the test probe is also relevant.

      3. It is not clear how the authors interpret the "joint representation" of past and future. Put aside "future" and "past" for a moment. If there are two elements in memory, both of which are associated with spatial bindings, the attentional focus might be a spatial average of the associated spatial indices. One might also view this as an interference effect, such that the location of the encoded location attracts spatial attention since it has not been fully deleted/removed from working memory. Again, for the impact of the encoded location to be exactly zero after the retrieval cue, requires zero interference or instantaneous decay of the bound location information. It would be helpful for the authors to expand their discussion to further explain how the results fit within a broader theoretical framework and how it fits with empirical data on how quickly an irrelevant feature of an object can be deleted from working memory.

    1. Reviewer #2 (Public Review):

      This MEG study used co-registered eye-tracking and Rapid Invisible Frequency Tagging (RIFT) to track the effects of semantic parafoveal preview during natural sentence reading. Unpredictable target words could either be congruent or incongruent with sentence context. This modulated the RIFT response already while participants were fixating on the preceding word. This indicates that the semantic congruency of the upcoming word modulates visual attention demands already in parafoveal preview.<br /> The quest for semantic parafoveal preview in natural reading has attracted a lot of attention in recent years, especially with the development of co-registered EEG and MEG. Evidence from dynamic neuroimaging methods using innovative paradigms as in this study is important for this debate.

      Major points:<br /> 1) The authors frame their study in terms of "congruency with sentence context". However, it is the congruency between adjective-noun pairs that determines congruency (e.g. "blue brother" vs "blue jacket", and examples p. 16 and appendix). This is confirmed by Suppl Figure 1, which shows a significantly larger likelihood of refixations to the pre-target word for incongruent sentences, probably because the pre-target word is most diagnostic for the congruency of the target word. The authors discuss some possibilities as to why there is variability in parafoveal preview effects in the literature. It is more likely to see effects for this simple and local congruency, rather than congruency that requires an integration and comprehension of the full sentence. I'm not sure whether the authors really needed to present their stimuli in a full-sentence context to obtain these effects. This should be explicitly discussed and also mentioned in the introduction (or even the abstract).

      2) The authors used MEG and provided a source estimate for the tagging response (Figure 2), which unsurprisingly is in the visual cortex. The most important results are presented at the sensor level. This does not add information about the brain sources of the congruency effect, as the RIFT response probably reflects top-down effects on visual attention etc. Was it necessary to use MEG? Would EEG have produced the same results? In terms of sensitivity, EEG is better than MEG as it is more sensitive to radial and deeper sources. This should be mentioned in the discussion and/or methods section.

      3) The earliest semantic preview effects occurred around 100ms after fixating the pre-target word (discussed around l. 323). This means that at this stage the brain must have processed the pre-target and the target word and integrated their meanings (at some level). Even in the single-word literature, semantic effects at 100 ms are provocatively early. Even studies that tried to determine the earliest semantic effects arrived at around 200 ms (e.g. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382728/, https://psycnet.apa.org/record/2013-17451-002). The present results need to be discussed in a bit more detail in the context of the visual word recognition literature.

      4) As in previous EEG/MEG studies, the authors found a neural but no behavioural preview effect. As before, this raises the question of whether the observed effect is really "critical" for sentence comprehension. The authors provide a correlation analysis with reading speed, but this does not allow causal conclusions: Some people may simply read slowly and therefore pay more attention and get a larger preview response. Some readers may hurry and therefore not pay attention and not get a preview response. In order to address this, one would have to control for reading speed and show an effect of RIFT response on comprehension performance (or vice versa, with a task that is not close to ceiling performance). The last sentence of the discussion is currently not justified by the results.

      5) L. 577f.: ICA components were selected by visual inspection. I would strongly recommend including EOG in future recordings when the control of eye movements is critical.

      6) The authors mention "saccade planning" a few times. I would suggest looking at the SWIFT model of eye movement control, which is less mechanistic than the dominant EZ-Reader model (https://psycnet.apa.org/record/2005-13637-003). It may be useful for the framing of the study and interpretation of the results (e.g. second paragraph of discussion).

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors combined inhibitory neurostimulation (continuous theta-burst stimulation, cTBS) with subsequent MRI measurements to investigate the impact of inhibition of the left anterior temporal lobe (ATL) on task-related activity and performance during a semantic task and link stimulation-induced changes to the neurochemical level by including MR spectroscopy (MRS). cTBS effects in the ATL were compared with a control site in the vertex. The authors found that relative to stimulation of the vertex, cTBS significantly increased the local GABA concentration in the ATL. cTBS also decreased task-related semantic activity in the ATL and potentially delayed semantic task performance by hindering a practice effect from pre to post. Finally, pooled data from their previous MRS study suggest an inverted U-shape between GABA concentration and behavioral performance. These results help to better understand the neuromodulatory effects of non-invasive brain stimulation on task performance.

      Strengths:<br /> Multimodal assessment of neurostimulation effects on the behavioral, neurochemical, and neural levels. In particular, the link between GABA modulation and behavior is timely and potentially interesting.

      Weaknesses:<br /> The analyses are not sound. Some of the effects are very weak and not all conclusions are supported by the data since some of the comparisons are not justified. There is some redundancy with a previous paper by the same authors, so the novelty and contribution to the field are overall limited. A network approach might help here.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study examined the longitudinal brain-behaviour link between attentional neural filtering and listening behaviour among a sample of aging individuals. The results based on the latent change score modeling showed that neither attentional neural filtering at T1 nor its T1-T2 change predicted individual two-year listening performance change. The findings suggest that neural filtering and listening behaviour may follow independent developmental trajectories. This study focuses on an interesting topic and has the potential to contribute a better understanding of the neurobiological mechanisms of successful communication across the lifespan.

      Strengths:<br /> Although research suggests that speech comprehension is neurally supported by an attention-guided filter mechanism, the evidence of their causal association is limited. This study addresses this gap by testing the longitudinal stability of neural filtering as a neural mechanism upholding listening performance, potentially shedding light on translational efforts aiming at the preservation of speech comprehension abilities among aging individuals.

      The latent change score modeling approach is appropriately used as a tool to examine key developmental questions and distinguish the complex processes underlying lifespan development in brain and behaviour with longitudinal data.

      Weaknesses:<br /> Although the paper does have strengths in principle, the weaknesses of the paper are that the findings are merely based on a single listening task. Since both neural and behavioral indicators are derived from the same task, the results may be applicable only to this specific task, and it is difficult to extrapolate them to cognitive and listening abilities measured by the other tasks. Therefore, more listening tasks are required to comprehensively measure speech comprehension and neural markers.

      The age span of the sample is relatively large. Although no longitudinal change from T1 to T2 was found at the group-level, from the cross-sectional and longitudinal change results (see Figure 3), individuals of different age groups showed different development patterns. Particularly, individuals over the age of 70 show a clear downward trend in both neural filtering index and accuracy. Therefore, different results may be found based on different age groups, especially older groups. However, due to sample limitations, this study was unable to examine whether age has a moderating effect on this brain-behaviour link.

      In the Dichotic listening task, valid and invalid cues were manipulated. According to the task description, the former could invoke selective attention, whereas the latter could invoke divided attention. It is possible that under the two conditions, the neural filtering index may reflect different underlying cognitive processes, and thus may differ in its predictive effect on behavioral performance. The author could perform a more in-depth data analysis on indicators under different conditions.

    1. Reviewer #2 (Public Review):

      Summary: The authors tried to identify novel adult functions of the classical Drosophila juvenile-adult transition axis (i.e. ptth-ecdysone). Surprisingly, larval ptth-expressing neurons expressed the sex-specific doublesex gene, thus belonging to the sexual dimorphic circuit. Lack of ptth during late larval development caused enhanced female sexual receptivity, an effect rescued by supplying ecdysone in the food. Among many other cellular players, pC1 neurons control receptivity by encoding the mating status of females. Interestingly, during metamorphosis, a subtype of pC1 neurons required Ecdysone Receptor A in order to regulate such female receptivity. A transcriptomic analysis using pC1-specific Ecdyone signaling down-regulation gives some hints of possible downstream mechanisms.

      Strengths: the manuscript showed solid genetic evidence that lack of ptth during development caused enhanced copulation rate in female flies, which includes ptth mutant rescue experiments by over-expressing ptth as well as by adding ecdysone-supplemented food. They also present elegant data dissecting the temporal requirements of ptth-expressing neurons by shifting animals from non-permissive to permissive temperatures, in order to inactivate neuronal function (although not exclusively ptth function). By combining different drivers together with a EcR-A RNAi line authors also identified the Ecdysone receptor requirements of a particular subtype of pC1 neurons during metamorphosis. Convincing live calcium imaging showed no apparent effect of EcR-A in neural activity, although some effect on morphology is uncovered. Finally, bulk RNAseq shows differential gene expression after EcR-A down-regulation.

      Weaknesses: the paper has three main weaknesses. The first one refers to temporal requirements of ptth and ecdysone signaling. Whereas ptth is necessary during larval development, the ecdysone effect appears during pupal development. ptth induces ecdysone synthesis during larval development but there is no published evidence about a similar role for ptth during pupal stages. Furthermore, larval and pupal ecdysone functions are different (triggering metamorphosis vs tissue remodeling). The second caveat is the fact that ptth and ecdysone loss-of-function experiments render opposite effects (enhancing and decreasing copulation rates, respectively). The most plausible explanation is that both functions are independent of each other, also suggested by differential temporal requirements. Finally, in order to identify the effect in the transcriptional response of down-regulating EcR-A in a very small population of neurons, a scRNAseq study should have been performed instead of bulk RNAseq.

      In summary, despite the authors providing convincing evidence that ptth and ecdysone signaling pathways are involved in female receptivity, the main claim that ptth regulates this process through ecdysone is not supported by results. More likely, they'd rather be independent processes.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The sleep patterns of animals are adaptable, with shorter sleep durations in the winter and longer sleep durations in the summer. Chen and colleagues conducted a study using Drosophila (fruit flies) and discovered that a circadian photoreceptor called cryptochrome (cry) plays a role in reducing sleep duration during day/night cycles resembling winter conditions. They also found that cry functions in specific GABAergic circadian pacemaker cells known as s-LNvs inhibit these neurons, thereby promoting wakefulness in the animals in the winter. They also identified l-LNvs, known as arousal-promoting cells, as the downstream neurons.

      Strengths:<br /> Detailed mapping of the neural circuits cry acts to mediate the shortened sleep in winter-like day/night cycles.

      Weaknesses:<br /> The supporting evidence for s-LNvs being GABAergic neurons is not particularly strong. Additionally, there is a lack of direct evidence regarding changes in neural activity for s-LNvs and l-LNvs under varying day/night cycles, as well as in cry mutant flies.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Klug et al. use monosynaptic rabies tracing of inputs to D1- vs D2-SPNs in the striatum to study how separate populations of cortical neurons project to D1- and D2-SPNs. They use rabies to express ChR2, then patch D1-or D2-SPNs to measure synaptic input. They report that cortical neurons labeled as D1-SPN-projecting preferentially project to D1-SPNs over D2-SPNs. In contrast, cortical neurons labeled as D2-SPN-projecting project equally to D1- and D2-SPNs. They go on to conduct pathway-specific behavioral stimulation experiments. They compare direct optogenetic stimulation of D1- or D2-SPNs to stimulation of MCC inputs to DMS and M1 inputs to DLS. In three different behavioral assays (open field, intra-cranial self-stimulation, and a fixed ratio 8 task), they show that stimulating MCC or M1 cortical inputs to D1-SPNs is similar to D1-SPN stimulation, but that stimulating MCC or M1 cortical inputs to D2-SPNs does not recapitulate the effects of D2-SPN stimulation (presumably because both D1- and D2-SPNs are being activated by these cortical inputs).

      Strengths:<br /> Showing these same effects in three distinct behaviors is strong. Overall, the functional verification of the consequences of the anatomy is very nice to see. It is a good choice to patch only from mCherry-negative non-starter cells in the striatum.

      Weaknesses:<br /> One limitation is that all inputs to SPNs are expressing ChR2, so they cannot distinguish between different cortical subregions during patching experiments. Their results could arise because the same innervation patterns are repeated in many cortical subregions or because some subregions have preferential D1-SPN input while others do not. There are also some caveats with respect to the efficacy of rabies tracing. Although they only patch non-starter cells in the striatum, only 63% of D1-SPNs receive input from D1-SPN-projecting cortical neurons. It's hard to say whether this is "high" or "low," but one question is how far from the starter cell region they are patching. Without this spatial indication of where the cells that are being patched are relative to the starter population, it is difficult to interpret if the cells being patched are receiving cortical inputs from the same neurons that are projecting to the starter population. Convergence of cortical inputs onto SPNs may vary with distance from the starter cell region quite dramatically, as other mapping studies of corticostriatal inputs have shown specialized local input regions can be defined based on cortical input patterns (Hintiryan et al., Nat Neurosci, 2016, Hunnicutt et al., eLife 2016, Peters et al., Nature, 2021). A caveat for the optogenetic behavioral experiments is that these optogenetic experiments did not include fluorophore-only controls. Another point of confusion is that other studies (Cui et al, J Neurosci, 2021) have reported that stimulation of D1-SPNs in DLS inhibits rather than promotes movement.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors investigate replay (defined as sequential reactivation) and clustered reactivation during retrieval of an abstract cognitive map. Replay and clustered reactivation were analysed based on MEG recordings combined with a decoding approach. While the authors state to find evidence for both, replay and clustered reactivation during retrieval, replay was exclusively present in low performers. Further, the authors show that reactivation strength declined with an increasing graph distance.

      Strengths:<br /> The paper raises interesting research questions, i.e., replay vs. clustered reactivation and how that supports retrieval of cognitive maps. The paper is well-written, well-structured, and easy to follow. The methodological approach is convincing and definitely suited to address the proposed research questions.

      The paper is a great combination between replicating previous findings (Wimmer et al. 2020) with a new experimental approach but at the same time presenting novel findings (reactivation strength declines as a function of graph distance).<br /> What I also want to positively highlight is their transparency. They pre-registered this study but with a focus on a different part of the data and outlined this explicitly in the paper.

      The paper has very interesting, individual findings but there are some shortcomings.

      Weaknesses:<br /> Even though the individual findings are interesting, it is not easy to grasp how they are related. For example, the authors show that replay is present in low but not in high performers with the assumption that high performers tend to simultaneously reactivate items. But then, the authors do not investigate clustered reactivation (= simultaneous reactivation) as a function of performance (due to ceiling effects for most participants).

      Unfortunately, the evidence for clustered reactivation is not well supported by the analysis approach and the observed evidence. The analysis approach still holds the possibility of replay driving the observed clustered reactivation effect.

      A third shortcoming is that at least some analyses are underpowered (very low number of trials, n = ~10, and for some analyses, very low number of participants, n = 14). In both cases (low trial number and low participant number) the n could be increased by including the learning part in the analyses as well. It is not clear to me why the authors restricted their analyses to the retrieval period only (especially given that participants also have to retrieve during learning).

    1. Reviewer #2 (Public Review):

      This work provides a novel design of implantable and high-density EMG electrodes to study muscle physiology and neuromotor control at the level of individual motor units. Current methods of recording EMG using intramuscular fine-wire electrodes do not allow for isolation of motor units and are limited by the muscle size and the type of behavior used in the study. The authors of myomatrix arrays had set out to overcome these challenges in EMG recording and provided compelling evidence to support the usefulness of the new technology.

      Strengths:<br /> • They presented convincing examples of EMG recordings with high signal quality using this new technology from a wide array of animal species, muscles, and behavior.<br /> • The design included suture holes and pull-on tabs that facilitate implantation and ensure stable recordings over months.<br /> • Clear presentation of specifics of the fabrication and implantation, recording methods used, and data analysis

      I am satisfied with the authors' response to my previous concerns on the weaknesses of the study.

    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.

    1. Reviewer #2 (Public Review):

      Summary:<br /> An article with lots of interesting ideas and questions regarding the evolution of timing of dormancy, emphasizing mammalian hibernation but also including ectotherms. The authors compare selective forces of constraints due to energy availability versus predator avoidance and requirements and consequences of reproduction in a review of between and within species (sex) differences in the seasonal timing of entry and exit from dormancy.

      Strengths:<br /> The multispecies approach including endotherms and ectotherms is ambitious. This review is rich with ideas if not in convincing conclusions.

      Weaknesses:<br /> The differences between physiological requirements for gameatogenesis between sexes that affect the timing of heterothermy and the need for euthermy during mammalian hibernator are significant issues that underlie but are under-discussed, in this contrast of selective pressures that determine seasonal timing of dormancy. Some additional discussion of the effects of rapid climate change on between and within species phenologies of dormancy would have been interesting.

    1. Reviewer #2 (Public Review):

      This study focuses on the eco-morphology, the feeding behaviors, and the co-evolution of feeding organs of longirostrine gomphotheres (Amebelodontidae, Choerolophodontidae, and Gomphotheriidae) which are characterised by their distinctive mandible and mandible tusk morphologies. They also have different evolutionary stages of food acquisition organs which may have co-evolve with extremely elongated mandibular symphysis and tusks. Although these three longirostrine gomphothere families were widely distributed in Northern China in the Early-Middle Miocene, the relative abundances and the distribution of these groups were different through time as a result of the climatic changes and ecosysytems.

      These three groups have different feeding behaviors indicated by different mandibular symphysis and tusk morphologies. Additionally, they have different evolutionary stages of trunks which are reflected by the narial region morphology. To be able to construct the feeding behavior and the relation between the mandible and the trunk of early elephantiformes, the authors examined the crania and mandibles of these three groups from the Early and Middle Miocene of northern China from three different museums and also made different analyses.

      The analyses made in the study are:<br /> 1. Finite Element (FE) analysis: They conducted two kinds of tests: the distal forces test, and the twig-cutting test. With the distal forces test, advantageous and disadvantageous mechanical performances under distal vertical and horizontal external forces of each group are established. With the twig-cutting test, a cylindrical twig model of orthotropic elastoplasity was posed in three directions to the distal end of the mandibular task to calculate the sum of the equivalent plastic strain (SEPS). It is indicated that all three groups have different mandible specializations for cutting plants.

      2. Phylogenetic reconstruction: These groups have different narial region morphology, and in connection with this, have different stages of trunk evolution. The phylogenetic tree shows the degree of specialization of the narial morphology. And narial region evolutionary level is correlated with that of character-combine in relation to horizontal cutting. In the trilophodont longirostrine gomphotheres, co-evolution between the narial region and horizontal cutting behaviour is strongly suggested.

      3. Enamel isotopes analysis: The results of stable isotope analysis indicate an open environment with a diverse range of habitats and that the niches of these groups overlapped without obvious differentiation.

      The analysis shows that different eco-adaptations have led to the diverse mandibular morphology and open-land grazing has driven the development of trunk-specific functions and loss of the long mandible. This conclusion has been achieved with evidence on palaecological reconstruction, the reconstruction of feeding behaviors, and the examination of mandibular and narial region morphology from the detailed analysis during the study.

      All of the analyses are explained in detail in the supplementary files. The 3D models and movies in the supplementary files are detailed and understandable and explain the conclusion. The conclusions of the study are well supported by data.

    1. Reviewer #2 (Public Review):

      This study addresses the catalytic activity of a Ras-like ROC GTPase domain of LRRK2 kinase, a Ser/Thr kinase linked to Parkinson's disease (PD). The enzyme is associated with gain-of-function variants that hyper-phosphorylate substrate Rab GTPases. However, the link between the regulatory ROC domain and activation of the kinase domain is not well understood.

      It is within this context that the authors detail the kinetics of the ROC GTPase domain of pathogenic variants of LRRK2, in comparison to the WT enzyme. Their data suggest that LRRK2 kinase activity negatively regulates the ROC GTPase activity and that PD variants of LRRK2 have differential effects on the Km and catalytic efficiency of GTP hydrolysis.

      Based on mutagenesis, kinetics, and biophysical experiments, the authors suggest a model in which autophosphorylation shifts the equilibrium toward monomeric LRRK2 (locked GTP state of ROC). The authors further conclude that T1343 is a crucial regulatory site, located in the P-loop of the ROC domain, which is necessary for the negative feedback mechanism. Unfortunately, the data do not support this hypothesis, and further experiments are required to confirm this model for the regulation of LRRK2 activity.

      Specific comments are below:

      - Although a couple of papers are cited, the rationale for focusing on the T1343 site is not evident to readers. It should be clarified that this locus, and perhaps other similar loci in the wider ROCO family, are likely important for direct interactions with the GTP molecule.

      - Similar to the above, readers are kept in the dark about auto-phosphorylation and its effects on the monomer/dimer equilibrium. This is a critical aspect of this manuscript and a major conceptual finding that the authors are making from their data. However, the idea that auto-phosphorylation is (likely) to shift the monomer/dimer equilibrium toward monomer, thereby inactivating the enzyme, is not presented until page 6, AFTER describing much of their kinetics data. This is very confusing to readers, as it is difficult to understand the meaning of the data without a conceptual framework. If the model for the LRRK2 function is that dimerization is necessary for the phosphorylation of substrates, then this idea should be presented early in the introduction, and perhaps also in the abstract. If there are caveats, then they should be discussed before data are presented. A clear literature trail and the current accepted (or consensus) mechanism for LRRK2 activity is necessary to better understand the context for these data.

      - Following on the above concepts, I find it interesting that the authors mention monomeric cyotosolic states, and kinase-active oligomers (dimers??), with citations. Again here, it would be useful to be more precise. Are dimers (oligomers?) only formed at the membrane? That would suggest mechanisms involving lipid or membrane-attached protein interactions. Also, what do the authors mean by oligomers? Are there more than dimers found localized to the membrane?

      - Fig 5 is a key part of their findings, regarding the auto-phosphorylation induced monomer formation of LRRK2. From these two bar graphs, the authors state unequivocally that the 'monomer/dimer equilibrium is abolished', and therefore, that the underlying mechanism might be increased monomerization (through maintenance of a GTP-locked state). My view is that the authors should temper these conclusions with caveats. One is that there are still plenty of dimers in the auto-phosphorylated WT, and also in the T1343A mutant. Why is that the case? Can the authors explain why only perhaps a 10% shift is sufficient? Secondly, the T1343A mutant appears to have fewer overall dimers to begin with, so it appears to readers that 'abolition' is mainly due to different levels prior to ATP treatment at 30 deg. I feel these various issues need to be clarified in a revised manuscript, with additional supporting data. Finally, on a minor note, I presume that there are no statistically significant differences between the two sets of bar graphs on the right panel. It would be wise to place 'n.s.' above the graphs for readers, and in the figure legend, so readers are not confused.

      - Figure 6B, Westerns of phosphorylation, the lanes are not identified and it is unclear what these data mean.

    1. Reviewer #2 (Public Review):

      Summary:<br /> HCN-4 isoform is found primarily in the sino-atrial node where it contributes to the pacemaking activity. LRMP is an accessory subunit that prevents cAMP-dependent potentiation of HCN4 isoform but does not have any effect on HCN2 regulation. In this study, the authors combine electrophysiology, FRET with standard molecular genetics to determine the molecular mechanism of LRMP action on HCN4 activity. Their study shows that parts of N- and C-termini along with specific residues in C-linker and S5 of HCN4 are crucial for mediating LRMP action on these channels. Furthermore, they show that the initial 224 residues of LRMP are sufficient to account for most of the activity. In my view, the highlight of this study is Fig. 7 which recapitulates LRMP modulation on HCN2-HCN4 chimera. Overall, this study is an excellent example of using time-tested methods to probe the molecular mechanisms of regulation of channel function by an accessory subunit.

      Weaknesses:<br /> 1. Figure 5A- I am a bit confused with this figure and perhaps it needs better labeling. When it states Citrine, does it mean just free Citrine, and "LRMP 1-230" means LRMP fused to Citrine which is an "LF" construct? Why not simply call it "LF"? If there is no Citrine fused to "LRMP 1-230", this figure would not make sense to me.

      2. Related to the above point- Why is there very little FRET between NF and LRMP 1-230? The FRET distance range is 2-8 nm which is quite large. To observe baseline FRET for this construct more explanation is required. Even if one assumes that about 100 amino are completely disordered (not extended) polymers, I think you would still expect significant FRET.

      3. Unless I missed this, have all the Cerulean and Citrine constructs been tested for functional activity?

    1. Reviewer #2 (Public Review):

      Summary and strengths:

      O'Brien et al. present a compelling strategy to both understand rare disease that could have a neuronal focus and discover drugs for repurposing that can affect rare disease phenotypes. Using C. elegans, they optimize the Brown lab worm tracker and Tierpsy analysis platform to look at the movement behaviors of 25 knockout strains. These gene knockouts were chosen based on a process to identify human orthologs that could underlie rare diseases. I found the manuscript interesting and a powerful approach to making genotype-phenotype connections using C. elegans. Given the rate at which rare Mendelian diseases are found and candidate genes suggested, human geneticists need to consider orthologous approaches to understand the disease and seek treatments on a rapid time scale. This approach is one such way. Overall, I have a few minor suggestions and some specific edits.

      Weaknesses:<br /> (1) Throughout the text on figures, labels are nearly impossible to read. I had to zoom into the PDF to determine what the figure was showing. Please make text in all figures a minimum of 10-point font. Similarly, the Figure 2D point type is impossible to read. Points should be larger in all figures. Gene names should be in italics in all figures, following C. elegans convention.

      (2) I have a strong bias against the second point in Figure 1A. Sequencing of trios, cohorts, or individuals NEVER identifies causal genes in the disease. This technique proposes a candidate gene. Future experiments (oftentimes in model organisms) are required to make those connections to causality. Please edit this figure and parts of the text.

      (3) How were the high-confidence orthologs filtered from 767 to 543 (lines 128-131)? Also, the choice of the final list of 25 genes is not well justified. Please expand more about how these choices were made.

      (4) Figures 3 and 4, why show all 8289 features? It might be easier to understand and read if only the 256 Tierpsy features were plotted in the heat maps.

      (5) The unc-80 mutant screen is clever. In the feature space, it is likely better to focus on the 256 less-redundant Tierpsy features instead of just a number of features. It is unclear to me how many of these features are correlated and not providing more information. In other words, the "worsening" of less-redundant features is far more of a concern than the "worsening" of 1000 correlated features.

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


      This manuscript builds from the interesting observation that local recruitment of the DHPH domain of the RhoGEF PRG can induce local retraction, protrusion, or neither. The authors convincingly show that these differential responses are tied to the level of expression of the PRG transgene. This response depends on the Rho-binding activity of the recruited PH domain and is associated with and requires (co?)-activation of Cdc42. This begs the question of why this switch in response occurs. They use a computational model to predict that the timing of protein recruitment can dictate the output of the response in cells expressing intermediate levels and found that, "While the majority of cells showed mixed phenotypes irrespectively of the activation pattern, in few cells (3 out of 90) we were able to alternate the phenotype between retraction and protrusion several times at different places of the cell by changing the frequency while keeping the same total integrated intensity (Figure 6F and Supp Movie)."


      The experiments are well-performed and nicely documented. However, the molecular mechanism underlying the shift in response is not clear (or at least clearly described). In addition, it is not clear that a prediction that is observed in ~3% of cells should be interpreted as confirming a model, though the fit to the data in 6B is impressive.

      Overall, the main general biological significance of this work is that RhoGEF can have "off target effects". This finding is significant in that an orthologous GEF is widely used in optogenetic experiments in drosophila. It's possible that these findings may likewise involve phenotypes that reflect the (co-)activation of other Rho family GTPases.


      The manuscript makes a number of untested assumptions and the underlying mechanism for this phenotypic shift is not clearly defined.

      This manuscript is missing a direct phenotypic comparison of control cells to complement that of cells expressing RhoGEF2-DHPH at "low levels" (the cells that would respond to optogenetic stimulation by retracting); and cells expressing RhoGEF2-DHPH at "high levels" (the cells that would respond to optogenetic stimulation by protruding). In other words, the authors should examine cell area, the distribution of actin and myosin, etc in all three groups of cells (akin to the time zero data from figures 3 and 5, with a negative control). For example, does the basal expression meaningfully affect the PRG low-expressing cells before activation e.g. ectopic stress fibers? This need not be an optogenetic experiment, the authors could express RhoGEF2DHPH without SspB (as in Fig 4G).

      Relatedly, the authors seem to assume ("recruitment of the same DH-PH domain of PRG at the membrane, in the same cell line, which means in the same biochemical environment." supplement) that the only difference between the high and low expressors are the level of expression. Given the chronic overexpression and the fact that the capacity for this phenotypic shift is not recruitment-dependent, this is not necessarily a safe assumption. The expression of this GEF could well induce e.g. gene expression changes.

      The third paragraph of the introduction, which begins with the sentence, "Yet, a large body of works on the regulation of GTPases has revealed a much more complex picture with numerous crosstalks and feedbacks allowing the fine spatiotemporal patterning of GTPase activities" is potentially confusing to readers. This paragraph suggests that an individual GTPase may have different functions whereas the evidence in this manuscript demonstrates, instead, that *a particular GEF* can have multiple activities because it can differentially activate two different GTPases depending on expression levels. It does not show that a particular GTPase has two distinct activities. The notion that a particular GEF can impact multiple GTPases is not particularly novel, though it is novel (to my knowledge) that the different activities depend on expression levels.

      These descriptions are not precise. What is the nature of the competition between RhoA and Cdc42? Is this competition for activation by the GEFs? Is it a competition between the phenotypic output resulting from the effectors of the GEFs? Is it competition from the optogenetic probe and Rho effectors and the Rho biosensors? In all likelihood, all of these effects are involved, but the authors should more precisely explain the underlying nature of this phenotypic switch. Some of these points are clarified in the supplement, but should also be explicit in the main text.

    1. Reviewer #2 (Public Review):

      Although Trabid missense mutations are identified across a range of neurodevelopmental disorders, its role in neurodevelopment is not understood. Here the authors study two different patient mutations and implicate defects in its deubiquitylating activity and interactions with STRIPAK. Knockin mice for these mutations impaired trafficking of APC to microtubule plus ends, with consequent defects in neuronal growth cone and neurite outgrowth.

      The authors focus on R438W and A451V, two missense mutations seen in patients. Recombinant fragments showed R438W is nearly completely DUB-dead whereas A451V showed normal activity but failed to efficiently precipitate STRIPAK. Knockin of these mutations showed a partially penetrant reduced cortical neuronal and glial cell numbers and reduced TH+ neurons and their neuronal processes. Cell culture demonstrated that both DUB and STRIPAK-binding activities of Trabid are required for efficient deubiquitylation of APC in cells, and alter APC transport along neurites. APC-tdTomato fluorescent reporter mice crossed with the Trabid mutants confirmed these results. The results suggest that Trabid's mechanism of action is to suppress APC ubiquitylation to regulate its intracellular trafficking and neurite formation.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The present work addresses the mechanisms linking the sex-dependent temporal GH secretion patterns to the robust sex differences in chromatin accessibility and transcription factor binding that ultimately regulate sexually dimorphic liver gene expression. Using DNAseq analysis genomic sites hypersensitive to cleavage by DNase I, DNase hypersensitive sites [DHS] were studied in hepatocytes from male and female mice. DHS in the genome correspond to accessible chromatin regions and encompass key regulatory elements, including enhancers, promoters, insulators, and silencers, often flanked by specific histone modifications, and all of these players were described in different settings of GH action. Importantly, the dynamics of sex-dependent and independent chromatin accessibility linked to STAT5 binding were evaluated. For that purpose, hepatic samples from mice were divided into STAT high and STAT low binding by EMSA screening. With this information changes in DHS related to STAT binding were calculated in both sexes, giving an approximation of chromatin opening in response to STAT5, or alternatively to hypophsectomy, or a single GH pulse. More the 800 male-biased DHS (from a total of more than 70000 DHS) regions were identified in the STAT5 high groups, implying that the binding of a plasma GH pulse activates STAT5, and evokes a dynamic cycle of male liver chromatin opening and closing at sites that comprised 31% of all male-biased DHS. This proves that the pulsatility of plasma GH stimulation confers significant male bias in chromatin accessibility, and STAT5 binding at a fraction of the genomic sites linked to sex-biased liver gene expression and liver disease. As a proof of concept, authors show that a single physiological replacement dose or pulse of GH given to hypophysectomized mice recapitulate, within 30 min, the pulsatile re-opening of chromatin seen in pituitary-intact male mouse liver.

      In another male-biased DHS set (69% of male-biased DHS), chromatin accessibility was static, that is unchanged across the peaks and valleys of GH-induced liver STAT5 activity and mapped to a set of target genes and processes distinct though sometimes overlapping those of the dynamic male-biased DHS.

      In view of these distinct dynamic and static DHS in males, authors evaluated key epigenetic features distinguishing the dynamic STAT5-driven mechanism of chromatin opening from that of static male-biased DHS, which are constitutively open in the male liver but closed in the female liver. The analysis of histone marks enriched at each class of sex-biased DHS indicated exquisite differences in the epigenetic mechanisms that mediate sex-specific gene repression in each sex. For example, H3K27me3 and H3K9me3, two widely used repressive histone marks, are used in a unique way in each sex to enforce sex differences in chromatin states at sex-biased DHS.

      Finally, the work recapitulates and explains the classifications of sex dimorphic genes made in previous works. Sex-biased and pituitary hormone-dependent DHS act as regulatory elements with a positive enhancer potential, to induce or maintain gene expression in the intact liver by sustaining an open chromatin in the case of class I male-biased DHS and class I male-biased genes in the male liver. Contrariwise DHS may participate in the inhibition of gene expression by maintaining a closed chromatin state, as in the case of class II male-biased DHS and class II female-biased genes in male liver.

      These results as a whole present a complex mechanism by which GH regulates the sexual dimorphism of liver genes in order to cope with the metabolic needs of each sex. In a complete story, the information on chromatin accessibility, histone modification, and transcription factor binding was integrated to elucidate the complex patterns of transcriptional regulation, which is sexually dimorphic in the liver.

      Strengths:<br /> The work presents a novel insight into the fundamental underlying epigenetic mechanisms of sex-biased gene regulation.<br /> Results are supported by numerous Tables, and Supplementary Tables with the raw data, which present the advantage that they may be reanalyzed in the future to prove new hypotheses.

      Weaknesses<br /> It is a complicated work to analyze, even though the main messages are clearly conveyed.

    1. Reviewer #2 (Public Review):


      The manuscript by Morel et al. aims to identify some potential mechano-regulators of transendothelial cell macro-aperture (TEM). Guided by the recognized role of caveolar invaginations in buffering the membrane tension of cells, the authors focused on caveolin-1 and associated regulator PTRF. They report a comprehensive in vitro work based on siRNA knockdown and optical imaging approach complemented with an in vivo work on mice, a biophysical assay allowing measurement of the mechanical properties of membranes, and a theoretical analysis inspired by soft matter physics.


      The authors should be complimented for this multi-faceted and rigorous work. The accumulation of pieces of evidence collected from each type of approach makes the conclusion drawn by the authors very convincing, regarding the new role of cavolin-1 as an individual protein instead of the main molecular component of caveolae. On a personal note, I was very impressed by the quality of STORM images (Fig. 2) which are very illuminating and useful, in particular for validating some hypotheses of the theoretical analysis.


      While this work pins down the key role of caveolin-, its mechanism remains to be further investigated. The hypotheses proposed by the authors in the discussions about the link between caveolin and lipids/cholesterol are very plausible though challenging. Even though we may feel slightly frustrated by the absence of data in this direction, the quality and merit of this paper remain.

      - The analogy with dewetting processes drawn to derive the theoretical model is very attractive. However, although part of the model has already been published several times by the same group of authors, the definition of the effective membrane rigidity of a plasma membrane including the underlying actin cortex, was very vague and confusing. Here, for the first time, thanks to the STORM analysis, the authors show that HUVECs intoxicated by ExoC3 exhibit a loose and defective cortex with a significantly increased mesh size. This argues in favor of the validity of Helfrich formalism in this context. Nonetheless, there remains a puzzle. Experimentally, several TEMs are visible within one cell. Theoretically, the authors consider a simultaneous opening of several pores and treat them in an additive manner. However, when one pore opens, the tension relaxes and should prevent the opening of subsequent pores. Yet, experimentally, as seen from the beautiful supplementary videos, several pores open one after the other. This would suggest that the tension is not homogeneous within an intoxicated cell or that equilibration times are long. One possibility is that some undegraded actin pieces of the actin cortex may form a barrier that somehow isolates one TEM from a neighboring one. Could the authors look back at their STORM data and check whether intoxicated cells do not exhibit a bimodal population of mesh sizes and possibly provide a mapping of mesh size at the scale of a cell? In particular, it is quite striking that while bending rigidity of the lipid membrane is expected to set the maximal size of the aperture, most TEMs are well delimited with actin rings before closing. Is it because the surrounding loose actin is pushed back by the rim of the aperture? Could the authors better explain why they do not consider actin as a player in TEM opening?

      - Instead of delegating to the discussion the possible link between caveolin and lipids as a mechanism for the enhanced bending rigidity provided by caveolin-1, it could be of interest for the readership to insert the attempted (and failed) experiments in the result section. For instance, did the authors try treatment with methyl-beta-cyclodextrin that extracts cholesterol (and disrupts caveolar and clathrin pits) but supposedly keeps the majority of the pool of individual caveolins at the membrane?

      - Tether pulling experiments on Plasma membrane spheres (PMS) are real tours de force and the results are quite convincing: a clear difference in bending rigidity is observed in controlled and caveolin knock-out PMS. However, one recurrent concern in these tether-pulling experiments is to be sure that the membrane pulled in the tether has the same composition as the one in the PMS body. The presence of the highly curved neck may impede or slow down membrane proteins from reaching the tether by convective or diffusive motion. Could the authors propose an experiment to demonstrate that caveolin-1 proteins are not restricted to the body of the PMS and can access to the nanometric tether?

    1. Reviewer #2 (Public Review):


      In the manuscript "Recall-Gated Consolidation: A Model for Learning and Memory in Neural Systems," the authors suggest a computational mechanism called recall-gated consolidation, which prioritizes the storage of previously experienced synaptic updates in memory. The authors investigate the mechanism with different types of learning problems including supervised learning, reinforcement learning, and unsupervised auto-associative memory. They rigorously analyse the general mechanism and provide valuable insights into its benefits.


      The authors establish a general theoretical framework, which they translate into three concrete learning problems. For each, they define an individual mathematical formulation. Finally, they extensively analyse the suggested mechanism in terms of memory recall, consolidation dynamics, and learnable timescales.

      The presented model of recall-gated consolidation covers various aspects of synaptic plasticity, memory recall, and the influence of gating functions on memory storage and retrieval. The model's predictions align with observed spaced learning effects.

      The authors conduct simulations to validate the recall-gated consolidation model's predictions, and their simulated results align with theoretical predictions. These simulations demonstrate the model's advantages over consolidating any memory and showcase its potential application to various learning tasks.

      The suggestion of a novel consolidation mechanism provides a good starting point to investigate memory consolidation in diverse neural systems and may inspire artificial learning algorithms.


      I appreciate that the authors devoted a specific section to the model's predictions, and point out how the model connects to experimental findings in various model organisms. However, the connection is rather weak and the model needs to make more specific predictions to be distinguishable from other theories of memory consolidation (e.g. those that the authors discuss) and verifiable by experimental data.

      While the article extensively discusses the strengths and advantages of the recall-gated consolidation model, it provides a limited discussion of potential limitations or shortcomings of the model, such as the missing feature of generalization, which is part of previous consolidation models. The model is not compared to other consolidation models in terms of performance and how much it increases the signal-to-noise ratio. It is only compared to a simple STM or a parallel LTM, which I understand to be essentially the same as the STM but with a different timescale (so not really an alternative consolidation model). It would be nice to compare the model to an actual or more sophisticated existing consolidation model to allow for a fairer comparison.

      The article is lengthy and dense and it could be clearer. Some sections are highly technical and may be challenging to follow. It could benefit from more concise summaries and visual aids to help convey key points.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors report the results of QM/MM simulations and kinetic measurements for the phosphoryl-transfer step in adenylate kinase. The main assertion of the paper is that a wide transition state ensemble is a key concept in enzyme catalysis as a strategy to circumvent entropic barriers. This assertion is based on the observation of a "structurally wide" set of energetically equivalent configurations that lie along the reaction coordinate in QM/MM simulations, together with kinetic measurements that suggest a decrease in the entropy of activation.

      Strengths:<br /> The study combines theoretical calculations and supporting experiments.

      Weaknesses:<br /> The role(s) of entropy in enzyme catalysis has been discussed extensively in the literature, from the Circe effect proposed by Jencks and many other works. The current paper hypothesizes a "wide" transition state ensemble as a catalytic strategy and key concept in enzyme catalysis. Overall, it is not clear the degree to which this hypothesis is supported by the data. The reasons are as follows:

      1. Enzyme catalysis reflects a rate enhancement with respect to a baseline reaction in solution. In order to assert that something is part of a catalytic strategy of an enzyme, it would be necessary to demonstrate from simulations that the activation entropy for the baseline reaction is indeed greater and the transition state ensemble less "wide". Alternatively stated, when indicating there is a "wide transition state ensemble" for the enzyme system - one needs to indicate that is with respect to the non-enzymatic reaction. However, these simulations were not performed and the comparisons were not demonstrated.

      2. The observation of a "wide conformational ensemble" is not a quantitative measure of entropy. In order to make a meaningful computational prediction of the entropic contribution to the activation of free energy, one would need to perform free energy simulations over a range of temperatures (for the enzymatic and non-enzymatic systems). Such simulations were not performed, and the entropy of activation was thus not quantified by the computational predictions.

      3. The authors indicate that lid-opening, essential for product release, and not P-transfer is the rate-limiting step in the catalytic cycle and Mg2+ accelerates both steps. How is it certain that the kinetic measurements are reporting on the chemical steps of the reaction, and not other factors such as metal ion binding or conformational changes?

      4. The authors explore different starting states for the chemical steps of the reaction (e.g., different metal ion binding and protonation states), and conclude that the most reactive enzyme configuration is the one with the more favorable reaction-free energy barrier. However, it is not clear what is the probability of observing the system in these different states as a function of pH and metal ion concentration without performing appropriate pKa and metal ion binding calculations. This was not done, and hence these results seem somewhat inconclusive.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this work, the authors sought to 1) establish a method for measuring muscle fiber subcellular structure (myofibrils) using common, non-specialized laboratory techniques and equipment, and 2) use this method to provide evidence on whether loading-induced muscle fiber growth was the result of myofibril growth (of existing myofibrils) or myofbrillogenesis (creation of new myofibrils) in mice and humans. The latter is a fundamental question in the muscle field. The authors succeeded in their aims and provided useful methods for the muscle field and detailed insight into muscle fiber hypertrophy; specifically, that loading-induced muscle fiber hypertrophy may be driven mostly by myofibrillogenesis.

      Strengths:<br /> 1) The usage of murine and human samples to provide evidence on myofibril hypertrophy vs myofibrillogenesis.<br /> 2) A nice historical perspective on myofibrillogenesis in skeletal muscle.<br /> 3) The description of a useful and tractable IHC imaging method for the muscle biology field supported by extensive validation against electron microscopy.<br /> 4) Fundamental information on how myofiber hypertrophy ensues.

      Weaknesses:<br /> 1) The usage of young growing mice (8-10 weeks) versus adult mice (>4 months) in the murine mechanical overload experiments, as well as no consideration for biological sex. The former point is partly curtailed by the adult human data that is provided (male only). Still, the usage of adult mice would be preferable for these experiments given that maturational growth may somehow affect the outcomes. For the latter point, it is not clear whether male or female mice were used.

      2) Information on whether myofibrillogenesis is dependent on hypertrophy induced by loading, or just hypertrophy in general. To provide information on this, the authors could use, for instance, inducible Myostatin KO mice (a model where hypertrophy and force production are not always in lockstep) to see whether hypertrophy independent from load induces the same result as muscle loading regarding myofibrillogenesis.

      3) Limited information on Type 1 fiber hypertrophy. A "dual overload" model is used for the mouse where the soleus is also overloaded, but presumably, the soleus was too damaged to analyze. Exploring hypertrophy of murine Type 1 fibers using a different model (weight pulling, weighted wheel running, or forced treadmill running) would be a welcome addition.

    1. Reviewer #2 (Public Review):

      With the data presented in this manuscript, the authors help complete the set of high-resolution HER2-associated complex heterodimer structures as well as HER4 homodimer structures in the presence of NRG1b and BTC. Purification of HER2-HER4 heterodimers appears to be inherently challenging due to the propensity of HER4 to form homodimers. The authors have used an effective scheme to isolate these HER2-HER4 heterodimers and have employed graphene-oxide grid chemistry to presumably overcome the issues of low sample yield for solving cryo-EM structures of these complexes. The authors conclude HER2-HER4 heterodimers with either ligand are conformationally homogeneous relative to the HER4 homodimers. The HER2-HER4 heterodimers also appear to be better stabilized compared to other published HER2 heterodimers. The ability to model glycans in the context of HER4 homodimers is exciting to see and provides a strong rationale for the stability of these structures. Overall, the work is of great interest and the methods described in this work would benefit a wide variety of structural biology projects.

      Major comments-<br /> 1. The HER2-HER4 heterodimer with BTC appears to be the lowest resolution of the reported structures. Although the authors claim the overall structure is similar to the HER2-HER4 heterodimer with NRG1b, it is therefore unclear whether the lower resolution of the BTC is due to challenging data collection conditions, sample preparation, or conformational dynamics not discernible due to the lower resolution. The authors should minimally clarify where they see the possible issues arising for the lower resolution as this is a key aspect of the work.

      2. For all maps, authors should display Euler angle plots from their final refinements to assess the degree of preferred orientation. Judging by the sphericity, it appears all the structures, except HER2-HER4-BTC, have well-sampled projection distributions. However, a formal clarification would be useful to the reader.

      3. The authors should also include map-model FSCs to ascertain the quality of the map with respect to model building, as this is currently missing in the submission.

      Minor comments-<br /> 1. With respect to complex formation, is there a reason why HER2 expression is dramatically lower than HER4?

      2. Figures S1e authors should clarify if HER2 substitutions are VR alone or do these include GD substitutions as well. These should be suitably clarified in the main text.

      3. The validation reports for all 4 reported structures suggest the user-provided FSC-derived resolutions are different from those calculated by the deposition server. Are the masks deposited significantly different compared to the ones generated within cryoSPARC?

      4. For interpretation regarding activation through phosphorylation in Figure 2e, have the authors considered HER4 could homodimerize as well? It appears from the data presented in Figure 4 and S12 that the propensity to form homodimers is greater for HER4 than to heterodimerize with HER2, despite the VR/IQ substitutions. This also appears to be supported by the reasonable amount of signal for pERK in lanes with HER4-IQ alone in the presence of NRG1b. It is recommended that the authors comment on this possibility.

      5. In the following line, "NRG1b-induced phosphorylation of HER2, HER4, ERK and AKT was not notably affected by substitution of the HER4 dimerization arm to a GS-arm relative to wild type receptors", it is unclear what the authors mean by wild-type receptors? There is presently no wild-type HER2 and/or HER4 tested in this blot.

      6. Considering the asparagine residues can potentially mediate stabilization of HER2-HER4 dimers through glycosylation, the authors should include western blot data for receptor-activation for mutants where glycosylation can be disrupted. This could minimally instruct the reader on how functionally relevant the identified interactions like N576-N358 are.

    1. Reviewer #2 (Public Review):

      In bacteria and mammals, metabolically generated aldehydes become toxic at high concentrations because they irreversibly modify the free amino group of various essential biological macromolecules. However, these aldehydes can be present in extremely high amounts in archaea and plants without causing major toxic side effects. This fact suggests that archaea and plants have evolved specialized mechanisms to prevent the harmful effects of aldehyde accumulation.

      In this study, the authors show that the plant enzyme DTD2, originating from archaea, functions as a D-aminoacyl-tRNA deacylase. This enzyme effectively removes stable D-aminoacyl adducts from tRNAs, enabling these molecules to be recycled for translation. Furthermore, they demonstrate that DTD2 serves as a broad detoxifier for various aldehydes in vivo, extending its function beyond acetaldehyde, as previously believed. Notably, the absence of DTD2 makes plants more susceptible to reactive aldehydes, while its overexpression offers protection against them. These findings underscore the physiological significance of this enzyme.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The manuscript entitled "A 2-HB-mediated feedback loop regulates muscular fatigue" by the Johnson group reports interesting findings with implications for the health benefits of exercise. The authors use a combination of metabolic/biochemical in vivo and in vitro assays to delineate a metabolic route triggered by 2-HB (a relatively stable metabolite induced by exercise in humans and mice) that controls branched-chain amino transferase enzymes and mitochondrial oxidative capacity. Mechanistically, the author shows that 2-HB is a direct inhibitor of BCAT enzymes that in turn control levels of SIRT4 activity and ADp-ribosylation in the nucleus targeting C/EBP transcription factor, affecting BCAA oxidation genes (see Fig 4i in the paper). Overall, these are interesting and novel observations and findings with relevance to human exercise, with the potential implication of using these metabolites to mimic exercise benefits, or conditions or muscular fatigue that occurs in different human chronic diseases including rheumatic diseases or long COVID.

      Weaknesses:<br /> There are several experiments/comments that will strengthen the manuscript-

      1- A final model in Figure 6 integrating the exercise/mechanistic findings, expanding on Fig 4i) will clarify the findings.

      2- In some of the graphs, statistics are missing (e.g Fig 6G).

      3- The conclusions on SIRT4 dependency should be carefully written, as it is likely that this is only one potential mechanism, further validation with mouse models would be necessary.

      4- One of the needed experiments to support the oxidative capacity effects that could be done in cultured cells, is the use of radiosotope metabolites including BCCAs to determine the ability to produce CO2. Alternatively or in combination metabolite flux using isotopes would be useful to strengthen the current results.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Jojoa-Cruz et al. determined a high-resolution cryo-EM structure in the Arabidopsis thaliana (At) OSCA3.1 channel. Based on a structural comparison between OSCA3.1 and OSCA1.2 and the difference between these two paralogs in their mechanosensitivity to poking and membrane stretch, the authors performed structural-guided mutagenesis and tested the roles of three structural domains, including an amphipathic helix, a beam-like domain, and a lipid fenestration site at the pore domain, for mechanosensation of OSCA channels.

      Strengths:<br /> The authors successfully determined a structure of the AtOSCA3.1 channel reconstituted in lipid nanodiscs by cryo-EM to a high resolution of 2.6 Å. The high-resolution EM map enabled the authors to observe putative lipid EM densities at various sites where lipid molecules are associated with the channel. Overall, the structural data provides the information for comparison with other OSCA paralogs.

      In addition, the authors identified OSCA1.2 mutants that exhibit differential responses to mechanical stimulation by poking and membrane stretch (i.e., impaired response to poke assay but intact response to membrane stretch). This interesting behavior will be useful for further study on differentiating the mechanisms of OSCA activation by distinct mechanical stimuli.

      Major weakness:<br /> 1. The major weaknesses of this study are the mutagenesis design and the functional characterization of the three structural domains - an amphipathic helix (AH), a beam-like domain (BLD), and the fenestration site at the pore, in OSCA mechanosensation.

      1) First of all, it is confusing to the reviewer, whether the authors set out to test these structural domains as a direct sensor(s) of mechanical stimuli or as a coupling domain(s) for downstream channel opening and closing (gating). The data interpretations are vague in this regard as the authors tend to interpret the effects of mutations on the channel 'sensitivity' to different mechanical stimuli (poking or membrane stretch). The authors ought to dissect the molecular bases of sensing mechanical force and opening/closing (gating) the channel pore domain for the structural elements that they want to study.

      Furthermore, the authors relied on the functional discrepancies between OSCA1.2 (sensitive to both membrane poking and stretch) and OSCA3.1 (little or weak sensitivity to poking but sensitive to membrane stretch). But the experimental data presented in the study are not clear to address the mechanisms of channel activation by poking vs. by stretch, and why the channels behave differently.

      2) The reviewer questions if the "apparent threshold" of poke-induced membrane displacement and the threshold of membrane stretch are good measures of the change in the channel sensitivity to the different mechanical stimuli.

      3) Overall, the mutagenesis design in the various structural domains lacks logical coherence and the interpretation of the functional data is not sufficient to support the authors' hypothesis. Essentially the authors mutated several residues on the hotspot domains, observed some effects on the channel response to poking and membrane stretch, then interpreted the mutated residues/regions are critical for OSCA mechanosensation. Examples are as follows.

      In the section "Mutation of key residues in the amphipathic helix", the authors mutated W75 and L80, which are located on the N- and C-terminal of the AH in OSCA1.2, and mutated Pro in the OSCA1.2 AH to Arg at the equivalent position in OSCA3.1 AH. W75 and L80 are conserved between OSCA 1.2 and OSCA3.1. Mutations of W75 and/or L80 impaired OSCA1.2 activation by poking, but not by membrane stretch. In comparison, the wildtype OSCA3.1 which contains W and L at the equivalent position of its AH exhibits little or weak response to poking. The loss of response to poking in the OSCA1.2 W/L mutants does not indicate their roles in poking-induced activation.

      Besides, the P2R mutation on OSCA1.2 AH showed no effect on the channel activation by poking, suggesting Arg in OSCA3.1 AH is not responsible for its weak response to poking. Together the mutagenesis of W75, L80, and P2R on OSCA1.2 AH does not support the hypothesis of the role of AH involved in OSCA mechanosensation.

      In the section "Replacing the OSCA3.1 BLD in OSCA1.2", the authors replaced the BLD in OSCA 1.2 with that from OSCA3.1, and only observed slightly stronger displacement by poking stimuli. The authors still suggest that BLD "appears to play a role" in the channel sensitivity to poke despite the evidence not being strong.

      OSCA1.2 has four Lys residues in TM4 and TM6b at the pore fenestration site, which were shown to interact with the lipid phosphate head group, whereas two of the equivalent residues in OSCA3.1 are Ile. In the section "Substitution of potential lipid-interacting lysine residues", the authors made K435I/K536I double mutant for OSCA1.2 to mimic OSCA3.1 and observed poor response to poking but an intact response to stretch. Did the authors mutate the Ile residues in OSCA3.1 to Lys, and did the mutation confer channel sensitivity to poking stimuli resembling OSCA1.2? The reviewer thinks it is necessary to perform such an experiment, to thoroughly suggest the importance of the four Lys residues in lipid interaction for channel mechanoactivation.

    1. Reviewer #2 (Public Review):

      Nucleosome structures inside cells remain unclear. Tan et al. tackled this problem using cryo-ET and 3-D classification analysis of yeast cells. The authors found that the fraction of canonical nucleosomes in the cell could be less than 10% of total nucleosomes. The finding is consistent with the unstable property of yeast nucleosomes and the high proportion of the actively transcribed yeast genome. The authors made an important point in understanding chromatin structure in situ. Overall, the paper is well-written and informative to the chromatin/chromosome field.

    2. Reviewer #2 (Public Review):

      Nucleosome structures inside cells remain unclear. Tan et al. tackled this problem using cryo-ET and 3-D classification analysis of yeast cells. The authors found that the fraction of canonical nucleosomes in the cell could be less than 10% of total nucleosomes. The finding is consistent with the unstable property of yeast nucleosomes and the high proportion of the actively transcribed yeast genome. The authors made an important point in understanding chromatin structure in situ. Overall, the paper is well-written and informative to the chromatin/chromosome field.

    1. Reviewer #2 (Public Review):


      The authors describe five-year outcomes of an internship program for graduate students and postdoctoral fellows at their institution spurred by pilot funding from an NIH BEST grant. They hypothesized that such a program would be beneficial to interns, internship hosts, and research advisors. The mixed methods study used surveys and focus groups to gather qualitative and quantitative data from the stakeholder groups, and the authors acknowledge the limitation that the study subjects were self-selected and also had research advisors who agreed to allow them to participate. Thus the generally favorable outcomes may not be applicable to students such as those who are struggling in the lab and/or lack career focus or supportive research advisors. Nonetheless, the overall findings support the hypothesis and also suggest additional benefits, including in some cases positive impact for the lab, improved communication between the intern and their research advisor, and an advantage for recruitment of students to the institution. The data refute one of the principal concerns of research advisors: that by taking students out of the lab, internships reduce individual and overall lab productivity. Students who did internships were significantly less likely to pursue postdoctoral fellowships before entering the biomedical workforce and were more likely to have science-related careers versus research careers than control students who did not do internships, although the study design cannot determine whether this was due to selection bias or to the internship.


      1. The sample size is good (123 internships).

      2. The internship program is well described. Outcomes are clearly defined.

      3. Methods and statistical analyses appear to be appropriate (although I am not an expert in mixed methods).

      4. "Take-home" lessons for institutions considering implementing internship programs are clearly stated.


      1. It is possible that interns, hosts, and research advisers with positive experiences were more likely to respond to surveys than those with negative experiences. The response rate and potential bias in responses should be discussed in the Results, not just given in a table legend in Methods.

      2. With regard to the biased selection of participants, do the authors know many subjects requested but were not permitted to do internships?

      3. While the authors mention internships in professional degree programs in fields such as law and business, some mention of internship practices in non-biomedical STEM PhD programs such as engineering or computer science would be helpful. Is biomedical science rediscovering lessons learned when it comes to internships?

      4. Figure 1 k, l - internships did not appear to change career goals, but are the 76% who agreed pre-internship the same individuals as the 75% who agreed post-internship? What percentage gave discordant responses?


      Overall the authors achieve their aims of describing outcomes of an internship program for graduate career development and offering lessons learned for other institutions seeking to create their own internship programs.


      The paper will be very useful for other institutions to dispel some of the concerns of research advisers about internships for PhD students (although not necessarily for postdoctoral fellows). In the long run, wider adoption of internships as part of PhD training will depend not only on faculty buy-in but also on the availability of resources and changes to the graduate school funding model so that such programs are not viewed as another "unfunded mandate" in graduate education. Perhaps the industry will be motivated to support internships by the positive outcomes for hosts reported in this paper. Additionally, NIH could allow a certain amount of F, T, or even RPG funds to be used to support internships for purposes of career development.

    1. Reviewer #2 (Public Review):

      Summary:<br /> In this paper, the authors point out that the standard approach of estimating LD is inefficient for datasets with large numbers of SNPs, with a computational cost of O(nm^2), where n is the number of individuals and m is the number of SNPs. Using the known relationship between the LD matrix and the genomic-relatedness matrix, they can calculate the mean level of LD within the genome or across genomic segments with a computational cost of O(n^2m). Since in most datasets, n<<br /> Strengths:<br /> Generally, for computational papers like this, the proof is in the pudding, and the authors appear to have been successful at their aim of producing an efficient computational tool. The most compelling evidence of this in the paper is Figure 2 and Supplementary Figure S2. In Figure 2, they report how well their X-LD estimates of LD compare to estimates based on the standard approach using PLINK. They appear to have very good agreement. In Figure S2, they report the computational runtime of X-LD vs PLINK, and as expected X-LD is faster than PLINK as long as it is evaluating LD for more than 8000 SNPs.

      Weakness:<br /> While the X-LD software appears to work well, I had a hard time following the manuscript enough to make a very good assessment of the work. This is partly because many parameters used are not defined clearly or at all in some cases. My best effort to intuit what the parameters meant often led me to find what appeared to be errors in their derivation. As a result, I am left worrying if the performance of X-LD is due to errors cancelling out in the particular setting they consider, making it potentially prone to errors when taken to different contexts.

      Impact:<br /> I feel like there is value in the work that has been done here if there were more clarity in the writing. Currently, LD calculations are a costly step in tools like LD score regression and Bayesian prediction algorithms, so a more efficient way to conduct these calculations would be useful broadly. However, given the difficulty I had following the manuscript, I was not able to assess when the authors' approach would be appropriate for an extension such as that.

    1. Reviewer #2 (Public Review):

      In this manuscript, Xiaoxuan Lin and colleagues provide new insights into the dynamics of prestin using H/D exchange coupled with mass spectrometry. The authors aim to reveal how local changes in folding upon anion binding sustain the unique electro-transduction capabilities of prestin.

      Prestin is an unusual member of the SLC26 family, that changes its cross-sectional area in the membrane upon binding of a chloride ion. In contrast to SLC26 homologs, prestin is not an anion transporter per se but requires an anion to sense voltage. Binding of Cl- at a conserved binding site located between the end of TM3 and TM10 drives the displacement of a conserved arginine (R399), that causes major conformational changes, transmitting the voltage sensing into a mechanical force exerted on the membrane.

      Cryo-EM structures are available for the protein bound to various anions, including Cl-, but these structures do not explain how a conserved couple of positive (R399) and negative (the Cl- anion) charge pair transforms voltage sensitivity into mechanical changes in the membrane. To address this challenge, the authors explore local dynamics of the anion binding site and compare it with that of a "real" anion transporter SLC26A9. The authors make a convincing case that the differences in local dynamics they measure are the molecular basis for voltage sensing and its translation into electromotility.

      Practically the authors make a thorough HDX-MS investigation of prestin in the presence of different anions Cl-, SO4-, salicylate as well as in the apo form, and provide insight mostly on local dynamics of the anion binding site. The experiments are well-designed and conducted and their quality and reproducibility allows for quantitative interpretation by deriving ΔΔG values of changes in dynamics at specific sites. Furthermore, the authors show by comparing the apo condition with Cl- bound condition that the absence of Cl- causes fraying of the TM3 and TM10 helices. They deduce that Cl- binding allows for directional helix structuration, leading to local structural changes that cause a rearrangement of the charge configuration at the anion binding site that lays the molecular basis for voltage sensitivity. They demonstrate based on a detailed analysis of their HDX data that such helix fraying is a specific feature of the binding site and differs from the cooperative unfolding happening elsewhere on the prestin.

      However, the main question that the authors are addressing is how voltage sensitivity translates at the molecular level in the requirement for a negative-positive charge pair. The interpretation that the binding site instability observed only for prestin is a feature required for this voltage dependent is a bit speculative. Could other lines of evidence support the claim that the charge ion gap is reduced upon Cl- binding and that this leads to cross-section area expansion? An obvious option that comes to mind is MD simulations There are differences in time-scale between HDX and simulations, but the propensity for H-bond destabilization can be quantified even at short timescales. It might be that such data is already available out there but it should be explicit in the discussion. The discussion section itself is a bit narrow in scope at the moment. Discussing the data in the context of the available structures would help the non-specialist reader.

    2. Reviewer #2 (Public Review):

      In this manuscript, Xiaoxuan Lin and colleagues provide new insights into the dynamics of prestin using H/D exchange coupled with mass spectrometry. The authors aim to reveal how local changes in folding upon anion binding sustain the unique electro-transduction capabilities of prestin.

      Prestin is an unusual member of the SLC26 family, that changes its cross-sectional area in the membrane upon binding of a chloride ion. In contrast to SLC26 homologs, prestin is not an anion transporter per se but requires an anion to sense voltage. Binding of Cl- at a conserved binding site located between the end of TM3 and TM10 drives the displacement of a conserved arginine (R399), that causes major conformational changes, transmitting the voltage sensing into a mechanical force exerted on the membrane.

      Cryo-EM structures are available for the protein bound to various anions, including Cl-, but these structures do not explain how a conserved couple of positive (R399) and negative (the Cl- anion) charge pair transforms voltage sensitivity into mechanical changes in the membrane. To address this challenge, the authors explore local dynamics of the anion binding site and compare it with that of a "real" anion transporter SLC26A9. The authors make a convincing case that the differences in local dynamics they measure are the molecular basis for voltage sensing and its translation into electromotility.

      Practically the authors make a thorough HDX-MS investigation of prestin in the presence of different anions Cl-, SO4-, salicylate as well as in the apo form, and provide insight mostly on local dynamics of the anion binding site. The experiments are well-designed and conducted and their quality and reproducibility allows for quantitative interpretation by deriving ΔΔG values of changes in dynamics at specific sites. Furthermore, the authors show by comparing the apo condition with Cl- bound condition that the absence of Cl- causes fraying of the TM3 and TM10 helices. They deduce that Cl- binding allows for directional helix structuration, leading to local structural changes that cause a rearrangement of the charge configuration at the anion binding site that lays the molecular basis for voltage sensitivity. They demonstrate based on a detailed analysis of their HDX data that such helix fraying is a specific feature of the binding site and differs from the cooperative unfolding happening elsewhere on the prestin.

      However, the main question that the authors are addressing is how voltage sensitivity translates at the molecular level in the requirement for a negative-positive charge pair. The interpretation that the binding site instability observed only for prestin is a feature required for this voltage dependent is a bit speculative. Could other lines of evidence support the claim that the charge ion gap is reduced upon Cl- binding and that this leads to cross-section area expansion? An obvious option that comes to mind is MD simulations There are differences in time-scale between HDX and simulations, but the propensity for H-bond destabilization can be quantified even at short timescales. It might be that such data is already available out there but it should be explicit in the discussion. The discussion section itself is a bit narrow in scope at the moment. Discussing the data in the context of the available structures would help the non-specialist reader.

    1. Reviewer #2 (Public Review):

      Summary:<br /> Bian et al studied creatine (Cr) in the context of central nervous system (CNS) function. They detected Cr in synaptic vesicles purified from mouse brains with anti-Synaptophysin using capillary electrophoresis-mass spectrometry. Cr levels in the synaptic vesicle fraction were reduced in mice lacking the Cr synthetase AGAT, or the Cr transporter SLC6A8. They provide evidence for Cr release within several minutes after treating brain slices with KCl. This KCl-induced Cr release was partially calcium-dependent and was attenuated in slices obtained from AGAT and SLC6A8 mutant mice. Cr application also decreased the excitability of cortical pyramidal cells in one third of the cells tested. Finally, they provide evidence for SLC6A8-dependent Cr uptake into synaptosomes, and ATP-dependent Cr loading into synaptic vesicles. Based on these data, the authors propose that Cr may act as a neurotransmitter in the CNS.

      Strengths:<br /> 1. A major strength of the paper is the broad spectrum of tools used to investigate Cr.<br /> 2. The study provides strong evidence that Cr is present in/loaded into synaptic vesicles.

      Weaknesses:<br /> (in sequential order)<br /> 1. Are Cr levels indeed reduced in Agat-/-? The decrease in Cr IgG in Agat-/- (and Agat+/-) is similar to the corresponding decrease in Syp (Fig. 3B). What is the explanation for this? Is the decrease in Cr in Agat-/- significant when considering the drop in IgG? The data should be normalized to the respective IgG control.<br /> 2. The data supporting that depolarization-induced Cr release is SLC6A8 dependent is not convincing because the relative increase in KCl-induced Cr release is similar between SLC6A8-/Y and SLC6A8+/Y (Fig. 5D). The data should be also normalized to the respective controls.<br /> 3. The majority (almost 3/4) of depolarization-induced Cr release is Ca2+ independent (Fig. 5G). Furthermore, KCl-induced, Ca2+-independent release persists in SLC6A8-/Y (Fig. 5G). What is the model for Ca2+-independent Cr release? Why is there Ca2+-independent Cr release from SLC6A8 KO neurons?<br /> How does this relate to the prominent decrease in Ca2+-dependent Cr release in SLC6A8-/Y (Fig. 5G)? They show a prominent decrease in Cr control levels in SLC6A8-/Y in Fig. 5D. Were the data shown in Fig. 5D obtained in the presence or absence of Ca2+? Could the decrease in Ca2+-dependent Cr release in SLC6A8-/Y (Fig. 5G) be due to decreased Cr baseline levels in the presence of Ca2+ (Fig. 5D)?<br /> 4. Cr levels are strongly reduced in Agat-/- (Fig. 6B). However, KCl-induced Cr release persists after loss of AGAT (Fig. 6B). These data do not support that Cr release is Agat dependent.<br /> 5. The authors show that Cr application decreases excitability in ~1/3 of the tested neurons (Fig. 7). How were responders and non-responders defined? What justifies this classification? The data for all Cr-treated cells should be pooled. Are there indeed two distributions (responders/non-responders)? Running statistics on pre-selected groups (Fig. 7H-J) is meaningless. Given that the effects could be seen 2-8 minutes after Cr application - at what time points were the data shown in Fig. 7E-J collected? Is the Cr group shown in Fig. 7F significantly different from the control group/wash?<br /> 6. Indirect effects: The phenotypes could be partially caused by indirect effects of perturbing the Cr/PCr/CK system, which is known to play essential roles in ATP regeneration, Ca2+ homeostasis, neurotransmission, intracellular signaling systems, axonal and dendritic transport... Similarly, high GAMT levels were reported for astrocytes (e.g., Schmidt et al. 2004; doi: 10.1093/hmg/ddh112), and changes in astrocytic Cr may underlie the phenotypes. Cr has been also reported to be an osmolyte: a hyperosmotic shock of astrocytes induced an increase in Cr uptake, suggesting that Cr can work as a compensatory osmolyte (Alfieri et al. 2006; doi: 10.1113/jphysiol.2006.115006). Potential indirect effects are also consistent with a trend towards decreased KCl-induced GABA (and Glutamate) release in SLC6A8-/Y (Fig. 5C). These indirect effects may in part explain the phenotypes seen after perturbing Agat, SLC6A8, and should be thoroughly discussed.<br /> 7. As stated by the authors, there is some evidence that Cr may act as a co-transmitter for GABAA receptors (although only at high concentrations). Would a GABAA blocker decrease the fraction of cells with decreased excitability after Cr exposure?<br /> 8. The statement "Our results have also satisfied the criteria of Purves et al. 67,68, because the presence of postsynaptic receptors can be inferred by postsynaptic responses." (l.568) is not supported by the data and should be removed.

    2. Reviewer #2 (Public Review):

      Bian et al studied creatine (Cr) in the context of central nervous system (CNS) function. They detected Cr in synaptic vesicles purified from mouse brains with anti-Synaptophysin using capillary electrophoresis-mass spectrometry. Cr levels in the synaptic vesicle fraction was reduced in mice lacking the Cr synthetase AGAT, or the Cr transporter SLC6A8. They provide evidence for Cr release within several minutes after treating brain slices with KCl. This KCl-induced Cr release was partially calcium dependent and was attenuated in slices obtained from AGAT and SLC6A8 mutant mice. Cr application also decreased the excitability of cortical pyramidal cells in one third of the cells tested. Finally, they provide evidence for SLC6A8-dependent Cr uptake into synaptosomes, and ATP-dependent Cr loading into synaptic vesicles. Based on these data, the authors propose that Cr may act as neurotransmitter in the CNS.


      1. A major strength of the paper is the broad spectrum of tools used to investigate Cr.<br /> 2. The study provides evidence that Cr is present in/loaded into synaptic vesicles.

      Weaknesses (resubmission):

      1. There is no significant decrease in Cr content pulled down by anti-Syp in AGAT-/- mice when normalized to IgG controls. Hence, blocking AGAT activity/Cr synthesis does not affect Cr levels in the synaptic vesicle fraction, arguing against a Cr enrichment.<br /> 2. There is no difference in KCl-induced Cr release between SLC6A8-/Y and SLC6A8+/Y when normalizing the data to the respective controls. Thus, the data are not consistent with the idea that depolarization-induced Cr release requires SLC6A8.<br /> 3. The rationale of grouping the excitability data into responders and non-responders is not convincing because the threshold of 10% decrease in AP rate is arbitrary. The data do therefore not support the conclusion that Cr reduces neuronal excitability.

    1. Reviewer #2 (Public Review):

      In this study the authors sought to understand the extent of similarity among species in intraspecific adaptation to environmental heterogeneity at the phenotypic and genetic levels. A particular focus was to evaluate if regions that were associated with adaptation within putative inversions in one species were also candidates for adaptation in another species that lacked those inversions. This study is timely for the field of evolutionary genomics, due to recent interest surrounding how inversions arise and become established in adaptation.

      Major strengths

      Their study system was well suited to addressing the aims, given that the different species of sunflower all had GWAS data on the same phenotypes from common garden experiments as well as landscape genomic data, and orthologous SNPs could be identified. Organizing a dataset of this magnitude is no small feat. The authors integrate many state-of-the-art statistical methods that they have developed in previous research into a framework for correlating genomic Windows of Repeated Association (WRA, also amalgamated into Clusters of Repeated Association based on LD among windows) with Similarity In Phenotype-Environment Correlation (SIPEC). The WRA/CRA methods are very useful and the authors do an excellent job at outlining the rationale for these methods.

      Major weaknesses

      The study results rely heavily on the SIPEC measure, but I found the values reported difficult to interpret biologically. For example, in Figure 4 there is a range of SIPEC from 0 to 0.03 for most species pairs, with some pairs only as high as ~0.01. This does not appear to be a high degree of similarity in phenotype-environment correlation. For example, given the equation on line 517 for a single phenotype, if one species has a phenotype-environment correlation of 1.0 and the other has a correlation of 0.02, I would postulate that these two species do not have similar evolutionary responses, but the equation would give a value of (1+0.02)*1*0.02/1 = 0.02 which is pretty typical "higher" value in Figure 4. I also question the logic behind using absolute values of the correlations for the SIPEC, because if a trait increases with an environment in one species but decreases with the environment in another species, I would not predict that the genetic basis of adaptation would be similar (as a side note, I would not question the logic behind using absolute correlations for associations with alleles, due to the arbitrary nature of signing alleles). I might be missing something here, so I look forward to reading the author's responses on these thoughts.

      An additional potential problem with the analysis is that from the way the analysis is presented, it appears that the 33 environmental variables were essentially treated as independent data points (e.g. in Figure 4, Figure 5). It's not appropriate to treat the environmental variables independently because many of them are highly correlated. For example in Figure 4, many of the high similarity/CRA values tend to be categorized as temperature variables, which are likely to be highly correlated with each other. This seems like a type of pseudo replication and is a major weakness of the framework.

      Below I highlight the main claims from the study and evaluate how well the results support the conclusions.

      * "We find evidence of significant genome-wide repeatability in signatures of association to phenotypes and environments" (abstract)<br /> * Given the questions above about SIPEC, I did not find this conclusion well supported with the way the data are presented in the manuscript.

      * "We find evidence of significant genome-wide repeatability in signatures of association to phenotypes and environments, which are particularly enriched within regions of the genome harbouring an  inversion in one species. " (Abstract) And "increased repeatability found in regions of the genome that harbour inversions" (Discussion)<br /> * These claims are supported by the data shown in Figure 4, which shows that haploblocks are enriched for WRAs. I want to clarify a point about the wording here, as my understanding of the analysis is that the authors test if *haploblocks* are enriched with *WRAs*, not whether *WRAs* are enriched for *haploblocks*. The wording of the abstract is claiming the latter, but I think what they tested was the former. Let me know if I'm missing something here.<br /> * Notwithstanding the concerns about highly correlated environments potentially inflating some of the patterns in the manuscript, to my knowledge this is the first attempt in the literature to try this kind of comparison, and the results does generally suggest that inversions are more likely capturing, rather than accumulating adaptive variation. However, I don't think the authors can claim that repeated signatures are enriched with haploblock regions, and the authors should take care to refrain from stating the relative importance of different regions of the genome to adaptation without an analysis.

      * "While a large number of genomic regions show evidence of repeated adaptation, most of the strongest signatures of association still tend to be species-specific, indicating substantial genotypic redundancy for local adaptation in these species." (Abstract)<br /> * Figure 3B certainly makes it look like there is very little similarity among species in the genetic basis of adaptation, which leaves the question as to how important the repeated signatures really are for adaptation if there are very few of them. (Is 3B for the whole genome or only that region?). This result seems to be at odds with the large number of CRAs and the claims about the importance of haploblock regions to adaptation, which extend from my previous point.

      * "we have shown evidence of significant repeatability in the basis of local adaptation (Figure 4, 5), but also an abundance of species-specific, non-repeated signatures (Figure 3)"<br /> * While the claim is a solid one, I am left wondering how much of these genomes show repeated vs. non-repeated signatures, how much of these genomes have haploblocks, and how much overlap there really is. Finding a way to intuitively represent these unknowns would greatly strengthen the manuscript.

      Overall, I think the main claims from the study, the statistical framework, and the results could be revised to better support each other.

      Although the current version of the manuscript has some potential shortcomings with regards to the statistical approaches, and the impact of this paper in its present form could be stifled because the biology tended to get lost in the statistics, these shortcomings may be addressed by the authors.

      With some revisions, the framework and data could have a high impact and be of high utility to the community.

    1. Reviewer #2 (Public Review):


      Reward and punishment learning have long been seen as emerging from separate networks of frontal and subcortical areas, often studied separately. Nevertheless, both systems are complimentary and distributed representations of rewards and punishments have been repeatedly observed within multiple areas. This raised the unsolved question of the possible mechanisms by which both systems might interact, which this manuscript went after. The authors skillfully leveraged intracranial recordings in epileptic patients performing a probabilistic learning task combined with model-based information theoretical analyses of gamma activities to reveal that information about reward and punishment was not only distributed across multiple prefrontal and insular regions, but that each system showed specific redundant interactions. The reward subsystem was characterized by redundant interactions between orbitofrontal and ventromedial prefrontal cortex, while the punishment subsystem relied on insular and dorsolateral redundant interactions. Finally, the authors revealed a way by which the two systems might interact, through synergistic interaction between ventromedial and dorsolateral prefrontal cortex.


      Here, the authors performed an excellent reanalysis of a unique dataset using innovative approaches, pushing our understanding on the interaction at play between prefrontal and insular cortex regions during learning. Importantly, the description of the methods and results is truly made accessible, making it an excellent resource to the community.

      This manuscript goes beyond what is classically performed using intracranial EEG dataset, by not only reporting where a given information, like reward and punishment prediction errors, is represented but also by characterizing the functional interactions that might underlie such representations. The authors highlight the distributed nature of frontal cortex representations and propose new ways by which the information specifically flows between nodes. This work is well placed to unify our understanding of the complementarity and specificity of the reward and punishment learning systems.


      The conclusions of this paper are mostly supported by the data, but whether the findings are entirely generalizable would require further information/analyses.

      First, the authors found that prediction errors very quickly converge toward 0 (less than 10 trials) while subjects performed the task for sets of 96 trials. Considering all trials, and therefore having a non-uniform distribution of prediction errors, could potentially bias the various estimates the authors are extracting. Separating trials between learning (at the start of a set) and exploiting periods could prove that the observed functional interactions are specific to the learning stages, which would strengthen the results.

      Importantly, it is unclear whether the results described are a common feature observed across subjects or the results of a minority of them. The authors should report and assess the reliability of each result across subjects. For example, the authors found RPE-specific interactions between vmPFC and lOFC, even though less than 10% of sites represent RPE or both RPE/PPE in lOFC. It is questionable whether such a low proportion of sites might come from different subjects, and therefore whether the interactions observed are truly observed in multiple subjects. The nature of the dataset obviously precludes from requiring all subjects to show all effects (given the known limits inherent to intracerebral recording in patients), but it should be proven that the effects were reproducibly seen across multiple subjects.

      Finally, the timings of the observed interactions between areas preclude one of the authors' main conclusions. Specifically, the authors repeatedly concluded that the encoding of RPE/PPE signals are "emerging" from redundancy-dominated prefrontal-insular interactions. However, the between-region information and transfer entropy between vmPFC and lOFC for example is observed almost 500ms after the encoding of RPE/PPE in these regions, questioning how it could possibly lead to the encoding of RPE/PPE. It is also noteworthy that the two information measures, interaction information and transfer entropy, between these areas happened at non overlapping time windows, questioning the underlying mechanism of the communication at play (see Figures 3/4). As an aside, when assessing the direction of information flow, the authors also found delays between pairs of signals peaking at 176ms, far beyond what would be expected for direct communication between nodes. Discussing this aspect might also be of importance as it raises the possibility of third-party involvement.

    1. Reviewer #2 (Public Review):

      Summary: This manuscript addresses what rapid molecular events underly the earliest responses after gravity-sensing via the sedimentation of starch-enriched amyloplasts in columella cells of the plant root cap. The LAZY or NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR) protein family is involved in this process and localizes to both the amyloplast and to the plasma membrane (PM) of columella cells.

      The current manuscript complements and extends Nishimura et al., Science, 2023. Kulich and colleagues describe the role of the LZY2 protein, also called NGR1, during this process, imaging its fast relocation and addressing additional novel points such as molecular mechanisms underlying NGR1 plasma membrane association as well as revealing the requirement of NGR1/LZY2, 3,4 for the polar localization of the AGCVIII D6 protein kinase at the PM of columella cells, in which NGR1/LZY2 acts redundantly with LZY3 and LZY4.

      The authors initially monitored relocalization of functional NGR1-GFP in columella cells of the ngr1 ngr2 ngr3 triple mutant after 180-degree reorientation of the roots. Within 10 -15 min NGR1-GFP signal disappeared from the upper PM after reorientation and reappeared at the lower PM of the reoriented cells in close proximity to the sedimented amyloplasts. Reorientation of NGR1-GFP occurred substantially faster than PIN3-GFP reorientation, at about the same time or slightly later than a rise in a calcium sensor (GCaMP3) just preceding a change in D2-Venus auxin sensor alterations. Reorientation of NGR1-GFP proved to be fast and not dependent on a brefeldin A-sensitive ARF GEF-mediated vesicle trafficking, unlike the trafficking of PIN proteins, like PIN3, or the AGCVIII D6 protein kinase. Strikingly, the PM association of NGR1-GFP was highly sensitive to pharmacological interference with sterol composition or concentration and phosphatidylinositol (4)kinase inhibition as well as dithiothreitol (DTT) treatment interfering with thioester bond formation e.g. during S-acylation. Indeed, combined mutation of a palmitoylation site and polybasic regions of NRG1 abolished its PM but not its amyloplast localization and rendered the protein non-functional during the gravitropic response, suggesting NRG1 PM localization is essential for the gravitropic response. Targeting the protein to the PM via an artificially introduced N-terminal myristoylation and an ROP2-derived polybasic region and geranylgeranylation site partially restored its functionality in the gravitropic response.

      Strengths: This timely work should be of broad interest to plant, cell and developmental biologists across the field as gravity sensing and signaling may well be of general interest. The point that NGR1 is rapidly responsive to gravistimulation, polarizes at the PM in the vicinity to amyloplast and that this is required for repolarization of D6 protein kinase, prior to PIN relocation is really compelling. The manuscript is generally well-written and accessible to a general readership. The figures are clear and of high quality, and the methods are sufficiently explained for reproduction of the experiments.

      Weaknesses: Statistical analysis has been performed for some figures but is lacking for most of the quantitative analyses in the figure legends.

      The title claims a bit more than what is actually shown in the manuscript: While auxin response reporter alterations are monitored, "rapid redirection of auxin fluxes" are not really directly addressed and, while D6PK can activate PIN proteins in other contexts, it is not explicitly shown in the manuscript that PIN3 is a target in the context of columella cells in vivo. A title such as "Rapid redirection of D6 protein kinase during Arabidopsis root gravitropism relies on plasma membrane translocation of NGR proteins" would reflect the results better.

      Fig. 4: The point that D6PK is transcytosed cannot be made here based on the data of these authors. They should have used a photoswitchable version of NGR1 to show that the same molecules observed at the upper PM are translocated to the lower PM. Nishimura and colleagues actually did that for NGR4. However, this is a lot of work and maybe for NGR1 that fusion would have too low fluorescence intensity (as it was the case for NGR3). So, I think a rewording would be sufficient such as NGR-dependent reorientation of D6PK plasma membrane localization" as this does not say, from where it comes to the lower PM. Theoretically, the signal could also be amyloplast-derived or newly synthesized (or just folded) NGR1-GFP.

      The authors make a model in which D6PK AGCVIII kinase-dependent on NGRs activates PIN3 to drive auxin fluxes. However, alterations in auxin responses are observed prior to PIN3 reorientation. They should explain this discrepancy better and clearly describe that this is a working hypothesis for the future rather than explicitly proven, yet.

    1. Reviewer #2 (Public Review):

      Summary:<br /> This study presents data from a broad range of methods (biochemical, EPR, SAXS, microscopy, etc.) on the large disordered protein FRQ relevant to circadian clocks and its interaction partners FRH and CK1, providing novel and fundamental insight into oligomerization state, local dynamics, and overall structure as a function of phosphorylation and association. Liquid-liquid phase separation is observed. These findings have bearings on the mechanistic understanding of circadian clocks, and on functional aspects of disordered proteins in general.

      Strengths:<br /> This is a thorough work that is well presented. The data are of overall high quality given the difficulty of working with an intrinsically disordered protein, and the conclusions are sufficiently circumspect and qualitative to not overinterpret the mostly low-resolution data.

      Weaknesses:<br /> None

    1. Reviewer #2 (Public Review):

      Summary:<br /> The authors use a dual optical trap instrument combined with 2-color fluorescence imaging to analyze the diffusion of RSC and ISW2 on DNA, both in the presence and absence of nucleosomes, as well as long-range nucleosome sliding by these remodelers. This allowed them to demonstrate that both enzymes can participate in 1D diffusion along DNA for rather long ranges, with ISW2 predominantly tracking the DNA strand, while RSC diffusion involves hopping. In an elegant two-color assay, the authors were able to analyze interactions of diffusing remodeler molecules, both of the same or different types, observing their collisions, co-diffusion, and bypassing. The authors demonstrate that nucleosomes act as barriers for remodeler diffusion, either repelling or sequestering them upon collision. In the presence of ATP, they observed surprisingly processive unidirectional nucleosome sliding with a strong bias in the direction opposite to where the remodeler approached the nucleosome from for ISW2. These results have fundamentally important implications for the mechanism of nucleosome positioning at promoters in vivo, will be of great interest to the scientific community, and will undoubtedly spark exciting future research.

      Strengths:<br /> The mechanism of target search for chromatin-interacting protein machines is a 'hot' topic, and this manuscript provides extremely important and timely new information about how RSC and ISW2 find the nucleosomes they slide. Intriguingly, although both remodelers analyzed in this study can diffuse along DNA, the diffusion mechanisms are substantially different, with extremely interesting mechanistic implications.<br /> The strong directional preference in nucleosome sliding by ISW2 dictated by the direction it approaches the nucleosomes from during 1D sliding on DNA is a very intriguing result with interesting implications for the regulation of nucleosome organization around promoters. It will be of great interest to the scientific community and will undoubtedly inspire future research.<br /> Relatively little is known about nucleosome sliding at longer ranges (>100bp), and this manuscript provides a unique view into such sliding and also establishes a versatile methodology for future studies.

      Weaknesses:<br /> All measurements were conducted at 5pN tension, which induces unwrapping of the outer DNA gyre from nucleosomes. This could potentially represent a limitation for experiments involving nucleosomes, since partial nucleosome unwrapping could affect the behavior of remodelers, especially their sliding of nucleosomes.

    1. Reviewer #2 (Public Review):

      In the manuscript the authors conduct a series of in vitro experiments using N-terminal and C-terminal BRAF fragments (SPR, HDX-MS, pull-down assays) to interrogate BRAF domain-specific autoinhibitory interactions and engagement by H- and KRAS GTPases. Of the three RAF isoforms, BRAF contains an extended N-terminal domain that has yet to be detected in X-ray and cryoEM reconstructions but has been proposed to interact with the KRAS hypervariable region. The investigators probe binding interactions between 4 N-terminal (NT) BRAF fragments (containing one more NT domain (BRS, RBD, and CRD)), with full-length bacterial expressed HRAS, KRAS as well as two BRAF C-terminal kinase fragments to tease out the underlying contribution of domain-specific binding events. They find, consistent with previous studies, that the BRAF BSR domain may negatively regulate RAS binding and propose that the presence of the BSR domain in BRAF provides an additional layer of autoinhibitory constraints that mediate BRAF activity in a RAS-isoform-specific manner. One of the fragments studied contains an oncogenic mutation in the kinase domain (BRAF-KDD594G). The investigators find that this mutant shows reduced interactions with an N-terminal regulatory fragment and postulate that this oncogenic BRAF mutant may promote BRAF activation by weakening autoinhibitory interactions between the N- and C-terminus.

      The manuscript is now significantly improved. The inclusion of additional controls and new experiments with KRAS strengthen the manuscript and aid in establishing RAS isoform-specific BRAF interactions.