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    1. Reviewer #1 (Public Review):

      The current manuscript focusses on the adenine phosphoribosyltransferase (Aprt) and how the lack of its function affects nervous system function. It puts it into the context of Lesch-Nyhan disease, a rare hereditary disease linked to hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Since HGPRT appears absent in Drosophila, the study focusses initially on Aprt and shows that aprt mutants have a decreased life-span and altered uric acid levels (the latter can be attenuated by allopurinol treatment). Moreover, aprt mutants show defects in locomotor reactivity behaviors. A comparable phenotype can be observed when specifically knocking down aprt in dopaminergic cells (in an adult-specific fashion). Interestingly, also glia-specific knock-down caused a similar behavioral defect, which could not be restored when re-expressing UAS-aprt, while neuronal re-expression did restore the mutant phenotype. Moreover, mutants, pan-neuronal and glia-specific RNAi for aprt caused sleep-defects. Based on immunostainings Dopamine levels are increased; UPLC shows that adenosine levels are reduced and PCR showed in increase of Ent2 levels are increased (but not AdoR). Moreover, aprt mutants display seizure-like behaviros, which can be partly restored by purine feeding (adenosine and N6-methyladenosine). Finally, expression of the human HGPRT also causes locomotor defects.

      The authors provide a wide range of genetic experimental data to assess behavior and some molecular assessment on how the defects may emerge. It is clearly written, and the arguments follow the experimental evidence that is provided.

      The findings provide a new example of how manipulating specific genes in the fruit fly allow the study of fundamental molecular processes that are linked to a human disease.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, Clark et. al. used electrophysiology approaches to measure MEC neuron activity while mice performed spatial memory tasks in one-dimensional virtual tracks, where the mice must stop in a specific reward zone for a reward. The authors identified that grid cell activity could either be anchored to the track reference frame ('task-anchored') or can maintain a periodic firing pattern independent of the track reference frame ('task-independent'). They found that in the task that requires path integration, good task performance is specifically associated with task-anchored grid cell activity.

      Strength:<br /> This study took advantage of the variation in neural activity and navigation task behaviors to answer an important question: how grid cell activity is associated with performance of spatial tasks. The mice performed individual trials where they must stop in a specific reward zone for a reward. Individual behavioral sessions could include three types of trials: (1) a visual cue at the reward location (beaconed trials), (2) no cue at the reward location (non-beaconed trials), and (3) no cue and no reward regardless of stopping (probe trials). The authors found that, interestingly, grid cell activity pattern could be anchored to task reference frame or maintain a periodic pattern independent of the reference frame. The anchoring of activity patterns could switch within a behavioral session. On the other hand, spatial firing of non-grid cells was either coherent with the grid population or was stably anchored to the task reference frame. Combining grid cell activity feature with task behaviors, they uncovered an association between the task-anchoring of grid cell activity with good performance in spatial navigation tasks that requires path integration (non-beaconed and probe trials). This work suggests the contribution of grid firing to path integration-dependent navigation.

      Weakness:<br /> It would be interesting to find out that on the trial-by-trial basis, whether the activity anchoring switched first, or the task behaviors altered first, or whether they happened within the same trial. This will potentially determine whether the encoding is causal for the behavior, or the other way around. However, based the authors explanation, their experimental design lacks sufficient statistical power to address the timing of mode switches within a trial, because task mode switching is relatively infrequent (so the n for switching is low) and only a subset of trials are uncued (making the relevant n even lower), while at a trial level the behavioral outcome is variable (increasing the required n for adequate power).

      In addition, the authors reported that the activity anchoring of some non-grid cells coherently switched with grid cells, while others do not. They propose that the MEC implement multiple coding schemes. However, it is unclear whether and how the coding scheme is associated with behavior. It would be interesting to further investigate this question.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This paper presents a compelling and comprehensive study of decision-making under uncertainty. It addresses a fundamental distinction between belief-based (cognitive neuroscience) formulations of choice behaviour with reward-based (behavioural psychology) accounts. Specifically, it asks whether active inference provides a better account of planning and decision-making, relative to reinforcement learning. To do this, the authors use a simple but elegant paradigm that includes choices about whether to seek both information and rewards. They then assess the evidence for active inference and reinforcement learning models of choice behaviour, respectively. After demonstrating that active inference provides a better explanation of behavioural responses, the neuronal correlates of epistemic and instrumental value (under an optimised active inference model) are characterised using EEG. Significant neuronal correlates of both kinds of value were found in sensor and source space. The source space correlates are then discussed sensibly, in relation to the existing literature on the functional anatomy of perceptual and instrumental decision-making under uncertainty.

      Strengths:<br /> The strengths of this work rest upon the theoretical underpinnings and careful deconstruction of the various determinants of choice behaviour using active inference. A particular strength here is that the experimental paradigm is designed carefully to elicit both information-seeking and reward-seeking behaviour; where the information-seeking is itself separated into resolving uncertainty about the context (i.e., latent states) and the contingencies (i.e., latent parameters), under which choices are made. In other words, the paradigm - and its subsequent modelling - addresses both inference and learning as necessary belief and knowledge-updating processes that underwrite decisions.

      The authors were then able to model belief updating using active inference and then look for the neuronal correlates of the implicit planning or policy selection. This speaks to a further strength of this study; it provides some construct validity for the modelling of belief updating and decision-making; in terms of the functional anatomy as revealed by EEG. Empirically, the source space analysis of the neuronal correlates licences some discussion of functional specialisation and integration at various stages in the choices and decision-making.

      In short, the strengths of this work rest upon a (first) principles account of decision-making under uncertainty in terms of belief updating that allows them to model or fit choice behaviour in terms of Bayesian belief updating - and then use relatively state-of-the-art source reconstruction to examine the neuronal correlates of the implicit cognitive processing.

      Weaknesses:<br /> The main weaknesses of this report lies in the communication of the ideas and procedures. Although the language is generally excellent, there are some grammatical lapses that make the text difficult to read. More importantly, the authors are not consistent in their use of some terms; for example, uncertainty and information gain are sometimes conflated in a way that might confuse readers. Furthermore, the descriptions of the modelling and data analysis are incomplete. These shortcomings could be addressed in the following way.

      First, it would be useful to unpack the various interpretations of information and goal-seeking offered in the (active inference) framework examined in this study. For example, it will be good to include the following paragraph:

      "In contrast to behaviourist approaches to planning and decision-making, active inference formulates the requisite cognitive processing in terms of belief updating in which choices are made based upon their expected free energy. Expected free energy can be regarded as a universal objective function, specifying the relative likelihood of alternative choices. In brief, expected free energy can be regarded as the surprise expected following some action, where the expected surprise comes in two flavours. First, the expected surprise is uncertainty, which means that policies with a low expected free energy resolve uncertainty and promote information seeking. However, one can also minimise expected surprise by avoiding surprising, aversive outcomes. This leads to goal-seeking behaviour, where the goals can be regarded as prior preferences or rewarding outcomes.

      Technically, expected free energy can be expressed in terms of risk plus ambiguity - or rearranged to be expressed in terms of expected information gain plus expected value, where value corresponds to (log) prior preferences. We will refer to both decompositions in what follows; noting that both decompositions accommodate information and goal-seeking imperatives. That is, resolving ambiguity and maximising information gain have epistemic value, while minimising risk or maximising expected value have pragmatic or instrumental value. These two kinds of values are sometimes referred to in terms of intrinsic and extrinsic value, respectively [1-4]."

      The description of the modelling of choice behaviour needs to be unpacked and motivated more carefully. Perhaps along the following lines:

      "To assess the evidence for active inference over reinforcement learning, we fit active inference and reinforcement learning models to the choice behaviour of each subject. Effectively, this involved optimising the free parameters of active inference and reinforcement learning models to maximise the likelihood of empirical choices. The resulting (marginal) likelihood was then used as the evidence for each model. The free parameters for the active inference model scaled the contribution of the three terms that constitute the expected free energy (in Equation 6). These coefficients can be regarded as precisions that characterise each subjects' prior beliefs about contingencies and rewards. For example, increasing the precision or the epistemic value associated with model parameters means the subject would update her beliefs about reward contingencies more quickly than a subject who has precise prior beliefs about reward distributions. Similarly, subjects with a high precision over prior preferences or extrinsic value can be read as having more precise beliefs that she will be rewarded. The free parameters for the reinforcement learning model included..."

      In terms of the time-dependent correlations with expected free energy - and its constituent terms - I think the report would benefit from overviewing these analyses with something like the following:

      "In the final analysis of the neuronal correlates of belief updating - as quantified by the epistemic and intrinsic values of expected free energy - we present a series of analyses in source space. These analyses tested for correlations between constituent terms in expected free energy and neuronal responses in source space. These correlations were over trials (and subjects). Because we were dealing with two-second timeseries, we were able to identify the periods of time during decision-making when the correlates were expressed.

      In these analyses, we focused on the induced power of neuronal activity at each point in time, at each brain source. To illustrate the functional specialisation of these neuronal correlates, we present whole-brain maps of correlation coefficients and pick out the most significant correlation for reporting fluctuations in selected correlations over two-second periods. These analyses are presented in a descriptive fashion to highlight the nature and variety of the neuronal correlates, which we unpack in relation to the existing EEG literature in the discussion. Note that we did not attempt to correct for multiple comparisons; largely, because the correlations observed were sustained over considerable time periods, which would be almost impossible under the null hypothesis of no correlations."

      There was a slight misdirection in the discussion of priors in the active inference framework. The notion that active inference requires a pre-specification of priors is a common misconception. Furthermore, it misses the point that the utility of Bayesian modelling is to identify the priors that each subject brings to the table. This could be easily addressed with something like the following in the discussion:

      "It is a common misconception that Bayesian approaches to choice behaviour (including active inference) are limited by a particular choice of priors. As illustrated in our fitting of choice behaviour above, priors are a strength of Bayesian approaches in the following sense: under the complete class theorem [5, 6], any pair of choice behaviours and reward functions can be described in terms of ideal Bayesian decision-making with particular priors. In other words, there always exists a description of choice behaviour in terms of some priors. This means that one can, in principle, characterise any given behaviour in terms of the priors that explain that behaviour. In our example, these were effectively priors over the precision of various preferences or beliefs about contingencies that underwrite expected free energy."

      (1) Oudeyer, P.-Y. and F. Kaplan, What is intrinsic motivation? a typology of computational approaches. Frontiers in Neurorobotics, 2007. 1: p. 6.<br /> (2) Schmidhuber, J., Formal Theory of Creativity, Fun, and Intrinsic Motivation (1990-2010). Ieee Transactions on Autonomous Mental Development, 2010. 2(3): p. 230-247.<br /> (3) Barto, A., M. Mirolli, and G. Baldassarre, Novelty or surprise? Front Psychol, 2013. 4: p. 907.<br /> (4) Schwartenbeck, P., et al., Computational mechanisms of curiosity and goal-directed exploration. Elife, 2019. 8: p. e41703.<br /> (5) Wald, A., An Essentially Complete Class of Admissible Decision Functions. Annals of Mathematical Statistics, 1947. 18(4): p. 549-555.<br /> (6) Brown, L.D., A Complete Class Theorem for Statistical Problems with Finite-Sample Spaces. Annals of Statistics, 1981. 9(6): p. 1289-1300.

    1. Reviewer #1 (Public Review):

      The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

      The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain. Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This technical report by Kugler et al., expands the application of a fluorescence-based reporter to study the conformational state of various kinases. This reporter, named KinCon (Kinase Conformation), interrogates the conformational state of a kinase (i.e., active vs. inactive) based on engineering complementary fusion proteins that fluoresce upon interaction. This assay has several advantages as it allows studying full-length kinases, that is, the kinase domain and regulatory domains, inside the cell and under various experimental conditions such as the presence of inhibitors or activator proteins, and in wildtype and mutants involved in disease states.


      One major strength of this study is that it is quite comprehensive. The authors use KinCon for four different kinases, BRAF, LKB1, RIP, and CDK4/6. These kinases have very different regulatory elements and associated proteins, which the authors explore to study their conformational state. Moreover, they use small molecule inhibitors or mutations to further dissect how the conformational state of the kinase in disease states. The collective set of results strongly suggests that KinCon is a versatile tool that can be used to study many kinases of biomedical and fundamental importance. Given that kinases are extensively studied by researchers in academia or industry, KinCon could have a broad impact as well.

      Weaknesses:<br /> This manuscript, however, also has several weaknesses. These include:

      - The manuscript is exceedingly long. For instance, the introduction provides background information for each kinase that is further expanded in the results section. I think the background information for each kinase in the Introduction and Results sections could be significantly reduced to highlight the major points. Otherwise, not only does the manuscript become too long, but the main points get diluted.

      - The figure legends are very long, providing information that is already in the main text or Methods. In the legend, the authors should provide only the essential information to understand the figure.

      - A major concern throughout the manuscript is the use of the word "dynamics," which is used in the text in various contexts. The authors should clarify what they understand about the dynamics of conformation. Are they measuring how the time-dependent process by which the kinase is interconverting between active and inactive states? It seems to me that the assays in this report evaluate a population of kinases that are in an open or close conformation (i.e., a particular state in each experimental condition) but there is no direct information on how the kinase goes from one state to the other. In that sense, the use of the word dynamics is unclear. Also, the use of the word dynamics in different sentences is ambiguous.

      - There are various other issues with terminology and presentation that also affect the overall level of impact of the manuscript.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This study provides convincing data showing that expression of the PIK3R1(delta Exon11) dominant negative mutation in Activated PI3K Delta Syndrome 1/2 (APDS1/2) patient-derived cells reduces AKT activation and p110δ protein levels. Using a 3T3-L1 model cell system, the authors show that overexpressed p85α delta Exon 11) displays reduced association with the p110α catalytic subunit but strongly interacts with Irs1/2. Overexpression of PIK3R1 dominant negative mutants inhibits AKT phosphorylation and reduces cellular differentiation of preadipocytes. The strength of this article is the clear results derived from Western blots analysis of cell signaling markers (e.g. pAKT1), and co-immunoprecipitation of PI3K holoenzyme complexes and associated regulatory factors (e.g. Irs1/2). The experimental design, interpretation, and quantification broadly support the authors' conclusions.

      Strengths:<br /> The authors analyze a variety of PIK3R1 mutants (i.e. delta Exon11, E489K, R649W, and Y657X), which reveals a range of phenotypes that support the proposed model for dominant negative activity. The use of clonal cell lines with doxycycline-induced expression of the PIK3R1 mutants (Exon 11, R649W, and Y657X) provides convincing experimental data concerning the relationship between p85α mutant expression and AKT phosphorylation in vivo. The authors convincingly show that p85α delta Exon11, R649W, or Y657X) is unable to associate with p110α but instead more strongly associates with Irs1/2 compared to wild type p85α. This helps explain why the authors were unable to purify the recombinant p110α/p85α delta Exon 11) heterodimeric complex from insect cells.

      Weaknesses:<br /> Future experimentation will be needed to reconcile the cell type specific differences (e.g. APDS2 patient-derived cells vs. the 3T3-L1 cell model system) in PIK3R1 mutant behavior reported by the authors. An unbiased proteomic study that broadly evaluates the cell signaling landscape could provide a more holistic understanding of the APDS2 and SHORT mutants compared to a candidate-based approach. Additional biochemical analysis of p110α/p85α delta Exon 11) complex is needed to explain why this mutant regulatory subunit does not strongly associate with the p110 catalytic subunit. It remains unclear why p85α delta Exon 11) expression reduces p110δ protein levels in APDS2 patient-derived dermal fibroblasts. This study would benefit from a more comprehensive biochemical analysis of the described p110α/p85α, p110β/p85α, and p110δ/p85α mutant protein complexes. The current limitation of this study to the use of a single endpoint assay to measure PI3K lipid kinase activity in the presence of a single regulatory input (i.e. RTK-derived pY peptide). A broader biochemical analysis of the mutant PI3K complexes across the canonical signaling landscape will be important for establishing how competition between wild-type and mutant regulatory subunits is regulated in different cell signaling pathways.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this manuscript, the authors suggest that Keratin 17 (K17) a component of intermediate filaments that is highly expressed in the more aggressive basal subtype of pancreatic cancer, is functionally involved in tumor promotion. They use mouse and human cell lines and overexposed wild type or mutant K17 (the latter a form that accumulates in the nuclei) and show a modest reduction in survival and increase in tumor size and metastasis. The authors use in vitro work to show that phosphorylation, through a PKC/MEK/RSK kinase cascade, leads to K17 phosphorylation and K17 disassembly.

      Strengths:<br /> K17 is an intriguing protein, as it becomes part of intermediate filaments but it has also been described to have a role in the nucleus. Whether K17 functionally drives the malignant phenotype of pancreatic cancer is unclear. Thus, the article addresses an important area of research.

      Weaknesses:<br /> Some shortcomings with the interpretation of results and the strength of the evidence provided are notes. Among those, evidence that nuclear K17 is a feature of basal pancreatic cancer in human tumors is missing. Further, the survival effects observed in the mouse experiments are modest, especially with the L3.6 cell line. Lastly, while the authors point at some potentially intriguing gene expression changes in pancreatic cancer cells expressing K17, such as the expression of genes related to epithelial mesenchymal transition (EMT) they do not provide evidence that these genes are K17 targets, not that they mediate the nuclear function of K17 in experimental models, nor that they are associated with K17-high human tumors.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This study is valuable in that it may lead to the discovery of future OA markers, etc., in that changes in glycan metabolism in chondrocytes are involved in the initiation of cartilage degeneration and early OA via hypertrophic differentiation of chondrocytes. However, more robust results would be obtained by analyzing the mechanisms and pathways by which changes in glycosylation lead to cartilage degeneration.

      Strengths:<br /> This study is important because it indicates that glycan metabolism may be associated with pre-OA and may lead to the elucidation of the cause and diagnosis of pre-OA.

      Weaknesses:<br /> More robust results would be obtained by analyzing the mechanism by which cartilage degeneration induced by changes in glycometabolism occurs.

    1. Review #1 (Public Review)

      Single-molecule visualization of chromatin remodelers on long chromatin templates-a long sought-after goal-is still in its infancy. This work describes the behaviors of two remodelers RSC and ISW2, from SWI/SNF and ISWI families respectively, with well-conducted experiments and rigorous quantitative analysis, thus representing a significant advance in the field of chromatin biology and biophysics.

    1. Reviewer #1 (Public Review):

      Summary: Here, the authors were attempting to use molecular simulation or probe the nature of how lipids, especially PIP lipids, bind to a medically-important ion channel. In particular, they look at how this binding impacts the function of the channel.

      Strengths: The study is very well written and composed. The techniques are used appropriately, with plenty of sampling and analysis. The findings are compelling, and provide clear insights into the biology of the system.

      Weaknesses: A few of the analyses are hard to understand/follow, and rely on "in house" scripts. This is particularly the case for the lipid binding events, which can be difficult to compute accurately. However the provision of these scripts on github means that these can be assessed by the reader if desired. Additionally, a lack of experimental validation, or coupling to existing experimental data, limits the study.

      It is my view that the authors have achieved their aims, and their findings are compelling and believable. Their findings should have impacts on how researchers understand the functioning of the Nav1.4 channel, as well as on the study of other ion channels and how they interact with membrane lipids.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Cyclic Nucleotide Binding (CNB) domains are pervasive structural components involved in signaling pathways across eukaryotes and prokaryotes. Despite their similar structures, CNB domains exhibit distinct ligand-sensing capabilities. The manuscript offers a thorough and convincing investigation that clarifies numerous puzzling aspects of nucleotide binding in Trypanosoma.

    1. Reviewer #1 (Public Review):

      Summary of Author's Objectives:<br /> The authors aimed to explore JMJD6's role in MYC-driven neuroblastoma, particularly in the interplay between pre-mRNA splicing and cancer metabolism, and to investigate the potential for targeting this pathway.

      Strengths:<br /> (1) The study employs a diverse range of experimental techniques, including molecular biology assays, next-generation sequencing, interactome profiling, and metabolic analysis. Moreover, the authors specifically focused on gained chromosome 17q in neuroblastoma, in combination with analyzing cancer dependency genes screened with Crispr/Cas9 library, analyzing the association of gene expression with prognosis of neuroblastoma patients with large clinical cohort. This comprehensive approach strengthens the credibility of the findings. The identification of the link between JMJD6-mediated pre-mRNA splicing and metabolic reprogramming in MYC-driven cancer cells is innovative.<br /> (2) The authors effectively integrate data from multiple sources, such as gene expression analysis, RNA splicing analysis, JMJD6 interactome assay, and metabolic profiling. This holistic approach provides a more complete understanding of JMJD6's role.<br /> (3) The identification of JMJD6 as a potential therapeutic target and its correlation with the response to indisulam have significant clinical implications, addressing an unmet need in cancer treatment.

      Weaknesses:<br /> It would be beneficial to explore whether treatment with JMJD6 inhibitors, both in vitro and in vivo, can effectively target the enhanced pre-mRNA splicing of metabolic genes in MYC-driven cancer cells. However, the authors have noted that there are currently no potent and selective JMJD6 inhibitors available.

      Appraisal of Achievement and Conclusion Support:<br /> The authors have effectively met their objectives by offering valuable insights into JMJD6's role in MYC-driven neuroblastoma. The results robustly underpin their conclusions about JMJD6's contribution to metabolic reprogramming through alternative splicing and its connection to the therapeutic response to indisulam.

      Likely Impact on the Field and Utility of Methods/Data:<br /> The study's findings have the potential to significantly impact the field of cancer research by identifying JMJD6 as a promising therapeutic target for MYC-driven cancers. The methods and data presented in the manuscript offer valuable resources to the research community for further investigations into cancer metabolism and splicing regulation.

      Additional Context for Interpretation:<br /> Understanding the complex interplay between cancer metabolism and splicing regulation is crucial for developing effective cancer treatments. This study sheds light on a previously poorly understood aspect of MYC-driven cancers and opens new avenues for targeted therapies. However, the transition from preclinical findings to clinical applications may face challenges, which should be considered in future research and clinical trials.

    1. Reviewer #1 (Public Review):

      The authors of this study seek to visualize NS1 purified from dengue virus infected cells. They infect vero cells with DV2-WT and DV2 NS1-T164S (a mutant virus previously characterized by the authors). The authors utilize an anti-NS1 antibody to immunoprecipitate NS1 from cell supernatants and then elute the antibody/NS1 complex with acid. The authors evaluate the eluted NS1 by SDS-PAGE, Native Page, mass spec, negative-stain EM, and eventually Cryo-EM. SDS-PAGE, mas spec, and native page reveal a >250 Kd species containing both NS1 and the proteinaceous component of HDL (ApoA1). The authors produce evidence to suggest that this population is predominantly NS1 in complex with ApoA1. This contrasts with recombinantly produced NS1 (obtained from a collaborator) which did not appear to be in complex with or contain ApoA1 (Figure 1C). The authors then visualize their NS1 stock in complex with their monoclonal antibody by CryoEM. For NS1-WT, the major species visualized by the authors was a ternary complex of an HDL particle in complex with an NS1 dimer bound to their mAB. For their mutant NS1-T164S, they find similar structures, but in contrast to NS1-WT, they visualize free NS1 dimers in complex with 2 Fabs (similar to what's been reported previously) as one of the major species. This highlights that different NS1 species have markedly divergent structural dynamics. It's important to note that the electron density maps for their structures do appear to be a bit overfitted since there are many regions with electron density that do not have a predicted fit and their HDL structure does not appear to have any predicted secondary structure for ApoA1. The authors then map the interaction between NS1 and ApoA1 using cross-linking mass spectrometry revealing numerous NS1-ApoA1 contact sites in the beta-roll and wing domain. The authors find that NS1 isolated from DENV infected mice is also present as a >250 kD species containing ApoA1. They further determine that immunoprecipitation of ApoA1 out of the sera from a single dengue patient correlates with levels of NS1 (presumably COIPed by ApoA1) in a dose-dependent manner.

      In the end, the authors make some useful observations for the NS1 field (mostly confirmatory) providing additional insight into the propensity of NS1 to interact with HDL and ApoA1. The study does not provide any functional assays to demonstrate activity of their proteins or conduct mutagenesis (or any other assays) to support their interaction predications. The authors assertion that higher-order NS1 exists primarily as a NS1 dimer in complex with HDL is not well supported as their purification methodology of NS1 likely introduces bias as to what NS1 complexes are isolated. While their results clearly reveal NS1 in complex with ApoA1, the lack of other NS1 homo-oligomers may be explained by how they purify NS1 from virally infected supernatant. Because NS1 produced during viral infection is not tagged, the authors use an anti-NS1 monoclonal antibody to purify NS1. This introduces a source of bias since only NS1 oligomers with their mAb epitope exposed will be purified. Further, the use of acid to elute NS1 may denature or alter NS1 structure and the authors do not include controls to test functionality of their NS1 stocks (capacity to trigger endothelial dysfunction or immune cell activation). The acid elution may force NS1 homo-oligomers into dimers which then reassociate with ApoA1 in a manner that is not reflective of native conditions. Conducting CryoEM of NS1 stocks only in the presence of full-length mAbs or Fabs also severely biases what species of NS1 is visualized since any NS1 oligomers without the B-ladder domain exposed will not be visualized. If the residues obscured by their mAb are involved in formation of higher-order oligomers then this antibody would functionally inhibit these species from forming. The absence of critical controls, use of one mAb, and acid elution for protein purification severely limits the interpretation of these data and do not paint a clear picture of if NS1 produced during infection is structurally distinct from recombinant NS1. Certainly there is novelty in purifying NS1 from virally infected cells, but without using a few different NS1 antibodies to purify NS1 stocks (or better yet a polyclonal population of antibodies) it's unclear if the results of the authors are simply a consequence of the mAb they selected.

      Data produced from numerous labs studying structure and function of flavivirus NS1 proteins provide diverse lines of evidence that the oligomeric state of NS1 is dynamic and can shift depending on context and environment. This means that the methodology used for NS1 production and purification will strongly impact the results of a study. The data in this manuscript certainly capture one of these dynamic states and overall support the general model of a dynamic NS1 oligomer that can associate with both host proteins as well as itself but the assertions of this manuscript are overall too strong given their data, as there is little evidence in this manuscript, and none available in the large body of existing literature, to support that NS1 exists only as a dimer associated with ApoA1. More likely the results of this paper are a result of their NS1 purification methodology.

      Comments on revised version:

      The authors have not adequately addressed my concerns from the original review. My major concerns are that the binding modality of NS1 to ApoA1/HDL was not validated using a mutagenesis approach and that the overarching conclusion drawn by the authors, that the major species of NS1 in vivo is a dimer in complex with ApoA1, is not supported by the data in this study given the methodology of using a single monoclonal antibody to immunoprecipitate NS1. Certainly, the structures in this manuscript are valuable in confirming that NS1 interacts with HDL and captures a snapshot of NS1/HDL interaction dynamics, but the use of only a single antibody is a major source of bias that makes it challenging to draw conclusions about the oligomeric state of NS1. Further on this point, a critically important control that is missing from this study is to determine if the anti-NS1 mAb 56.2 prevents NS1 from interacting with cells, triggering the release of proinflammatory cytokines from immune cells, or mediating endothelial dysfunction of endothelial cells. If this antibody inhibits these NS1-triggered events (linked to pathogenesis), it would suggest that the NS1 within this ternary complex is not active. Presumably some protective anti-NS1 antibodies may function by modulating the oligomeric state of NS1.

    1. Reviewer #1 (Public Review):

      Summary<br /> In this study, Xu et al. provide insights into the substrate divergence of caspase 3 and 7 (CASP3 and CASP7) for gasdermin E (GSDME) cleavage and activation during evolution in vertebrates. Using a diverse set of biochemical assays, domain swapping, site-directed mutagenesis, and bioinformatics tools, the authors demonstrate that the human GSDME C-terminal region and the S234 residue of human CASP7 are the key determinants that impede the cleavage of human GSDME by human CASP7. Their findings suggest that mutations affecting the function of caspases have enabled the functional divergence of distinct caspase family members to specialize in controlling complicated cellular functions in mammals.

      Strengths<br /> The authors made an important contribution to the field by demonstrating how human CASP7 has functionally diverged to lose the ability to cleave GSDME and showing that reverse-mutations in CASP7 can restore GSDME cleavage. The use of multiple methods to support their conclusions strengthens the authors' findings. The unbiased mutagenesis screen performed to identify S234 in huCASP7 as the determinant of its GSDME cleavability is also a strength.

      Weaknesses<br /> While the authors employed a comprehensive experimental setup to investigate the CASP7-mediated GSDME cleavage across evolution, future studies will be required to fully understand the physiological implications of this evolutionary divergence.

    1. Joint Public Review:

      Previous findings by authors show that heliomycin induces autophagy to inhibit cancer progression, while its water-soluble analogs induce apoptosis. Here, they show that one of the analogs, 4-dmH, binds to tNOX, a NADH oxidase which supports SirT1 activity, in addition to SirT1, while heliomycin only binds to SirtT1 but not tNOX, using CETSA and in silico molecular docking studies, in human oral cancer cells. The additional binding activity of 4-dmH to tNOX might explain the different biological outcome from heliomycin. 4-dmH induces ubiquitination and degradation of tNOX protein, in dependent of p53 status. The tumor suppressive effect of 4-dmH (by intra-tumoral injections) is better than heliomycin. TCGA data base analysis suggests that high tNOX mRNA expression is correlated with poor prognosis of oral cancer patients.

      This group has been a leading lab of chemical and biological characterization of heliomycin and its analogs. Their findings are interesting and advance their previous findings. The revised manuscript well responded to the reviewers' concerns.

    1. l’article L.121-1 du code del’éducationa prévu que les établissements scolaires «assurent une mission d’information sur les violences et une éducation à la sexualité ainsi qu’une obligation de sensibilisation des personnelsenseignants aux violences sexistes et sexuelles et à la formation au respect du non-consentement29»
    1. Reviewer #1 (Public Review):


      - The paper is clearly written, and all the conclusions stem from a set of 3 principles: circular topology, rotational symmetry, and noise minimization. The derivations are sound and such rigor by itself is commendable.

      - The authors provide a compelling argument on why evolution might have picked an eight-column circuit for path-integration, which is a great example of how theory can inform our thinking about the organization of neural systems for a specific purpose.

      - The authors provide a self-consistency argument on how cosine-like activity supports cosine-like connectivity with a simple Hebbian rule. However, their framework doesn't answer the question of how this system integrates angular velocity with the correct gain in the absence of allothetic cues to produce a heading estimate (more on that on point 3 below).


      - The authors make simplifying assumptions to arrive at the cosine activity/cosine connectivity circuit. Among those are the linear activation function, and cosine driving activity u. The authors provide justification for the linearization in methods 3.1, however, this ignores the well-established fact that bump amplitude is modulated by angular velocity in the fly head direction system (Turner-Evans et al 2017). In such a case, nonlinearities in the activation function cannot be ignored and would introduce harmonics in the activity. Furthermore, even though activity has been reported to be cosine-like, in fact in the fruit fly it takes the form of a somewhat concentrated activity bump (~80-100 degrees, Seelig & Jayaraman 2015; Turner-Evans et al 2017), and one has to take into account the smoothing effect of calcium dynamics too which might make the bump appear more cosine-like. So in general, it would be nice to see how the conclusions extend if the driving activity is more square-like, which would also introduce further harmonics. Overall, it would be interesting to see whether, despite the harmonics introduced by these two factors interacting in the learning rule, Oja's rule can still pick up the "base" frequency and produce sinusoidal weights (as mentioned in methods 3.8). At this point, the examples shown in Figure 5 (tabula rasa and slightly perturbed weights) are quite simple. Such a demonstration would greatly enhance the generality of the results.

      - The match of the theoretical prediction of cosine-like connectivity profiles with the connectivity data is somewhat lacking. In the locust the fit is almost perfect, however, the low net path count combined with the lack of knowledge about synaptic strengths makes this a motivating example in my opinion. In the fruit fly, the fit is not as good, and the function-fitting comparison (Methods Figure 6) is not as convincing. First, some function choices clearly are not a good fit (f1+2, f2). Second, the profile seems to be better fit by a Gaussian or other localized function, however the extra parameter of the Gaussian results in the worst AIC and AICc. To better get at the question of whether the shape of the connectivity profile matches a cosine or a Gaussian, the authors could try for example to fix the width of the Gaussian (e.g. to the variance of the best-fit cosine, which seems to match the data very well even though it wasn't itself fit), and then fit the two other parameters to the data. In that case, no AIC or AICc is needed. And then do the same for a circular distribution, e.g. von Mises. In addition, the theoretical prediction of cosine-like connectivity is not clearly stated in the abstract, introduction, or discussion. As a prediction, I believe it should be center forward, as it might be revisited again in the future in lieu of e.g. new experimental data.

      - I find the authors' claim that Oja's rule suffices to learn the insect head direction circuit (l. 273-5) somewhat misleading/vague. The authors seem to not be learning angular integration here at all. First, it is unclear to me what is the form of u(t). Is it the desired activity in the network at time t given angular velocity? This is different than modelling a population of PEN neurons jointly tuned to head direction and angular velocity, and learning weights so as to integrate angular velocity with the correct gain (Vafidis et al 2022). The learning rule here establishes a self-consistency between sinusoidal weights and activity, however, it does not learn the weights from PEN to EPG neurons so as to perform angular integration. Similar simple Hebbian rules have been used before to learn angular integration (Stringer et al 2002), however, they failed to learn the correct gain. Therefore, the authors should limit the statement that their simpler learning rule is enough to learn the circuit (l. 273-5), making sure to outline differences with the current literature (Vafidis et al 2022).

    1. Reviewer #1 (Public Review):

      Summary:<br /> Wang and co-workers characterise the fossil of Beretella spinosa from the early Cambrian, Yanjiahe Formation, South China. Combining morphological analyses with phylogenetic reconstructions, the authors conclude that B. spinosa is closely related to Saccorhytus, an enigmatic fossil recently ascribed to Ecdysozoa, or moulting animals, as an extinct "basal" lineage. Finding additional representatives of the clade Saccorhytida strengthens the idea that there existed a diversity of body plans previously underappreciated in Ecdysozoa, which may have implications for our understanding of the earliest steps in the evolution of this major animal group.

      Strengths:<br /> I'm not a paleobiologist; therefore, I cannot give an expert opinion on the descriptions of the fossils. However, the similarities with Saccorhytus seem evident, and the phylogenetic reconstructions are adequate. Evolutionary interpretations are generally justified, and the consolidation of Saccorhytida as the extinct sister lineage to extant Ecdysozoans will have significant implications for our understanding of this major animal clade.

      Weaknesses:<br /> While I generally agree with the author's interpretations, the idea of Saccorhytida as a divergent, simplified off-shot is slightly contradictory with a probably non-vermiform ecdysozoan ancestor. The author's analyses do not discard the possibility of a vermiform ecdysozoan ancestor (importantly, Supplementary Table 4 does not reconstruct that character), and outgroup comparison with Spiralia (and even Deuterostomia for Protostomia as a whole) indicates that a more or less anteroposteriorly elongated (i.e., vermiform) body is likely common and ancestral to all major bilaterian groups, including Ecdysozoa. Indeed, Figure 4b depicts the potential ancestor as a "worm". The authors argue that the simplification of Saccorhytida from a vermiform ancestor is unlikely "because it would involve considerable anatomical transformations such as the loss of vermiform organization, introvert, and pharynx in addition to that of the digestive system". However, their data support the introvert as a specialisation of Scalidophora (Figure 4a and Supplementary Table 4), and a pharyngeal structure cannot be ruled out in Saccorhytida. Likewise, loss of an anus is not uncommon in Bilateria. Moreover, this can easily become a semantics discussion (to what extent can an animal be defined as "vermiform"? Where is the limit?). Therefore, I suggest to leave the evolutionary scenario more open. Supporting Saccorhytida as a true group at the early steps of Ecdysozoa evolution is important and demonstrates that animal body plans are more plastic than previously appreciated. However, with the current data, it is unlikely that Saccorhytida represents the ancestral state for Ecdysozoa (as the authors admit), and a vermiform nature is not ruled out (and even likely) in this animal group. Suggesting that the ancestral Ecdysozoan might have been small and meiobenthic is perhaps more interesting and supported by the current data (phylogeny and outgroup comparison with Spiralia).

    1. Reviewer #1 (Public Review):

      Summary:<br /> The evolution of non-shivering thermogenesis is of fundamental importance to understand. Here, in small mammals, the contractile apparatus of the muscle is shown to increase energy expenditure upon a drop in ambient temperature. Additionally, in the state of torpor, small hibernators did not show an increase in energy expenditure under the same challenge.

      Strengths:<br /> The authors have conducted a very well-planned study that has sampled the muscles of large and small hibernators from two continents. Multiple approaches were then used to identify the state of the contractile apparatus, and its energy expenditure under torpor or otherwise.

      Weaknesses:<br /> There was only one site of biopsy from the animals used (leg). It would be interesting to know if non-shivering thermogenesis is something that is regionally different in the animal, given the core body and distal limbs have different temperatures.

    1. Reviewer #1 (Public Review):

      Ps observed 24 objects and were asked which afforded particular actions (14 action types). Affordances for each object were represented by a 14-item vector, values reflecting the percentage of Ps who agreed on a particular action being afforded by the object. An affordance similarity matrix was generated which reflected similarity in affordances between pairs of objects. Two clusters emerged, reflecting correlations between affordance ratings in objects smaller than body size and larger than body size. These clusters did not correlate themselves. There was a trough in similarity ratings between objects ~105 cm and ~130 cm, arguably reflecting the body size boundary. The authors subsequently provide some evidence that this clear demarcation is not simply an incidental reflection of body size, but likely causally related. This evidence comes in the flavour of requiring Ps to imagine themselves as small as a cat or as large as an elephant and showing a predicted shift in the affordance boundary. The manuscript further demonstrates that ChatGPT (theoretically interesting because it's trained on language alone without sensorimotor information; trained now on words rather than images) showed a similar boundary.

      The authors also conducted a small MRI study task where Ps decide whether a probe action was affordable (graspable?) and created a congruency factor according to the answer (yes/no). There was an effect of congruency in posterior fusiform and superior parietal lobule for objects within body size range, but not outside. No effects in LOC or M1.

      The major strength of this manuscript in my opinion is the methodological novelty. I felt the correlation matrices were a clever method for demonstrating these demarcations, the imagination manipulation was also exciting, and the ChatGPT analysis provided excellent food for thought. These findings are important for our understanding of the interactions between action and perception, and hence for researchers from a range of domains of cognitive neuroscience.

      The major element that limits conclusions is that an MRI study with 12 P in this context can really only provide pilot data. Certainly the effects are not strong enough for 12 P to generate much confidence. The others of my concerns have been addressed in the revision.

    1. Reviewer #1 (Public Review):

      Herzog and colleagues investigated the interactions between working memory (WM) task condition (updating, maintenance) and BMI (body-mass-index), while considering selected dopaminergic genes (COMT, Taq1A, C957T, DARPP-32). Emerging evidence suggests that there might be a specific negative association with BMI in the updating but not maintenance condition, with potential bearings to reversal reward learning in obesity. The inclusion of multiple dopaminergic genes is a strength in the present study, considering the complexity of the interactions between tonic and phasic dopamine across the brain that may distinctly associate with the component processes of WM. Here, the finding was that BMI was negatively associated with WM performance regardless of the condition (updating, maintenance), but in models including moderation by either Taq1A or DARPP-32 (but not by COMT and C957T) an interaction by task condition was observed. Furthermore, a two-way interaction effect between BMI and genotype was observed exclusively in the updating condition. These findings are in line with the accounts by which striatal dopamine as reflected by Taq1A and DARPP-32 play an important role in working memory updating, while cortical dopamine as reflected by COMT is mainly associated with maintenance. The authors conclude that the genetic moderation reflects a compound negative effect of having high BMI and a risk allele in Taq1A or DARPP-32 to working memory updating specifically.

      These data increment the accumulating evidence that the dopamine system may play an important role in obesity, but some of the claims in the present work are not entirely supported by the data and analysis presented. In particular, theoretical analysis of the extant evidence and formulation of the hypothesis remains elusive in terms of the potential mechanisms of updating/maintaining balance in obesity, and as such the interpretation of the present findings in the light of dopaminergic moderation warrants some caution. The result that Taq1A and DARPP-32 moderated the interaction between WM condition and BMI requires intricate post hoc analysis to understand the bearings to update. The authors found that Taq1A or DARPP-32 genotype moderated the negative association between BMI and WM exclusively in the update condition (significant two-way interaction effect), suggesting that the BMI-WM associations in other conditions were similar across genotypes. Importantly, visual inspection of the relationship between WM and BMI (Fig 4 & 5) suggests more prevalent positive effects of the putatively advantageous Taq1A-A1 and DARPP-32-AA genotypes to the overall negative relationship between WM and BMI in updating, but not in the other conditions. Given that an overall negative relationship was statistically supported across all conditions (model 1), a plausible interpretation would be that the updating condition stands out in terms of a positive moderation by putative advantageous genotypes, rather than compound negative consequences of BMI and genotype in updating. Critically, this interpretation stands in stark contrast with the interpretation put forth by the authors suggesting a specifically negative association between BMI and WM updating.

      In conclusion, in its current form the title of the present work is ambivalent in terms of 1) the use of the term "impaired" in the context of cognitively normal individuals, 2) a BMI group difference specifically in the updating condition, and 3) the dopaminergic mechanisms based on observational data.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors sought to understand the stage-dependent regulation of exophergenesis, a process thought to contribute to promoting neuronal proteostasis in C. elegans. Focusing on the ALMR neuron, they show that the frequency of exopher production correlates with the timing of reproduction. Using many genetic tools, they dissect the requirements of this pathway to eventually find that occupancy of the uterus acts as a signal to induce exophergenesis. Interestingly, the physical proximity of neurons to the egg zone correlates with exophergenesis frequency. The authors conclude that communication between the uterus and proximal neurons occurs through the sensing of mechanic forces of expansion normally provided by egg occupancy to coordinate exophergenesis with reproduction.

      Strengths:<br /> The genetic data presented is thorough and solid, and the observation is novel.

      Weaknesses:<br /> The main weakness of the study is that the detection of exophers is based on the overexpression of a fluorescent protein in touch neurons, and it is not clear whether this process is actually stimulated in wild-type animals, or if neurons have accumulated damaged proteins in relatively young day 2 animals.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Kimura et al performed a saturation mutagenesis study of CDKN2A to assess the functionality of all possible missense variants and compare them to previously identified pathogenic variants. They also compared their assay result with those from in silico predictors.

      Strengths:<br /> CDKN2A is an important gene that modulates cell cycle and apoptosis, therefore it is critical to accurately assess the functionality of missense variants. Overall, the paper reads well and touches upon major discoveries in a logical manner.

      Weaknesses:<br /> The paper lacks proper details for experiments and basic data, leaving the results less convincing. Analyses are superficial and do not provide variant-level resolution.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In the manuscript entitled "Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding," Chugh and co-workers utilize a suite of NMR relaxation methods to probe the dynamic landscape of the TAR RNA binding protein (TRBP) double-stranded RNA-binding domain 2 (dsRBD2) and compare these to their previously published results on TRBP dsRBD1. The authors show that, unlike dsRBD1, dsRBD2 is a rigid protein with minimal ps-ns or us-ms time scale dynamics in the absence of RNA. They then show that dsRBD2 binds to canonical A-form dsRNA with a higher affinity compared to dsRBD1 and does so without much alteration in protein dynamics. Using their previously published data, the authors propose a model whereby dsRBD2 recognizes dsRNA first and brings dsRBD1 into proximity to search for RNA bulge and internal loop structures.

      Strengths:<br /> The authors expertly use a variety of NMR techniques to probe protein motions over six orders of magnitude in time. Other NMR titration experiments and ITC data support the RNA-binding model.

      Weaknesses:<br /> The data collection and analysis are sound. The only weakness in the manuscript is the lack of context with the much broader field of RNA-binding proteins. For example, many studies have shown that RNA recognition motif (RRM) domains have similar dynamic characteristics when binding diverse RNA substrates. Furthermore, there was no discussion about the entropy of binding derived from ITC. It might be interesting to compare with dynamics from NMR.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This study investigated the co-option of IGF2BP2, an RNA-binding protein by ZIKV proteins. Designed experiments evaluated if IFG2BP2 co-localized to sites of viral RNA replication, interacted with ZIKV proteins, and how ZIKV infection changed the IGF2BP2 interactome.

      Strengths:<br /> The authors have used multiple interdisciplinary techniques to address several questions regarding the interaction of ZIKV proteins and IGF2BP2.<br /> The findings could be exciting, specifically regarding how ZIKV infection alters the interactome of IGF2BP2.

      Weaknesses:<br /> Significant concerns regarding the current state of the figures, descriptions in the figure legends, and the quality of the immunofluorescence and electron microscopy exist.

    1. Reviewer #1 (Public Review):

      The issue:<br /> The ciliates are a zoo of genetic codes, where there have been many reassignments of stop codons, sometimes with conditional meanings which include retention of termination function, and thus > 1 meaning. Thus ciliate coding provides a hotspot for the study of genetic code reassignments.

      The particular issue here is the suggestion that translation of a stop (UGA) in Blastocritihidia has been attributed to a joint change in the protein release factor that reads UGA's and also breaking a base pair at the top of the anticodon stem of tRNATrp (Nature 613, 751, 2023).

      The work:<br /> However, Swart, et al have looked into this suggestion, and find that the recently suggested mechanism is overly complicated.

      The broken pairing at the top of the anticodon stem of tRNATrp indeed accompanies the reading of UGA as Trp as previously suggested. It changes the codon translated even though the anticodon remains CCA, complementary to UGG. A compelling point is that this misreading matches previous mutational studies of E coli tRNA's, in which breaking the same base pair in a mutant tRNATrp suppressor tRNA stimulated the same kind of miscoding.

      But the amino acid change in release factor eRF1, the protein that catalyzes termination of protein biosynthesis at UGA is broadly distributed. There are about 9 organisms where this mutation can be compared with the meaning of UGA, and the changes are not highly correlated with a change in the meaning of the codon. Therefore, because UGA can be translated as Trp with or without the eRF1 mutation, Swart et al suggest that the tRNA anticodon stem change is the principal cause of the coding change.

      The review:<br /> Swart et al have a good argument. I would only add that eRF1 participation is not ruled out, because finding that UGA encodes Trp does not distinguish between encoding Trp 90% of the time and encoding it 99% of the time. The release factor could still play a measurable quantitative role, but the major inference here seems convincing.

    1. Reviewer #1 (Public Review):

      Summary: In this manuscript, the authors performed single nucleus RNA-seq for perirenal adipose tissue (PRAT) at different ages. They concluded a distinct subpopulation of adipocytes arises through beige-to-white conversion and can convert to a thermogenic phenotype upon cold exposure.

      Strengths: PRAT adipose tissue has been reported as an adipose tissue that undergoes browning. This study confirms that beige-to-white and white-to-beige conversions also exist in PRAT, as previously reported in the subcutaneous adipose tissue.

      Weaknesses:<br /> (1) There is overall a disconnection between single nucleus RNA-seq data and the lineage chasing data. No specific markers of this population have been validated by staining.<br /> (2) It would be nice to provide more evidence to support the conclusion shown in lines 243 to 245 "These results indicated that new BAs induced by cold exposure were mainly derived from UCP1- adipocytes rather than de novo ASPC differentiation in puPRAT". Pdgfra-negative progenitor cells may also contribute to these new beige adipocytes.<br /> (3) The UCP1Cre-ERT2; Ai14 system should be validated by showing Tomato and UCP1 co-staining right after the Tamoxifen treatment.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In their revised manuscript, the authors analyze the evolution of the gasdermin family and observe that the GSDMA proteins from birds, reptiles and amphibians does not form a clade with the mammalian GSDMAs. Moreover, the non-mammalian GSDMA proteins share a conserved caspase-1 cleavage motif at the predicted activation site. The authors provide several series of experiments showing that the non-mammalian GSDMA proteins can indeed be activated by caspase-1 and that this activation leads to cell death (in human cells). They also investigate the role of the caspase-1 recognition tetrapeptide for cleavage by caspase-1 and for the pathogen-derived protease SpeB.

      Strengths:<br /> The evolutionary analysis performed in this manuscript appears to use a broader data basis than what has been used in other published work. An interesting result of this analysis is the suggestion that GSDMA is evolutionary older than the main mammalian pyroptotic GSDMD, and that birds, reptiles and amphibians lack GSDMD but use GSDMA for the same purpose. The consequence that bird GSDMA should be activated by an inflammatory caspase (=caspase1) is convincingly supported by the experiments provided in the manuscript.

      The changes made by the authors in response to the previous reviewer comments are (in my opinion) sufficient.

    1. Reviewer #1 (Public Review):


      Spinal cord injury (SCI) causes immediate and prolonged bladder dysfunction, for which there are poor treatments. Following up on evidence that AMPA glutamatergic receptors play a key role in bladder function, the authors induced spinal cord injury and its attendant bladder dysfunction and examined the effects of graded doses of allosteric AMPA receptor activators (ampakines). They show that ampakines ameliorate several prominent derangements in bladder function resulting from SCI, improving voiding intervals and pressure thresholds for voiding and sphincter function.


      Well performed studies on a relevant model system. The authors induced SCI reproducibly and showed that they had achieved their model. The drugs revealed clear and striking effects. Notably, in some mice which had such bad SCI that they could not void, the drug appeared to restore voiding function.


      The studies are well conducted, but it would be helpful to include information on the kinetics of the drugs used, their half-life and how long they are present in rats after administration. What blood levels of the drugs are achieved after infusion? How do these compare with blood levels achieved when these drugs are used in humans?

    1. Reviewer #1 (Public Review):

      Summary:<br /> Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in the HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.

      Strengths:<br /> (1) Both human and mouse samples were examined.<br /> (2) The HA model was used.<br /> (3) Genome-wide DNA methylation analysis was performed.

      Weaknesses:<br /> (1) In some experiments, the selection of the control groups was not ideal.<br /> (2) More details on analyzing methods and information on replicates need to be included.<br /> (3) Discussion can be improved by comparing findings to other relevant studies.<br /> (4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.

    1. Reviewer #2 (Public Review):

      Summary:<br /> The manuscript by Morel et al. aims at identifying 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 to measure the mechanical properties of membranes and a theoretical analysis inspired from soft matter physics.

      The authors should be complimented for this multi-facetted and rigorous work. The accumulation of pieces of evidence collected from each type of approach makes very convincing the conclusion drawn by the authors on 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-1, 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, and although part of the model has already been published several times by the same group of authors, the validity of the Helfrich formalism is a key assumption that has to be explained clearly. Here, for the first time, thanks to these STORM analysis, the authors show that HUVECs intoxicated by ExoC3 exhibit a loose and defective cortex with a significantly increase mesh size, which supports this hypothesis.

    1. Reviewer #1 (Public Review):

      Summary<br /> A new method, tCFS, is introduced to offer richer and more efficient measurement of interocular suppression. It generates a new index, the suppression depth, based on the contrast difference between the up-ramped contrast for the target to breakthrough suppression and the down-ramped contrast for the target to disappear into suppression. A uniform suppression depth regardless of image types (e.g., faces, gratings and scrambles) was discovered in the paper, favoring an early-stage mechanism involving in CFS. Discussions about claims of unconscious processing and the related mechanisms.

      Strength<br /> The tCFS method adds to the existing bCFS paradigms by providing the (re-)suppression threshold and thereafter the depression depth. Benefiting from adaptive procedures with continuous trials, the tCFS is able to give fast and efficient measurements. It also provides a new opportunity to test theories and models about how information is processed outside visual awareness.

      Weakness:<br /> This paper reports the surprising finding of uniform suppression depth over a variety of stimuli. This is novel and interesting. But given the limited samples being tested, the claim of uniformity suppression depth needs to be further examined, with respect to different complexities and semantic meanings.<br /> From an intuitive aspect, the results challenged previous views about "preferential processing" for certain categories, though it invites further research to explore what exactly could suppression depth tell us about unconscious visual processing. The authors discussed about the possibility of gaining awareness according to different CRF functions in V1 and V4 neurons. But it confuses me about how the logic goes, especially from Line 713 to Line 718.

    1. Reviewer #1 (Public Review):


      This manuscript introduces an exciting way to measure SARS-CoV-2 aerosolized shedding using a disposable exhaled breath condensate collection device (EBCD). The paper draws the conclusion that the contagious shedding of the virus via aerosol route persists at a high level 8 days after symptoms.


      The methodology is potentially of high importance and the paper is clearly written. The study design is clever. If aerosolized viral load kinetics truly differed from those of nasal swabs, then this would be a very important finding.


      The study conclusions are not entirely supported by the data for several reasons:

      (1) Most data points in the study are relatively late during infection when viral loads from other compartments (nasal and oral swabs) are typically much lower than peak viral loads which often occur in the pre-symptomatic or early symptomatic phase of infection. Moreover, the generation time for SARS-CoV-2 has been estimated to be 3-4 days on average meaning that most infections occur before or very early during symptoms. Therefore, the available epidemiologic data does not support 12 days of infection (day 8 symptoms) as important for most transmissions. Therefore, many of the measurement timepoints in this study may not be relevant for transmission.

      (2) Fig 1A would be more powerful as a correlation plot between viral load from nasal samples (x-axis) and aerosol (y-axis). One would expect at least a rough correlation (as has been seen between viral loads in oral and nasal samples) and deviations from this correlation would provide crucial information about how and when aerosol shedding is discordant from nasal samples (ie early vs late time points, low versus high viral loads< etc...). It is too strong to state correspondence is 100% when viral load is only measured in one compartment and nasal swabs are reduced to the oversimplified "positive or negative".

      (3) Results are reported in RNA copies which is fine but particle-forming units (pfu, or quantitative culture) are likely a more accurate surrogate of infectivity. It is quite possible that all of these samples would have been negative for pfu given that the ratio of RNA: pfu is often >1000 (though also dynamic over time during infection). This could be another indicator that most samples in the study were collected too late during infection to represent contagious time points.

      (4) Individual kinetic curves should be shown for participants with more than three time points to demonstrate whether there are clear kinetic trends within individuals that would help further validate this approach. The inclusion of single samples from individuals is less informative.

      (5) The S-shaped model in 2A is somewhat misleading as it is fit to means but there is tremendous variability within the data. Therefore the 8-day threshold should be listed clearly as a mean but not a rule for all individuals. The statement that viral RNA copies do not decrease until 8 days from symptom onset is unlikely to be true for all infected people and can't be made based on the available data in this study given that many people contributed only one datapoint.

      (6) The incubation period for SARS-CoV-2 is highly variable. Therefore duration of symptoms is a rather poor correlate of the duration of infection. This further diminishes the interpretive value of positive samples from individuals who were only sampled once.

    1. Reviewer #1 (Public Review):

      This manuscript uses 3 large neuroimaging datasets - which together span childhood to late adulthood - to model the relationship between birthweight (BW) and cortical anatomy over time. The authors separately consider BW associations with the "height" of cortical anatomy trajectories (intercept effects) vs. BW associations with trajectory shape. They authors also distinguish between BW associations with cortical surface area (SA) and cortical thickness (CT), which together determine cortical volume (CV). Prior studies have firmly established robust positive associations between BW and cortical SA, but this study adds evidence for the protracted lifespan persistence of these associations, and the degree to which BW associations with cortical change over time are much weaker.

      The study has several strengths including: clearly motivation of this work in the Introduction and contextualization of the results in Discussion; use of three large neuroimaging datasets; inclusion of sensible sensitivity analyses; disambiguation of SA and CT findings; and use of formal spatial analysis to quantify the reproducibility of effects across cohorts.

      The primary way in which this work seeks to extend beyond established findings is to determine if BW is associated with differences in cortical change over time. The results presented clearly establish that such BW-change associations are much more localized and less consistent across cohorts that BW-intercept associations. The authors use multiple complementary approaches to verify the robustness of this inference to dataset subsampling and variation in statistical methods.

      Overall, this work provides a valuable new data point in our understanding of the profound and protracted influences that prenatal developmental features can have on postnatal outcomes.

    1. Reviewer #1 (Public Review):

      Wang and colleagues recently demonstrated the essential role of RBM24 (RNA-binding motif protein 24a) in the development of mouse hair cells (source: https://doi.org/10.1002/jcp.31003). In this study, they further expand on their findings by revealing that Rbm24 expression is absent in Pou4f3 mutant mice but not in Gfi1 mutant mice. This observation suggests that POU4F3 acts as an upstream regulator of Rbm24. The researchers effectively demonstrate that POU4F3 can bind to and regulate Rbm24 through three distant enhancers, which are located in open chromatin regions and are bound by POU4F3. Lastly, Wang and colleagues discovered that ectopic expression of Rbm24 was unable to prevent the degeneration of POU4F3 null hair cells.

      The findings in this manuscript hold great significance as they provide additional insights into the transcriptional cascades crucial for hair cell development. The discovery of enhancers capable of driving transgene expression specifically in hair cells holds promising therapeutic implications. The figures presented in the study are of excellent quality, the employed techniques are state-of-the-art, the data are accurately represented without exaggeration, and the study demonstrates a high level of rigor.

    1. Reviewer #1 (Public Review):

      Summary: This is a very meticulous and precise anatomical description of the external sensory organs in Drosophila larvae. It generates an integral and accurate map. The authors revise all the data for the abdominal and thoracic segments and describe in detail, for the first time, the head and tail segments.

      Strengths: It is a very thorough anatomical description of the external sensory organs of the genetically amenable fruitfly. This study represents a very useful tool for the research community that will definitely be used it as a reference paper. It will allow us to investigate sensory processing in depth. The discussion places the anatomical data into a functional and developmental frame.

    1. Reviewer #1 (Public Review):

      This study by Paoli et al. used a resonant scanning multiphoton microscope to examine olfactory representation in the projection neurons (PNs) of the honeybee with improved temporal resolution. PNs were classified into 9 groups based on their response patterns. Authors found that excitatory repose in the PNs precedes the inhibitory responses for ~40ms, and ~50% of PN responses contain inhibitory components. They built the neural circuit model of the mushroom body (MB) with evolutionally conserved features such as sparse representation, global inhibition, and a plasticity rule. This MB model fed with the experimental data could reproduce a number of phenomena observed in experiments using bees and other insects, including dynamical representations of odor onset and offset by different populations of Kenyon cells, prolonged representations of after-smell, different levels of odor-specificity for early/delay conditioning, and shift of behavioral timing in delay conditioning. The trace conditioning was not modeled and tested experimentally. Also, the experimental result itself is largely confirmatory to preceding studies using other organisms. Nonetheless, the experimental data and the model provide a solid basis for future studies.

    1. Reviewer #2 (Public Review):

      The present manuscript investigates the implication of locus coeruleus-noradrenaline system in the stress-induced transcriptional changes of dorsal and ventral hippocampus, combining pharmacological, chemogenetic, and optogenetic techniques. Authors have revealed that stress-induced release of noradrenaline from locus coeruleus plays a modulatory role in the expression of a large scale of genes in both ventral and dorsal hippocampus through activation of β-adrenoreceptors. Similar transcriptional responses were observed after optogenetic and chemogenetic stimulation of locus coeruleus. Among all the genes analysed, authors identified the most affected ones in response to locus coeruleus-noradrenaline stimulation as being Dio2, Ppp1r3c, Ppp1r3g, Sik1, and Nr4a1. By comparing their transcriptomic data with publicly available datasets, authors revealed that these genes were upregulated upon exposure to different stressors. Additionally, authors found that upregulation of Ppp1r3c, Ppp1r3g, and Dio2 genes following swim stress was sustained from 90 min up to 2-4 hours after stress and that it was predominantly restricted to hippocampal astrocytes, while Sik1 and Nr4a1 genes showed a broader cellular expression and a sharp rise and fall in expression, within 90 min of stress onset.

      The paper is well written and provides a useful inventory of dorsal and ventral hippocampal gene expression upregulated by activation of LC-NA system, which can be used as starting point for more functional studies related to the effects of stress-induced physiological and pathological changes. Sex-differences were also explored which represents a strength of the study.

    1. Reviewer #1 (Public Review):

      Summary: This paper suggests to apply intrinsically-motivated exploration for the discovery of robust goal states in gene regulatory networks.

      Strengths:<br /> The paper is well written. The biological motivation and the need for such methods are formulated extraordinarily well. The battery of experimental models is impressive.

      Weaknesses:<br /> (1) The proposed method is compared to the random search. That says little about the performance with regard to the true steady-state goal sets. The latter could be calculated at least for a few simple ODE (e.g., BIOMD0000000454, `Metabolic Control Analysis: Rereading Reder'). The experiment with 'oscillator circuits' may not be directly interpolated to the other models.

      The lack of comparison to the ground truth goal set (attractors of ODE) from arbitrary initial conditions makes it hard to evaluate the true performance/contribution of the method. A part of the used models can be analyzed numerically using JAX, while there are models that can be analyzed analytically.

      "...The true versatility of the GRN is unknown and can only be inferred through empirical exploration and proxy metrics....": one could perform a sensitivity analysis of the ODEs, identifying stable equilibria. That could provide a proxy for the ground truth 'versatility'.

      (2) The proposed method is based on `Intrinsically Motivated Goal Exploration Processes with Automatic Curriculum Learning', which assumes state action trajectories [s_{t_0:t}, a_{t_0:t}], (2.1 Notations and Assumptions' in the IMGEP paper). However, the models used in the current work do not include external control actions, but rather only the initial conditions can be set. It is not clear from the methods whether IMGEP was adapted to this setting, and how the exploration policy was designed w/o actual time-dependent actions. What does "...generates candidate intervention parameters to achieve the current goal...."<br /> mean considering that interventions 'Sets the initial state...' as explained in Table 2?

      (3) Fig 2 shows the phase space for (ERK, RKIPP_RP) without mentioning the typical full scale of ERK, RKIPP_RP. It is unclear whether the path from (0, 0) to (~0.575, ~3.75) at t=1000 is significant on the typical scale of this phase space. is it significant on the typical scale of this phase space?

      (4) Table 2:<br /> a. Where is 'effective intervention' used in the method?<br /> b. in my opinion 'controllability', 'trainability', and 'versatility' are different<br /> terms. If their correspondence is important I would suggest to extend/enhance the column "Proposed Isomorphism". otherwise, it may be confusing. I don't see how this table generalizes generalizes "concepts from dynamical complex systems and behavioral sciences under a common navigation task perspective".

    1. Reviewer #1 (Public Review):

      Jiang et al. demonstrated that ablating Neurexins results in alterations to glycinergic transmission and its calcium sensitivity, utilizing a robust experimental system. Specifically, the authors employed rAAV-Cre-EGFP injection around the MNTB in Nrxn1/2/3 triple conditional mice at P0, measuring Glycine receptor-dependent IPSCs from postsynaptic LSO neurons at P13-14. Notably, the authors presented a clear reduction of 60% and 30% in the amplitudes of opto- and electric stimulation-evoked IPSCs, respectively. Additionally, they observed changes in kinetics, alterations in PPR, and sensitivity to lower calcium and the calcium chelator, EGTA, indicating solid evidence for changes in presynaptic properties of glycinergic transmission.

      Furthermore, the authors uncovered an unexpected increase in sIPSC frequency without altering amplitude. Despite the reduction in evoked IPSC, immunostaining revealed an increase in GlyT2 and VGAT in TKO mice, supporting the notion of an increase in synapse number. However, the reviewer expresses caution regarding the authors' conclusion that "glycinergic neurotransmission likely by promoting the synapse formation/maintenance, which is distinct from the phenotypes observed in glutamatergic and GABAergic neurons (Chen et al., 2017; Luo et al., 2021)", as outlined in lines 173-175. The reviewer suggests that this statement may be overstated, pointing out the authors' own discussion in lines 254-265, which acknowledges multiple possibilities, including the potential that the increase in synapses is a consequence rather than a causal effect of Nrxn deletion.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Compared with conventional SQUID-MEG, OPM-MEG offers theoretical advantages of sensor configurability (that is, sizing to suit the head size) and motion tolerance (the sensors are intrinsically in the head reference frame). This study purports to be the first to experimentally demonstrate these advantages in a developmental study from age 2 to age 34.

      In short, while the theoretical advantages of OPM-MEG are attractive - both in terms of young child sensitivity and in terms of motion tolerance - neither was in fact demonstrated in this manuscript. We are left with a replication of SQUID-MEG observations, which certainly establishes OPM-MEG as "substantially equivalent" to conventional technology but misses the opportunity to empirically demonstrate the much-discussed theoretical advantages/opportunities.

      Strengths:<br /> A replication of SQUID-MEG observations, which certainly establishes OPM-MEG as "substantially equivalent" to conventional technology but misses the opportunity to empirically demonstrate the much-discussed theoretical advantages/opportunities.

      Weaknesses:<br /> The authors describe 64 tri-axial detectors, which they refer to as 192 channels. This is in keeping with some of the SQUID-MEG description, but possibly somewhat disingenuous. For the scientific literature, perhaps "64 tri-axial detectors" is a more parsimonious description.

      A small fraction (<20%) of trials were eliminated for analysis because of "excess interference" - this warrants further elaboration.

      Figure 3 shows a reduced beta ERD in the youngest children. Although the authors claim that OPM-MEG would be similarly sensitive for all ages and that SQUID-MEG would be relatively insensitive to young children, one trivial counterargument that needs to be addressed is that OPM has NOT in fact increased the sensitivity to young child ERD. This can possibly be addressed by analogous experiments using a SQUID-based system. An alternative would be to demonstrate similar sensitivity across ages using OPM to a brain measure such as evoked response amplitude. In short, how does Figure 3 demonstrate the (theoretical) sensitivity advantage of OPM MEG in small heads ?

      The data do not make a compelling case for the motion tolerance of OPM-MEG. Although an apparent advantage of a wearable system, an empirical demonstration is still lacking. How was motion tracked in these participants?

      Furthermore, while the introduction discusses at some length the phenomenon of PMBR, there is no demonstration of the recording of PMBR (or post-sensory beta rebound). This is a shame because there is literature suggesting an age-sensitivity to this, that the optimal sensitivity of OPM-MEG might confirm/refute. There is little evidence in Figure 3 for adult beta rebound. Is there an explanation for the lack of sensitivity to this phenomenon in children/adolescents ? Could a more robust paradigm (button-press) have shed light on this?

      Data on functional connectivity are valuable but do not rely on OPM recording. They further do not add strength to the argument that OPM MEG is more sensitive to brain activity in smaller heads - in fact, the OPM recordings seem plagued by the same insensitivity observed using conventional systems.

      The discussion of burst vs oscillations, while highly relevant in the field, is somewhat independent of the OPM recording approach and does not add weight to the OPM claims.

      In short, while the theoretical advantages of OPM-MEG are attractive - both in terms of young child sensitivity and in terms of motion tolerance, neither was in fact demonstrated in this manuscript. We are left with a replication of SQUID-MEG observations, which certainly establishes OPM-MEG as "substantially equivalent" to conventional technology but misses the opportunity to empirically demonstrate the much-discussed theoretical advantages/opportunities.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In the manuscript titled "Coevolution due to physical interactions is not a major driving force behind evolutionary rate covariation" by Little et al., explores the potential contribution of physical interaction between correlated evolutionary rates among gene pairs. They find that physical interaction is not the main driving of evolutionary rate covariation (ECR). This finding is similar to a previous report by Clark et al. (2012), Genome Research, wherein the authors stated that "direct physical interaction is not required to produce ERC." The previous study used 18 Saccharomycotina yeast species, whereas the present study used 332 Saccharomycotina yeast species and 11 outgroup taxa. As a result, the present study is better positioned to evaluate the interplay between physical interaction and ECR more robustly.

      Strengths & Weaknesses:<br /> Various analyses nicely support the authors' claims.

    1. Reviewer #1 (Public Review):

      The manuscript by Park et. al. examines the interaction of macrophages with SARS-CoV-2 spike protein and subsequent inflammatory reactions. The authors demonstrate that following intranasal delivery of spike it rapidly accumulates in alveolar macrophages. Inflammation associated with internalized spike recruits neutrophils to the lung, where they undergo a cell death process consistent with NETosis. The authors demonstrate that modifications spike to contain high mannose reduces uptake of spike protein and limits the inflammation induced. This finding could have implications for vaccine development, as vaccines containing modified spike could be safer and better tolerated.

      The authors use a number of different techniques, including in vivo modeling, imaging, human and murine systems to interrogate their hypotheses. These systems provide robust supporting information for their conclusions. There are two key aspects from the current manuscript which would add key evidence. The authors suggest that neutrophils exposed to spike protein undergo a process of NETosis. To confirm this hypothesis inhibitors of NETosis should be used to demonstrate that the cell death is prevented. Additionally, vaccination of a murine model with the modified spike protein would add additional support to the conclusion that modified spike protein would be less inflammatory while maintaining its utility as a vaccine antigen.

    1. Joint Public Review:

      Using Ts65Dn - the most commonly used mouse model of Down syndrome (DS) - the goal of this study is two-pronged: 1) to conduct a thorough assessment of DS-related genotypic, physiological, behavioral, and phenotypic measures in a longitudinal manner; and 2) to measure the effects of chronic GTE-EGCG on these measures in the Ts65Dn mouse model. Corroborating results from several previous studies on Ts65Dn mice, findings of this study show confirm the Ts65Dn mouse model exhibits the suite of traits associated with DS. The findings also suggest that the mouse model might have experienced drift, given the milder phenotypes than those reported by earlier studies. Results of the GTE-EGCG treatment do not support its therapeutic use and instead show that the treatment exacerbated certain DS-related phenotypes.


      The authors performed a rigorous assessment of treatment and examined treatment and genotypic alterations at multiple time points during growth and aging. Detailed analysis shows differences in genotype during aging as well as genotype with treatment. This study is solid in the overarching methodological approach (with the exception of RNAseq, described below). The biggest strength of the study is its approach and dataset, which corroborate results from a multitude of past studies on Ts65Dn mice, albeit on adult specimens.

      Comments on revised submission:

      The authors have made numerous changes to address the concerns of the reviewers. The strengths remain: a large, longitudinal data set for the Ts65Dn mouse model across multiple organ systems. The results also clearly show the impact of GTE-EGCG treatment and do not support its therapeutic use.

      The authors should report their a priori power calculations that they used when designing their experiment. This should be added to either the Animals or Statistics subsections of the Methods.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, Yan et al. investigate the molecular bases underlying mating type recognition in Tetrahymena thermophila. This model protist possesses a total of 7 mating types/sexes and mating occurs only between individuals expressing different mating types. The authors aimed to characterize the function of mating type proteins (MTA and MTB) in the process of self- and non-self recognition, using a combination of elegant phenotypic assays, protein studies, and imaging. They showed that the presence of MTA and MTB in the same cell is required for the expression of concavalin-A receptors and for tip transformation - two processes that are characteristic of the costimulation phase that precedes cell fusion. Using protein studies, the authors identify a set of additional proteins of varied functions that interact with MTA and MTB and are likely responsible for the downstream signaling processes required for mating. This is a description of a fascinating self- and non-self-recognition system and, as the authors point out, it is a rare example of a system with numerous mating types/sexes. This work opens the door for the further understanding of the molecular bases and evolution of these complex recognition systems within and outside protists.

      The results shown in this study point to the unequivocal requirement of MTA and MTB proteins for mating. Nevertheless, some of the conclusions regarding the mode of functioning of these proteins are not fully supported and require additional investigation.

      Strengths:<br /> (1) The authors have established a set of very useful knock-out and reporter lines for MT proteins and extensively used them in sophisticated and well-designed phenotypic assays that allowed them to test the role of these proteins in vivo.

      (2) Despite their apparent low abundance, the authors took advantage of a varied set of protein isolation and characterization techniques to pinpoint the localization of MT proteins to the cell membrane, and their interaction with multiple other proteins that could be downstream effectors. This opens the door for the future characterization of these proteins and further elucidation of the mating type recognition cascade.

      Weaknesses:<br /> The manuscript is structured and written in a very clear and easy-to-follow manner. However, several conclusions and discussion points fall short of highlighting possible models and mechanisms through which MT proteins control mating type recognition:

      (1) The authors dismiss the possibility of a "simple receptor-ligand system", even though the data does not exclude this possibility. The model presented in Figure 2 S1, and on which the authors based their hypothesis, assumes the independence of MTA and MTB proteins in the generation of the intracellular cascade. However, the results presented in Figure 2 show that both proteins are required to be active in the same cell. Coupled with the fact that MTA and MTB proteins interact, this is compatible with a model where MTA would be a ligand and MTB a receptor (or vice-versa), and could thus form a receptor-ligand complex that could potentially be activated by a non-cognate MTA-MTB receptor-ligand complex, leading to an intracellular cascade mediated by the identified MRC proteins. As it stands, it is not clear what is the proposed working model, and it would be very beneficial for the reader for this to be clarified by having the point of view of the authors on this or other types of models.

      (2) The presence of MTA/MTB proteins is required for costimulation (Figure 2), and supplementation with non-cognate extracellular fragments of these proteins (MTAxc, or MTBxc) is a positive stimulator of pairing. However, alone, these fragments do not have the ability to induce costimulation (Figure 5). Based on the results in Figures 5 and 6 the authors suggest that MT proteins mediate both self and non-self recognition. Why do MTAxc and MTBxc not induce costimulation alone? Are any other components required? How to reconcile this with the results of Figure 2? A more in-depth interpretation of these results would be very helpful, since these questions remain unanswered, making it difficult for the reader to extract a clear hypothesis on how MT proteins mediate self- and non-self-recognition.

    1. Langes Interview mit Hans Joachim Schellnhuber im Standard, under anderem zu Kipppunkten und der Möglichkeit, dass wir uns schon auf dem Weg in ein „neues Klimaregime“ befinden. Schellnhuber geht davon aus, dass auch das 2°-Ziel überschritten werden wird. Der „Königsweg“, um der Atmosphäre danach wieder CO<sub>2</sub> zu entziehen, sei der weltweite Ersatz von Zement durch Holz beim Bauen, den er als Direktor des IIASA vor allem erforschen wolle. Die Wahrscheinlichkeit dafür, dass „noch alles gutgehen" werde, sei gering. https://www.derstandard.at/story/3000000204635/klimaforscher-schellnhuber-werden-auch-ueber-das-zwei-grad-ziel-hinausschiessen

  2. Feb 2024
    1. Reviewer #1 (Public Review):

      Summary:<br /> The manuscript, titled "Allosteric Modulation of the CXCR4:CXCL12 Axis by Targeting Receptor Nanoclustering via the TMV-TMVI Domain," presents a compelling investigation into the development of a potential anti-cancer therapeutic agent. The study focuses on targeting specific CXCR4 intermolecular interactions via an allosteric antagonist which binds proximal to the orthosteric ligand binding site. The novel compounds developed aim to mitigate tumor dissemination, proliferation, and metastasis in transgenic Zebrafish models implanted with HeLa cells.

      Strengths:<br /> The study holds significant promise, offering a novel approach to addressing the targeted modulation of CXCR4. The multidisciplinary methodology employed is commendable, providing a comprehensive understanding of the underlying molecular interactions. The proposed workflow, although requiring some adjustments, is reasonable and has the potential to make a substantial impact in the field.

      Weaknesses:<br /> Despite the brilliance of the concept and its potential impact, the computational approach appears somewhat superficial and lacks essential considerations. A comprehensive revision of the computational methodology is strongly recommended, with a focus on addressing key points. Additionally, the experimental section should be modified accordingly to align with the refined results. While the study's foundations are promising, its current state warrants a thorough revision to enhance its scientific rigor and overall robustness.

    1. Reviewer #1 (Public Review):


      This study presents fundamental new insights into vesicular monoamine transport and the binding pose of the clinical drug tetrabenazine (TBZ) to the mammalian VMAT2 transporter. Specifically, this study reports the first structure for the mammalian VMAT (SLC18) family of vesicular monoamine transporters. It provides insights into the mechanism by which this inhibitor traps VMAT2 into a 'dead-end' conformation. The structure also provides some evidence for a novel gating mechanism within VMAT2, which may have wider implications for understanding the mechanism of transport in the wider SLC18 family.


      The structure is high quality, and the method used to determine the structure via fusing mVenus and the anti-GFP nanobody to the amino and carboxyl termini is novel. The binding and transport data are convincing and provide new insights into the role of conserved side chains within the SLC18 members. The binding position of TBZ is of high value, given its role in treating Huntington's chorea and for being a 'dead-end' inhibitor for VMAT2.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Jin et al. investigated how the bacterial DNA damage (SOS) response and its regulator protein RecA affect the development of drug resistance under short-term exposure to beta-lactam antibiotics. Canonically, the SOS response is triggered by DNA damage, which results in the induction of error-prone DNA repair mechanisms. These error-prone repair pathways can increase mutagenesis in the cell, leading to the evolution of drug resistance. Thus, inhibiting the SOS regulator RecA has been proposed as a means to delay the rise of resistance.

      In this paper, the authors deleted the RecA protein from E. coli and exposed this ∆recA strain to selective levels of the beta-lactam antibiotic, ampicillin. After an 8-hour treatment, they washed the antibiotic away and allowed the surviving cells to recover in regular media. They then measured the minimum inhibitory concentration (MIC) of ampicillin against these treated strains. They note that after just 8-hour treatment with ampicillin, the ∆recA had developed higher MICs towards ampicillin, while by contrast, wild-type cells exhibited unchanged MICs. This MIC increase was also observed in subsequent generations of bacteria, suggesting that the phenotype is driven by a genetic change.

      The authors then used whole genome sequencing (WGS) to identify mutations that accounted for the resistance phenotype. Within resistant populations, they discovered key mutations in the promoter region of the beta-lactamase gene, ampC; in the penicillin-binding protein PBP3 which is the target of ampicillin; and in the AcrB subunit of the AcrAB-TolC efflux machinery. Importantly, mutations in the efflux machinery can impact the resistance towards other antibiotics, not just beta-lactams. To test this, they repeated the MIC experiments with other classes of antibiotics, including kanamycin, chloramphenicol, and rifampicin. Interestingly, they observed that the ∆recA strains pre-treated with ampicillin showed higher MICs towards all other antibiotics tested. This suggests that the mutations conferring resistance to ampicillin are also increasing resistance to other antibiotics.

      The authors then performed an impressive series of genetic, microscopy, and transcriptomic experiments to show that this increase in resistance is not driven by the SOS response, but by independent DNA repair and stress response pathways. Specifically, they show that deletion of the recA reduces the bacterium's ability to process reactive oxygen species (ROS) and repair its DNA. These factors drive the accumulation of mutations that can confer resistance to different classes of antibiotics. The conclusions are reasonably well-supported by the data, but some aspects of the data and the model need to be clarified and extended.

      Strengths:<br /> A major strength of the paper is the detailed bacterial genetics and transcriptomics that the authors performed to elucidate the molecular pathways responsible for this increased resistance. They systemically deleted or inactivated genes involved in the SOS response in E. coli. They then subjected these mutants to the same MIC assays as described previously. Surprisingly, none of the other SOS gene deletions resulted in an increase in drug resistance, suggesting that the SOS response is not involved in this phenotype. This led the authors to focus on the localization of DNA PolI, which also participates in DNA damage repair. Using microscopy, they discovered that in the RecA deletion background, PolI co-localizes with the bacterial chromosome at much lower rates than wild-type. This led the authors to conclude that deletion of RecA hinders PolI and DNA repair. Although the authors do not provide a mechanism, this observation is nonetheless valuable for the field and can stimulate further investigations in the future.

      In order to understand how RecA deletion affects cellular physiology, the authors performed RNA-seq on ampicillin-treated strains. Crucially, they discovered that in the RecA deletion strain, genes associated with antioxidative activity (cysJ, cysI, cysH, soda, sufD) and Base Excision Repair repair (mutH, mutY, mutM), which repairs oxidized forms of guanine, were all downregulated. The authors conclude that down-regulation of these genes might result in elevated levels of reactive oxygen species in the cells, which in turn, might drive the rise of resistance. Experimentally, they further demonstrated that treating the ∆recA strain with an antioxidant GSH prevents the rise of MICs. These observations will be useful for more detailed mechanistic follow-ups in the future.

      Weaknesses:<br /> Throughout the paper, the authors use language suggesting that ampicillin treatment of the ∆recA strain induces higher levels of mutagenesis inside the cells, leading to the rapid rise of resistance mutations. However, as the authors note, the mutants enriched by ampicillin selection can play a role in efflux and can thus change a bacterium's sensitivity to a wide range of antibiotics, in what is known as cross-resistance. The current data is not clear on whether the elevated "mutagenesis" is driven ampicillin selection or by a bona fide increase in mutation rate.

      Furthermore, on a technical level, the authors employed WGS to identify resistance mutations in the treated ampicillin-treated wild-type and ∆recA strains. However, the WGS methodology described in the paper is inconsistent. Notably, wild-type WGS samples were picked from non-selective plates, while ΔrecA WGS isolates were picked from selective plates with 50 μg/mL ampicillin. Such an approach biases the frequency and identity of the mutations seen in the WGS and cannot be used to support the idea that ampicillin treatment induces higher levels of mutagenesis.

      Finally, it is important to establish what the basal mutation rates of both the WT and ∆recA strains are. Currently, only the ampicillin-treated populations were reported. It is possible that the ∆recA strain has inherently higher mutagenesis than WT, with a larger subpopulation of resistant clones. Thus, ampicillin treatment might not in fact induce higher mutagenesis in ∆recA.

    1. Reviewer #1 (Public Review):

      In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

      Comments on revised version:

      For Figure 4, immunofluorecent staining of LBH following intracavernous injections with lentiviruses is required to justify overexpression and tissue specificity.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors utilize fluid-structure interaction analyses to simulation fluid flow within and around the Cambrian cnidarian Quadrapyrgites to reconstruct feeding/respiration dynamics. Based on vorticity and velocity flow patterns, the authors suggest that the polyp expansion and contraction ultimately develop vortices around the organism that are like what modern jellyfish employ for movement and feeding. Lastly, the authors suggest that this behavior is likely a prerequisite transitional form to swimming medusae.

      Strengths:<br /> While fluid-structure-interaction analyses are common in engineering, physics, and biomedical fields, they are underutilized in the biological and paleobiological sciences. Zhang et al. provide a strong approach to integrating active feeding dynamics into fluid flow simulations of ancient life. Based on their data, it is entirely likely the described vortices would have been produced by benthic cnidarians feeding/respiring under similar mechanisms. However, some of the broader conclusions require additional justification.


      1. The claim that olivooid-type feeding was most likely a prerequisite transitional form to jet-propelled swimming needs much more support or needs to be tailored to olivooids. This suggests that such behavior is absent (or must be convergent) before olivooids, which is at odds with the increasing quantities of pelagic life (whose modes of swimming are admittedly unconstrained) documented from Cambrian and Neoproterozoic deposits. Even among just medusozoans, ancestral state reconstruction suggests that they would have been swimming during the Neoproterozoic (Kayal et al., 2018; BMC Evolutionary Biology) with no knowledge of the mechanics due to absent preservation.<br /> 2. While the lack of ambient flow made these simulations computationally easier, these organisms likely did not live in stagnant waters even within the benthic boundary layer. The absence of ambient unidirectional laminar current or oscillating current (such as would be found naturally) biases the results.<br /> 3. There is no explanation for how this work could be a breakthrough in simulation gregarious feeding as is stated in the manuscript.

      Despite these weaknesses the authors dynamic fluid simulations convincingly reconstruct the feeding/respiration dynamics of the Cambrian Quadrapyrgites, though the large claims of transitionary stages for this behavior are not adequately justified. Regardless, the approach the authors use will be informative for future studies attempting to simulate similar feeding and respiration dynamics.

      The following text is directly in response to the revised version of the manuscript.<br /> Dynamic simulations of feeding and respiration of the early Cambrian periderm-bearing cnidarian polyps

      Revision 1

      I think this manuscript has been improved by the authors, and I appreciate their time and effort in considering my earlier comments. While most of my line by line comments have been incorporated, I do feel that some of my larger points have been insufficiently addressed. Those are repeated with additional clarifications below.

      Original comment: The claim that olivooid-type feeding was most likely a prerequisite transitional form to jet-propelled swimming needs much more support or needs to be tailored to olivooids. This suggests that such behavior is absent (or must be convergent) before olivooids, which is at odds with the increasing quantities of pelagic life (whose modes of swimming are admittedly unconstrained) documented from Cambrian and Neoproterozoic deposits. Even among just medusozoans, ancestral state reconstruction suggests that they would have been swimming during the Neoproterozoic (Kayal et al., 2018; BMC Evolutionary Biology) with no knowledge of the mechanics due to absent preservation.

      Author response: Thanks for your suggestions. Yes, we agree with you that the ancestral swimming medusae may appear before the early Cambrian, even at the Neoproterozoic deposits. However, discussions on the affinities of Ediacaran cnidarians are severely limited because of the lack of information concerning their soft anatomy. So, it is hard to detect the mechanics due to absent preservation. Olivooids found from the basal Cambrian Kuanchuanpu Formation can be reasonably considered as cnidarians based on their radial symmetry, external features, and especially the internal anatomies (Bengtson and Yue 1997; Dong et al. 2013; 2016; Han et al. 2013; 2016; Liu et al. 2014; Wang et al. 2017; 2020; 2022). The valid simulation experiment here was based on the soft tissue preserved in olivooids.

      Reviewer response: This response does not sufficiently address my earlier comment. While the authors are correct that individual Ediacaran affinities are an area of active research and that Olivooids can reasonably be considered cnidarians, this doesn't address the actual critique in my comment. Most (not all) Ediacaran soft-bodied fossils are considered to have been benthic, but pelagic cnidarian life is widely acknowledged to at least be present during later White Sea and Nama assemblages (and earlier depending on molecular clock interpretations). The authors have certainly provided support for the mechanics of this type of feeding being co-opted for eventual jet-propulsion swimming in Olivooids. They have not provided sufficient justifications within the manuscript for this to be broadened beyond this group.

      Original comment: There is no explanation for how this work could be a breakthrough in simulation gregarious feeding as is stated in the manuscript.

      Author response: Thanks for your suggestion. We revised the section "Perspectives for future work and improvements" (lines 396-404 in our revised version of MS). Conducting simulations of gregarious active feeding behavior generally need to model multi (or clustered) organisms, which is beyond the present computational capability. However, exploiting the simulation result and thus building a simplified model can be possible to realize that, as we may apply an inlet or outlet boundary condition to the peridermal aperture of Quadrapyrgites with corresponding exhale or inhale flow velocity profiles collected in this work. By doing this we can obtain a simplified version of an active feeding Quadrapyrgites model without using computational expensive moving mesh feature. Such a model can be used solely or in cluster to investigate gregarious feeding behavior incorporated with ambient current. Those above are explicit explanations for how this work could be a "breakthrough" in simulation gregarious feeding. However, we modified the corresponding description in section "Perspectives for future work and improvements" to make it more appropriate.

      Reviewer response: I think I understand where the authors are trying to take this next step. If the authors were to follow up on this study with the proposed implementation of inhalant/exhalent velocities profiles (or more preferably velocity/pressure fields), then that study would be a breakthrough in simulating such gregarious feeding. Based on what has been done within the present study, I think the term "breakthrough" is instead overly emphatic.<br /> An additional note on this. The authors are correct that incorporating additional models could be used to simulation a population (as has been successfully done for several Ediacaran taxa despite computational limitations), but it's not the only way. The authors might explore using periodic boundary conditions on the external faces of the flow domain. This could require only a single Olivooid model to assess gregarious impacts - see the abundant literature of modeling flow through solar array fields.

      Original comment: L446: two layers of hexahedral elements is a very low number for meshing boundary layer flow

      Author response: Many thanks for your question. We agree that an appropriate hexahedral elements mesh for boundary layer is essential to recover boundary flow, especially in cases where turbulence model incorporated with wall function is adopted such as the standard k-epsilon model. In this case, the boundary flow is not the main point since the velocity profile was collected above periderm aperture rather than near no-slip wall region. What else, we do not need drag (related to sheer stress and pressure difference) computations in this case, which requires a more accurate flow velocity reconstruction near no-slip walls as what previous palaeontological CFD simulations have done. Thus, we think two layers of hexahedral elements are enough. What else, hexahedral elements added to periderm aperture domain, as illustrated in figure below, can let the velocity near wall vary smoothly and thus can benefit the convergency of simulations.

      Reviewer response: As the authors point out in the main text, these organisms are small (millimeters in scale) and certainly lived within the boundary layer range of the ocean. While the boundary layer is not the main point, it still needs to be accurately resolved as it should certainly affect the flow further towards the far field at this scale. I'm not suggesting the authors need to perfectly resolve the boundary layer or focus on using turbulence models more tailored to boundary layer flows (such as k-w), but the flow field still needs sufficient realism for a boundary bounded flow. The authors really should consider quantitatively assessing the number of hexahedral elements within their mesh refinement study.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The goal of Pawel et al. is to provide a more rigorous and quantitative approach for judging whether or not an initial null finding (conventionally with p >= 0.05) has been replicated by a second similarly null finding. They discuss important objections to relying on the qualitative significant/non-significant dichotomy to make this judgement. They present two complementary methods (one frequentist and the other Bayesian) which provide a superior quantitative framework for assessing the replicability of null findings.

      Strengths:<br /> Clear presentation; illuminating examples drawn from the well-known Reproducibility Project: Cancer Biology data set; R-code that implements suggested analyses. Using both methods as suggested provides a superior procedure for judging the replicability of null findings.

      Weaknesses:<br /> The frequentist and the Bayesian methods can be used to make binary assessments of an original finding and its replication. The authors clarify, though, that they can also be used to make continuous quantitative judgements about strength of evidence. I believe that most will use the methods in a binary fashion, but the availability of more nuanced assessments is welcome. This revision has addressed what I initially considered a weakness.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Theriot et al. are proposing here a technically very impressive screening method. Their optimization of single-cell sgRNA barcode sequencing/reading is fundamental progress towards the use of CRISPRi technology in phenotypic screening.

      The biology side of the manuscript focuses on cell morphology and cytoskeleton. For this, others are also proposing innovative methods for phenotypic quantification and analysis. The output of the phenotypic analysis shows interesting hit correlations between the methods used and identifies well-known hit genes. Nevertheless, the strength and the validity of the results are yet difficult to assess. The complexity and the amount of features extracted from the cell images do not always seem justified. Indeed, the visual conclusion from the authors at the end mostly refers to basic features (cell size, shape, nuclear localization, actin network polarity), which in my opinion could be quantified in a more straightforward way, which then would facilitate the ultimate goal of such a work, which is the biological interpretation of the screening campaign.

      Strengths:<br /> A very impressive technology work on molecular biology, microscopy, image analysis, and data analysis. The investment of such efforts seems fundamental for the development of phenotypic and CRISPR screens.

      Weaknesses:<br /> The phenotypic analysis method seems too complex in regard to the actual output. The biological interpretation of the screen is therefore suffering from this complexity. Having said that the quantification of cell morphology and actin network phenotype is a very risky and complex task.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, the authors investigate the tolerance of aminoglycosides in E. coli mutants deleted in the Krebs cycle and respiratory chain enzymes. The motivation for this study is unclear. Transport of aminoglycosides is pmf-dependent, as the authors correctly note, and knocking out energy-producing components leads to tolerance of aminoglycosides, this has been well established. In S. aureus, clinically relevant "small colony" strains selected for in the course of therapy with aminoglycosides acquire null mutations in the biosynthesis of heme or ubiquinone, and have been studied in detail. In E. coli, such knockouts have not been reported in clinical isolates, probably due to severe fitness costs. At the same time, single-cell analysis has shown that individual cells with a decrease in the expression of Krebs cycle enzymes are tolerant of antibiotics and have lower ATP (Manuse et al., PLoS Biol 19: e3001194). The authors of the study under review report that knocking out ICD, isocitrate dehydrogenase that catalyzes the rate-limiting step in the Krebs cycle, has little effect on aminoglycoside tolerance and actually leads to an increase in the level of ATP over time. This observation does not seem to make much sense and contradicts previous reports, specifically that E. coli ICD is tolerant of antibiotics and, not surprisingly, produces Less ATP (Kabir and Shimizu, Appl Micro-biol Biotechnol. 2004; 65(1):84-96; Manuse et al., PLoS Biol 19: e3001194). Mutations in other Krebs cycle enzymes, unlike ICD, do lead to a dramatic increase in tolerance of aminoglycosides according to the paper under review. This is all very confusing.

      Apart from the confusing data, it is not clear what useful information may be obtained from the choice of the experimental system. The authors examine exponentially growing cells of E. coli for tolerance of aminoglycosides. The population at this stage of growth is highly susceptible to aminoglycosides, and only some rare persister cells can survive. However, the authors do not study persisters. A stationary population of E. coli is tolerant of aminoglycosides, and this is clinically relevant, but this is not the subject of the study.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This is well-performed research with solid results and thorough controls. The authors did a good job of finding the relationship between the 5-HT1A receptor and megakaryocytopoiesis, which demonstrated the potential of vilazodone in the management of thrombocytopenia. The paper emphasizes the regulatory mechanism of 5-HT1A receptor signaling on hematopoietic lineages, which could further advance the field of thrombocytopenia for therapeutic purposes.

      Strengths:<br /> This is comprehensive and detailed research using multiple methods and model systems to determine the pharmacological effects and molecular mechanisms of vilazodone. The authors conducted in vitro experiments using HEL and Meg-01 cells and in vivo experiments using Zebrafish and Kunming-irradiated mice. The experiments and bioinformatics analysis have been performed with a high degree of technical proficiency. The authors demonstrated how vilazodone binds to 5-HTR1A and regulates the SRC/MAPK pathway, which is inhibited by particular 5-HTR1A inhibitors. The authors determined this to be the mechanistic underpinning for the effects of vilazodone in promoting megakaryocyte differentiation and thrombopoiesis.

      Weaknesses:<br /> 1. Which database are the drug test sets and training sets for the creation of drug screening models obtained from? What criteria are used to grade the results?

      2. What is the base of each group in Figure 3b for the survival screening of zebrafish? The positivity rate of GFP-labeled platelets is too low, as indicated by the quantity of eGFP+ cells. What gating technique was used in Figure 3e?

      3. In Figure 4C, the MPV values of each group of mice did not show significant downregulation or upregulation. The possible reasons for this should be explained.

      4. The PPI diagram and the KEGG diagram in Figure 6 both provide a possible mechanism pathway for the anti-thrombocytopenia effect of vilazodone. How can the authors analyze the differences in their results?

      5. 5-HTR1A protein expression is measured only in the Meg-01 cells assay. Similar quantitation through western blot is not shown in other cell models.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors attempt to understand how cells forage for spatially heterogeneous complex polysaccharides. They aimed to quantify the foraging behavior and interrogate its genetic basis. The results show that cells aggregate near complex polysaccharides, and disperse when simpler byproducts are added. Dispersing cells tend to move towards the polysaccharide. The authors also use transcriptomics to attempt to understand which genes support each of these behaviors - with motility and transporter-related genes being highly expressed during dispersal, as expected.

      Strengths:<br /> The paper is well written and builds on previous studies by some of the authors showing similar behavior by a different species of bacteria (Caulobacter) on another polysaccharide (xylan). The conceptual model presented at the end encapsulates the findings and provides an interesting hypothesis. I also find the observation of chemotaxis towards the polysaccharide in the experimental conditions interesting.

      Weaknesses:<br /> Much of the genetic analysis, as it stands, is quite speculative and descriptive. I found myself confused about many of the genes (e.g., quorum sensing) that pop up enriched during dispersal quite in contrast to my expectations. While the authors do mention some of this in the text as worth following up on, I think the analysis as it stands adds little insight into the behaviors studied. However, I acknowledge that it might have the potential to generate hypotheses and thus aid future studies. Further, I found the connections to the carbon cycle and marine environments in the abstract weak --- the microfluidics setup by the authors is nice, but it provides limited insight into naturalistic environments where the spatial distribution and dimensionality of resources are expected to be qualitatively different.

    1. Reviewer #1 (Public Review):

      Questions and concerns:

      The abstract is hard to follow. The authors there refer to a previous experiment showing that "overnight fasting diminishes excessive avoidance and speeds up fear extinction by decreasing subjective relief during threat omissions" (L26). They go on to say that "relief tracks the reward prediction error signal that governs safety learning" (L28). This is puzzling. While getting less relief/safety from avoidance actions will surely diminish avoidance (because avoidance actions are less reinforced), getting less relief/safety from omissions of an unconditioned stimulus (US) in fear extinction should slow down (not speed up) fear extinction. In the same vein, why are "lower activations [in fMRI] in the ventromedial prefrontal cortex and nucleus accumbens in response to threat omissions signaled by a safe cue" (L34) associated with "increased effective avoidance and sped up fear extinction" (L33)? This clearly goes against the existing literature on reward prediction errors (PEs) in fear learning paradigms, where these PEs in the mesolimbic dopamine system drive extinction, that is, they are associated with better extinction (and should therefore also be associated with more avoidance). For instance, in the rodent, Luo et al., 2018 (DOI: 10.1038/s41467-018-04784-7) and Salinas-Hernandez et al., 2018 (DOI: https://doi.org/10.7554/eLife.388181 of 25RESEARCH ARTICLE) and 2023 (https://doi.org/10.1016/j.neuron.2023.08.025ll) have in various constellations optogenetically enhanced and diminished, respectively, the PE signal at the time of US omission in extinction in either VTA or nucleus accumbens and thereby sped up and slowed down, respectively, extinction learning. If the results of the current experiment contradict established knowledge, the reader must be clearly informed about this. By contrast, the abstract gives the impressions as if the current results were to be expected and in line with the literature ("since relief tracks the reward prediction error signal ..., we hypothesized ...").

      It would also help the reader if it was clarified that the finding of "increased effective avoidance" (L33) went counter to the hypothesis, e.g., by saying "Contrary to our hypothesis, we observed ...".


      L51: The presentation of exposure therapy is a bit misleading and may create confusion. While it is probably correct that exposure works by "promoting safety learning", this is generally thought to be the case only for Pavlovian associations (CS-US), that is, for extinction (where safety learning creates the new association of CS and "no US"). It is, however, not generally considered to be the case for the instrumental action-outcome associations that underlie avoidance learning ("I do this or that, then I do not have to experience the feared object or situation"). Therapists try to prevent this type of learning from happening, exactly by promoting the confrontation with fear objects or situations in the absence of any avoidance action.

      Generally, I think the introduction suffers from the absence of a short explanation of what avoidance and extinction learning are, behaviorally, and what types of mechanisms are believed to drive them, and that the one (avoidance) is thought to contribute to the maintenance of fears whereas the other (extinction) reduces fear. The non-specialist reader is somehow left in the dark.

      In the same vein, on L63, presenting the results of their previous fasting study that serves as a discovery study for the present experiment, the authors make a distinction between "unnecessary avoidance during a signal of safety" and "effective avoidance during a signal of upcoming threat". It is really expecting too much from the reader that they will understand at this stage that a CS can become a signal of safety through extinction or that a CS not paired with a US during conditioning (a "CS-") is a safety signal and that it is not necessary to avoid such a signal, whereas a non-extinguished CS (signaling threat) may well be avoided. (At least, this is how I understood the distinction.)

      I was then really confused by the following statement (L65) that "the decrease in unnecessary avoidance was mediated by lower levels of relief ... during omissions of threat". If a CS is already extinguished (has no remaining or only little threat value, that is, is a safety stimulus), there is no longer threat omission when the US does not occur, and no relief. There should also be no relief to US omission after a CS-. More importantly even, if fasted participants reported lower levels of relief from threat omission, why did they not also show less effective avoidance (which is driven by the reinforcement provided by the relief that occurs when a successful avoidance action has prevented a US from occurring after or during the CS)?

      L69: Also the statement "a faster decline in relief ... ratings during ... extinction, suggesting faster decrease of threat expectancies" can only be understood by the reader if they already know what a PE is and by what rules PE-driven learning is governed (that is, essentially, if they know Rescorla-Wagner). I think the authors must explain, in order to allow a non-specialist reader to follow their text, that the PE (supposed to be indexed by the relief rating) reflects the discrepancy between the magnitude of an outcome expectation (e.g., here, expectation of the US) and the obtained outcome (here, US or not); that, therefore, a PE is generated when a subject expects a US (as a result of prior conditioning) but does not get it; that this leads to a proportional update (reduction) of the US expectation in the next trial; and that this in turn leads to a diminished PE when the US again does not occur. Notably, the reader must be made aware that the higher the PE, the higher the reduction and the faster the extinction (proportionality).

      The reader must also be made aware that the update is additionally determined by some multiplicatory "transmission" function or constant (e.g., learning rate in Rescorla-Wagner) that defines the size of the relationship between the magnitude of the PE and the magnitude of the update (reduction). Hence, in two individuals, even if the magnitude of the PE is identical, the magnitude of the update may differ because of individual differences in the learning rate (to take the Rescorla-Wagner implementation). The authors, however, seem to ignore the possibility that fasting changes the learning rate.

      Both the dynamics of the PE and the learning rate, of course, add complexity to the interpretation of the past and present data. But I think the authors cannot avoid this when they want to make sense of a treatment (fasting) that they believe affects safety learning. Speaking of "lower levels of relief" (L66) must be qualified by whether these lower ratings were observed initially (when the first PEs were registered at initial threat omissions, meaning that safety learning should be relatively slowed down by fasting) or on average or later during a safety learning experiment (which could indicate that learning under fasting was relatively quicker/more successful).

      Following upon this, in L74, the conclusion from observations of lower levels of relief during avoidance and faster decline in relief during extinction in the previous study that "overnight fasting decreased the reward value of safety (less relief pleasantness)" may be wrong if the faster decline and the resulting lower average levels of relief were the consequence of a higher initial PE in the fasting group, as would be expected from the Rescorla-Wagner rule. If the latter were the case, this would suggest that subjects actually registered more safety (a higher discrepancy to their threat expectation) in early trials. This could also explain why fasting sped up extinction in that study (see Abstract). It might also explain why "effective avoidance" (L64) was at least maintained (although it should actually also be sped up). It might make less parsimonious explanations ("fasting biases .. to focus on food at the expense of safety", L79), requiring the presence of a food source and a utility function of accepting a threat in the obtainment of food, unnecessary.

      All this, however, rests on whether I think I have understood what the authors want to say about their relief measurements and the way the operationalized avoidance in the previous study.

      More unclarities due to not giving full information: L91: "... extinction and avoidance learning. Accordingly, human fMRI studies have found ... activations in the ventral striatum and the VTA during threat omissions that might contribute to establishing a new safety CS-->noUS memory that reduces the initial fear response." However, in avoidance, it is an action that is reinforced by the US omission and hence an action-->noUS memory that is being formed. The CS keeps its threat value acquired during the preceding conditioning phase, and the reduction of fear during CS presentations is contingent upon the exertion of the avoidance action.

      L99: "Because overnight fasting decreased relief rating particularly during omissions after safety signals". Again, if a US is omitted after a safety signal (an extinguished CS or a CS-), there should be no PE and no relief. If there were still relief ratings at US omission after a safety signal, this would suggest extinction did not fully work or differential conditioning was not successful. In any case, it is not clear at all why relief was specifically decreased during omissions after safety signals and not (and much more so) during omissions after threat signals, where there is clearly a PE. If this was not the case, one has to wonder if something went wrong in the discovery study.

      The paragraph starting L103 and the associated figure 1 could be a bit more precise and give a bit more information in order to provide the reader a proper understanding of key experimental manipulations, in particular the ART task. Please define abbreviations "CS+unav", "CS+av". L108 ff.: One gets the impression there is only one CS+, whereas there are two. Say explicitly that one CS+ remains unavoidable during the Avoidance phase (CS+unav). What is the purpose of this stimulus? Do participants learn during the Avoidance phase that the CS+unav is unavoidable and the CS+av is avoidable or is this instructed? Do participants have to press the button within a certain time after CS+unav onset in order to avoid the US, or with a certain force? Is avoidance in case of successful button pressing deterministic or probabilistic? Say that the frame with the non-lit lamp is the ITI.

      Relief ratings (Figure 1b): The rating says "How pleasant was the relief that you felt?". That is, the experimenter insinuates that the participant will have felt relief and only wants to know how pleasant that relief was. The subjects has no chance to indicate there was no relief. This may be the reason why, in the discovery study, subjects indicated relief to safe stimuli, see above. Why did the authors not simply ask about the degree of relief felt, which would give a subject the chance to say there was no relief? I think this is a major flaw.

      L119: "We previously found that overnight fasting reduces avoidance and relief mostly to a safe CS-." If this is really the only thing that the authors found, then the fasting manipulation in their previous study failed to modulate avoidance of CS+s and the PE signaling at the time of US omissions after CS+s, that is, after actual threat stimuli. The procedure then clearly is not suited to study influences of fasting on avoidance learning. Whatever it does manipulate, it is not relief-based avoidance learning.

      L130: It makes absolutely no sense to hypothesize that a manipulation reducing relief in extinction learning will decrease activation in the neural PE circuitry at the time of US omission more after the CS- than after the CS+. Of course, the PE is highest when the US is not given after the CS+, and this is where any relief manipulation should have an effect. As said above, the authors must also specify their hypothesis with respect of timing (early or late extinction? See the animal papers cited above.)

    1. Reviewer #2 (Public Review):

      The formation of long-term memory representations requires the continuous updating of ongoing representations. Various studies have shown that the left angular gyrus (AG) may support this cognitive operation. However, this study demonstrates that this brain region plays a causal role in the formation of long-term memory representations, affecting both the neural and behavioural measures of information binding.

      A significant strength of this work is that it is the first one to test the hypothesis that the left angular gyrus has a causal role in the reconfiguration and binding of long-term memory representations by comparing when insights are primarily derived from direct observation versus imagination. Consequently, the results from this manuscript have the potential to be informative for all areas of cognitive research, including basic perception, language cognition and memory.

      Furthermore, this study presents a comprehensive set of measurements on the same individuals, encompassing various task-related behavioural measures, EEG data, and questionnaire responses.

      A weakness of the manuscript is the use of different groups of participants for the key TMS intervention.

    1. Reviewer #1 (Public Review):

      This manuscript reports on the behavior of participants playing a game to measure exploration. Specifically, participants completed a task with blocks of exploratory choices (choosing between two 'tables', and within each table, two 'card decks', each of which had a specific probability of showing cards with one color versus another) and test choices, where participants were asked to choose which of the two decks per table had a higher likelihood of one color. Blocks differed on how long (how many trials) the exploration phase lasted. Participants' choices were fit to increasingly complex models of next-trial exploration. Participants' choices were best fit by an intermediate model where the difference in uncertainty between tables influenced the choice. Next, the authors investigated factors affecting whether participants sought out or avoided uncertainty, their choice reaction times, and the relationship of these measures with performance during the test phase of each block. Participants were uncertainty-seeking (exploratory) under most levels of overall uncertainty but became less uncertainty-seeking at high levels of total uncertainty. Participants with a stronger tendency to approach uncertainty at lower levels of total uncertainty were more accurate in the test phase, while the tendency to avoid uncertainty when total uncertainty was high was also weakly positively related to test accuracy. In terms of reaction times, participants whose reaction times were more related to the level of uncertainty, and who deliberated longer, performed better. The individual tendency to repeat choices was related to avoidance of uncertainty under high total uncertainty and better test performance. Lastly, choices made after a longer lag were less affected by these measures.

      The authors note that their paradigm, which does not provide immediate rewarding feedback, is novel. However, the resulting behavior appears similar to other exploratory learning tasks, so it's unclear what this task design adds - besides perhaps showing that exploratory behavior is similar across types of reward environments. Several papers have shown that cognitive constraints modulate exploration (PMIDs: 30667262, 24664860, 35917612, 35260717); although this paper provides novel insights, it does not situate its findings in the context of this prior literature. As a result, what it adds to the literature is difficult to discern.

      Other methodological questions include whether the same model provides the best fit for all participants and whether possible individual differences in models used relate to individual differences in exploration and performance; how some analyses were carried out that currently lack sufficient detail in the manuscript; and how the two stages of choice behavior (tables versus card decks) were accounted for in the analyses.

    1. Reviewer #1 (Public Review):

      In this paper, the effects of two sensory stimuli (visual and somatosensory) on fMRI responsiveness during absence seizures were investigated in GEARS rats with concurrent EEG recordings. SPM analysis of fMRI showed a significant reduction in whole-brain responsiveness during the ictal period compared to the interictal period under both stimuli, and this phenomenon was replicated in a structurally constrained whole-brain computational model of rat brains.

      The conclusion of this paper is that whole-brain responsiveness to both sensory stimuli is inhibited and spatially impeded during seizures.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this manuscript, Rohde et al. discuss how single cells isolated from the presomitic mesoderm of the zebrafish embryo follow a cell-autonomous differentiation "programme", which is dependent on the initial anteroposterior position in the embryo.

      Strengths:<br /> This work and in particular the comparison to cellular behaviour in vivo presents a detailed description of the oscillatory system that brings the developmental biology forward in their understanding of somitogenesis.<br /> The main novelty lies in the direct comparison of these isolated single cells to single cells tracked within the developing embryo. This allows them to show that isolated cells follow a similar path of differentiation without direct contact to neighbours or the presence of external morphogen gradients. Based on this, the authors propose an internal timer that starts ticking as cells traverse the presomitic mesoderm, while external signals modify this behaviour.

      Weaknesses:<br /> There are a few things that would clarify the current statement or might be added in a reasonable amount of time to further increase the relevance of this study:<br /> - My main point of concern is the precision of dissection. The authors distinguish cells isolated from the tailbud and different areas in the PSM. They suggest that the cell-autonomous timer is initiated, as cells exit the tailbud.<br /> This is also relevant for the comparison of single cells isolated from the embryo and cells within the embryo. The dissection will always be less precise and cells within the PSM4 region could contain tailbud cells (as also indicated in Figure 1A), while in the analysis of live imaging data cells can be selected more precisely based on their location. This could therefore contribute to the difference in noise between isolated single cells and cells in the embryo. This could also explain why there are "on average more peaks" in isolated cells (p. 6, l. 7).<br /> This aspect should be considered in the interpretation of the data and mentioned at least in the discussion.<br /> (It does not contradict their finding that more anterior cells oscillate less often and differentiate earlier than more posterior ones.)

      - Here, the authors focus on the question of how cells differentiate. The reverse question is not addressed at all. How do cells maintain their oscillatory state in the tailbud? One possibility is that cells need external signals to maintain that as indicated in Hubaud et al. 2014. In this regard, the definition of tailbud is also very vague. What is the role of neuromesodermal progenitors? The proposal that the timer is started when cells exit the tailbud is at this point a correlation and there is no functional proof, as long as we do not understand how cells maintain the tailbud state. These are points that should be considered in the discussion.

      - The authors observe that the number of oscillations in single cells ex vivo is more variable than in the embryo. This is presumably due to synchronization between neighbouring cells via Notch signalling in the embryo. Would it be possible to add low doses of Notch inhibitor to interfere with efficient synchronization, while at the same time keeping single cell oscillations high enough to be able to quantify them?

      In the same direction, it would be interesting to test if variation is decreased, when the number of isolated cells is increased, i.e. if cells are cultured in groups of 2,3 or 4 cells, for instance.

      - It seems that the initiation of Mesp2 expression is rather reproducible and less noisy (+/- 2 oscillation cycles), while the number of oscillations varies considerably (and the number of cells continuing to oscillate after Mesp2 expression is too low to account for that). How can the authors explain this apparent discrepancy?

      - The observation that some cells continue oscillating despite the upregulation of Mesp2 should be discussed further and potential mechanism described, such as incomplete differentiation.

      - Fig. 3 supplement 3 B missing

    1. Reviewer #1 (Public Review):


      The authors present a neural network (NN)-based approach to computationally cheaper emulation of simulations of biophysically relatively detailed cardiac cell models based on systems of ordinary differential equations. Relevant case studies are used to demonstrate the performance in prediction of standard action potentials, as well as action potentials manifesting early depolarizations. Application to the "reverse problem" (inferring the effect of pharmacological compounds on ion channels based on action potential data before and after drug treatment) is also explored, which is a task of generally high interest.


      This is a well-designed study, which explores an area that many in the cardiac simulation community will be interested in. The article is well written and I particularly commend the authors on transparency of methods description, code sharing, etc. - it feels rather exemplary in this regard and I only wish more authors of cardiac simulation studies took such an approach. The training speed of the network is encouraging and the technique is accessible to anyone with a reasonably strong GPU, not needing specialized equipment.


      Below are several points that I consider to be weaknesses and/or uncertainties of the work:

      1. The scope for acceleration of single cell simulations is not vast, as it is easy to simulate tens of thousands of cells per day on a workstation computer, using simulation conditions similar to those of the authors. While this covers a large part of what is needed in the field, I agree with the authors that there are applications where the presented technology is helpful. In such cases, e.g., in uncertaintly quantification, it will enable studies that would be difficult to carry out previously. In addition, any application involving long-term pre-pacing of a large number of cells will benefit greatly from the reported tool.

      An area which is definitely in need of acceleration is simulations of whole ventricles or hearts, but it is not clear how much potential for speedup would the presented technology bring there. I can imagine interesting applications of rapid emulation in such a setting, some of which could be hybrid in nature (e.g. using simulation for the region around the wavefront of propagating electrical waves, while emulating the rest of the tissue, which is behaving more regularly/predictable, and is likely to be emulated well), but this is definitely beyond of the scope of this article.

      2. The exact speed-up achieved by the NN emulation is somewhat context-dependent. In particular, the reported speedup critically depends on the number of beats in the simulation. The emulator learns to directly estimate the state of the cell after X beats (where X is decided by the operator of training). The speedup appears to be relatively marginal when a single beat is simulated versus emulated - but when 1000 beats are simulated, this takes 1000fold more time for simulation, but unchanged time for emulation.

      While the initial submission did not communicate the practical speedup entirely clearly, this was addressed well by the authors in the revised version.

      3. It appears that the accuracy of emulation drops off relatively sharply with increasing real-world applicability/relevance of the tasks it is applied to. That said, the authors are to be commended on declaring this transparently, rather than withholding such analyses. I particularly enjoyed the discussion of the not always amazing results of the inverse problem on the experimental data. The point on low parameter identifiability is an important one, and serves as a warning against overconfidence in our ability to infer cellular parameters from action potentials alone. On the other hand, I'm not that sure the difference between small tissue preps and single cells which authors propose as another source of the discrepancy will be that vast beyond the AP peak potential (probably much of the tissue prep is affected by the pacing electrode?), but that is a subjective view only. The influence of coupling could be checked if the simulated data were generated from 2D tissue samples/fibres, e.g. using the Myokit software.

      In summary, I believe the range of tasks where the emulator provides a major advance is relatively narrow, particularly given the relatively limited need for further speedup compared to simulations. However, this does not make the study uninteresting in the slightest - on the contrary, it explores something that many of us are thinking about, and it is likely to stimulate further development in the direction of computationally efficient emulation of relatively complex simulations.

    1. Reviewer #1 (Public Review):


      There is a long-believed dogma in the malaria field; a mosquito infected with a single oocyst is equally infectious to humans as another mosquito with many oocysts. This belief has been used for goal setting (and modeling) of malaria transmission-blocking interventions. While recent studies using rodent malaria suggest that the dogma may not be true, there was no such study with human P. falciparum parasites. In this study, the numbers of oocysts and sporozoite in the mosquitoes and the number of expelled sporozoites into artificial skin from the infected mosquito was quantified individually. There was a significant correlation between sporozoite burden in the mosquitoes and expelled sporozoites. In addition, this study showed that highly infected mosquitoes expelled sporozoites sooner.


      • The study was conducted using two different parasite-mosquito combinations; one was lab-adapted parasites with Anopheles stephensi and the other was parasites, which were circulated in infected patients, with An. coluzzii. Both combinations showed statistically significant correlations between sporozoite burden in mosquitoes and the number of expelled sporozoites.

      • Usually, this type of study has been done in group bases (e.g., count oocysts and sporozoites at different time points using different mosquitoes from the same group). However, this study determined the numbers in individual bases after multiple optimization and validation of the approach. This individual approach significantly increases the power of correlation analysis.


      • In a natural setting, most mosquitoes have less than 5 oocysts. Thus, the conclusion is more convincing if the authors perform additional analysis for the key correlations (Fig 3C and 4D) excluding mosquitoes with very high total sporozoite load (e.g., more than 5-oocyst equivalent load).

      • As written as the second limitation of the study, this study did not investigate whether all expelled sporozoites were equally infectious. For example, Day 9 expelled sporozoites may be less infectious than Day 11 sporozoites, or expelled sporozoites from high-burden mosquitoes may be less infectious because they experience low nutrient conditions in a mosquito. Ideally, it is nice to test the infectivity by ex vivo assays, such as hepatocyte invasion assay, and gliding assay at least for salivary sporozoites. But are there any preceding studies where the infectivity of sporozoites from different conditions was evaluated? Citing such studies would strengthen the argument.

      • Since correlation analyses are the main points of this paper, it is important to show 95%CI of Spearman rank coefficient (not only p-value). By doing so, readers will understand the strengths/weaknesses of the correlations. The p-value only shows whether the observed correlation is significantly different from no correlation or not. In other words, if there are many data points, the p-value could be very small even if the correlation is weak.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This is a manuscript describing outbreaks of Pseudomonas aeruginosa ST 621 in a facility in the US using genomic data. The authors identified and analysed 254 P. aeruginosa ST 621 isolates collected from a facility from 2011 to 2020. The authors described the relatedness of the isolates across different locations, specimen types (sources), and sampling years. Two concurrently emerged subclones were identified from the 254 isolates. The authors predicted that the most recent common ancestor for the isolates can be dated back to approximately 1999 after the opening of the main building of the facility in 1996. Then the authors grouped the 254 isolates into two categories: 1) patient-to-patient; or 2) environment-to-patient using SNP thresholds and known epidemiological links. Finally, the authors described the changes in resistance gene profiles, virulence genes, cell wall biogenesis, and signaling pathway genes of the isolates over the sampling years.

      Strengths:<br /> The major strength of this study is the utilisation of genomic data to comprehensively describe the characteristics of a long-term Pseudomonas aeruginosa ST 621 outbreak in a facility. This fills the data gap of a clone that could be clinically important but easily missed from microbiology data alone.

      Weaknesses:<br /> The work would further benefit from a more detailed discussion on the limitations due to the lack of data on patient clinical information, ward movement, and swabs collected from healthcare workers to verify the transmission of Pseudomonas aeruginosa ST 621, including potential healthcare worker to patient transmission, patient-to-patient transmission, patient-to-environment transmission, and environment-to-patient transmission. For instance, the definition given in the manuscript for patient-to-patient transmission could not rule out the possibility of the existence of a shared contaminated environment. Equally, as patients were not routinely swabbed, unobserved carriers of Pseudomonas aeruginosa ST 621 could not be identified and the possibility of misclassifying the environment-to-patient transmissions could not be ruled out. Moreover, reporting of changes in rates of resistance to imipenem and cefepime could be improved by showing the exact p-values (perhaps with three decimal places) rather than dichotomising the value at 0.05. By doing so, readers could interpret the strength of the evidence of changes.

      Impact of the work:<br /> First, the work adds to the growing evidence implicating sinks as long-term reservoirs for important MDR pathogens, with direct infection control implications. Moreover, the work could potentially motivate investments in generating and integrating genomic data into routine surveillance. The comprehensive descriptions of the Pseudomonas aeruginosa ST 621 clones outbreak is a great example to demonstrate how genomic data can provide additional information about long-term outbreaks that otherwise could not be detected using microbiology data alone. Moreover, identifying the changes in resistance genes and virulence genes over time would not be possible without genomic data. Finally, this work provided additional evidence for the existence of long-term persistence of Pseudomonas aeruginosa ST 621 clones, which likely occur in other similar settings.

    1. Reviewer #1 (Public Review):

      The authors have generated a set of yeast S. cerevisiae strains containing different numbers of chromosomes.<br /> Elimination of telomerase activates homologous recombination (HR) to maintain telomeres in cells containing the original 16 chromosomes. However, elimination of telomerase leads to circularization of cells containing a single or two chromosomes. The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They found that the subtelomeric sequences X and Y' are dispensable for cell proliferation and HR-mediated telomere maintenance in telomerase-minus SY12 cells. They conclude that subtelomeric X and Y' sequences do not play essential roles in both telomerase-proficient and telomerase-null cells and propose that these sequences represent remnants of genome evolution.<br /> Interestingly, telomerase-minus SY12 generate survivors that are different from well-established Type I or Type II survivors. The authors uncover atypical telomere formation which does not depend on the Rad52 homologous recombination pathway.

      Strengths: The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes. They show that subtelomeres do not have essential roles in telomere maintenance and cell proliferation.

      Weaknesses:<br /> It is not fully addressed how atypical survivors are generated independently of Rad52-mediated homologous recombination.<br /> It remains possible that X and Y elements influence homologous recombination, type 1 and type 2 (type X), at telomeres. In particular, the presence of X and Y elements appears to be important for promoting type 1 recombination, although the authors conclude "Elimination of subtelomeric repeat sequences exerts little effect on telomere functions".

    1. Reviewer #1 (Public Review):

      Summary:<br /> The current manuscript by Hajra et al deals with the role of the prominent Sirtuins SIRT1 and -3 during infection of macrophages with Salmonella Typhimurium (ST). Apparently, ST infection induces upregulation of host cell SRTs to aid its own metabolism during the intracellular lifestyle and to help reprogramming macrophage polarization. The manuscript has two parts, namely one part that deals with Salmonella infection in cells, where RAW 264.7 murine macrophage-like cells, sharing some features with primary macrophages, were employed. Infected RAW cells displayed a tendency to polarize towards wound-healing M2 and not inflammatory M1 macrophages, which was dependent on SRT. Consequently, the inflammatory response in RAW was more robust in the absence of SRT. Moreover, loss of SRTs leads to impaired bacterial proliferation in these cells, which was attributed to defects in metabolic adaption of the bacteria in the absence of SRT-activity and to the increased M1 inflammatory response.

      Unfortunately, the line of argumentation remains incomplete because corresponding assays in mice showed the opposite result as compared to the experiments using RAW 264.7 cells. i.e. loss of SRTs leads to increased bacterial load in animals (versus impaired proliferation in RAW 264.7 cells). The authors cannot explain this discrepancy.

      Strengths:<br /> Extensive analysis of Salmonella infection in RAW macrophage-like cells and mice in the context of SRT1/3 function.

      Weaknesses:<br /> Lack of connection between the cell-based and organismic data, which are not supportive of each other.

    1. Reviewer #1 (Public Review):

      Pathogenic mutations of mTOR pathway genes have been identified in patients with malformation of cortical development and intractable epilepsy. Nguyen et al., established an in vivo rodent model to investigate the impact of different mTOR pathway gene dysfunction on neuronal intrinsic membrane excitability and cortical network activity. The results demonstrate that activation of mTORC1 activators or inactivation of mTORC1 repressors leads to convergent mTOR pathway activation and alterations of neuronal morphology, the key pathological feature of human FCD and hemimegalencephaly. However, different mTOR pathway gene mutations also exhibited variations in modulating Ih current and synaptic activity in rodent cortical neurons. These findings provide novel insights into the mechanism of seizure generation associated with cortical malformation.

    1. Reviewer #2 (Public Review):

      The authors aimed to investigate the microbiota present in the fallopian tubes (FT) and its potential association with ovarian cancer (OC). They collected swabs intraoperatively from the FT and other surgical sites as controls to profile the FT microbiota and assess its relationship with OC.

      They observed a clear shift in the FT microbiota of OC patients compared to non-cancer patients. Specifically, the FT of OC patients had more types of bacteria typically found in the gastrointestinal tract and the mouth. In contrast, vaginal bacterial species were more prevalent in non-cancer patients. Serous carcinoma, the most common OC subtype, showed a higher prevalence of almost all FT bacterial species compared to other OC subtypes.

      The strengths of the study include its large sample size, rigorous collection methods, and use of controls to identify the possible contaminants. Additionally, the study employed advanced sequencing techniques for microbiota analysis. However, there are some weaknesses to consider. The study relied on swabs collected intraoperatively, which may not fully represent the microbiota in the FT during normal physiological conditions. The study also did not establish causality between the identified bacteria and OC but rather demonstrated an association. Regardless, the findings are important and these questions need to be addressed by future studies. A few additions in data representation and analysis are instead recommended.

      Overall, the authors achieved their aims of identifying the FT microbiota and assessing its relationship with OC. The results support the conclusion that there is a clear shift in the FT microbiota in OC patients, paving the way for further investigations into the role of these bacteria in the pathogenesis of ovarian cancer.

      The identification of specific bacterial species associated with OC could contribute to the development of novel diagnostic and therapeutic approaches. The study design and the data generated here can be valuable to the research community studying the microbiota and its impact on cancer development. However, further research is needed to validate these findings and elucidate the underlying mechanisms linking the FT microbiota shift and OC.

    1. Reviewer #1 (Public Review):

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

    1. Reviewer #1 (Public Review):

      In the article "Temporal transcriptomic dynamics in developing macaque neocortex", Xu et al. analyze the cellular composition and transcriptomic profiles of the developing macaque parietal cortex using single-cell RNA sequencing. The authors profiled eight prenatal rhesus macaque brains at five timepoints (E40, E50, E70, E80, and E90) and obtained a total of around 53,000 high-quality cells for downstream analysis. The dataset provides a high-resolution view into the developmental processes of early and mid-fetal macaque cortical development and will potentially be a valuable resource for future comparative studies of primate neurogenesis and neural stem cell fate specification. Their analysis of this dataset focused on the temporal gene expression profiles of outer and ventricular radial glia and utilized pesudotime trajectory analysis to characterize the genes associated with radial glial and neuronal differentiation. The rhesus macaque dataset presented in this study was then integrated with prenatal mouse and human scRNA-seq datasets to probe species differences in ventricular radial glia to intermediate progenitor cell trajectories. Additionally, the expression profile of macaque radial glia across time was compared to those of mouse apical progenitors to identify conserved and divergent expression patterns of transcription factors.

      The main findings of this paper corroborate many previously reported and fundamental features of primate neurogenesis: deep layer neurons are generated before upper layer excitatory neurons, the expansion of outer radial glia in the primate lineage, conserved molecular markers of outer radial glia, and the early specification of progenitors. Furthermore, the authors show some interesting divergent features of macaque radial glial gene regulatory networks as compared to mouse. Overall, despite some uncertainties surrounding the clustering and annotations of certain cell types, the manuscript provides a valuable scRNA-seq dataset of early prenatal rhesus macaque brain development. The dynamic expression patterns and trajectory analysis of ventricular and outer radial glia provide valuable data and lists of differentially expressed genes (some consistent with previous studies, others reported for the first time here) for future studies.

    1. Reviewer #1 (Public Review):

      Summary<br /> Liao et al leveraged two powerful genomics techniques-CUT&RUN and RNA sequencing-to identify genomic regions bound by and activated or inactivated by SMAD1, SMAD5, and the progesterone receptor during endometrial stromal cell decidualization. Additionally, the authors generated novel knock-in HA-SMAD1 and PA-SMAD5 tagged mice to combat antibody issues facing the field, generating a novel model to advance the study of BMP signaling in the female reproductive tract. During decidualization in a murine model, SMAD1/5 are bound to many genomic sites of genes important in decidualization and pregnancy and coregulated responses with progesterone receptor signaling.

      Strengths<br /> The authors utilized powerful next generation sequencing and identified important transcriptional mechanisms of SMAD1/5 and PGR during decidualization in vivo.

      Weaknesses<br /> None.

      Overall, the manuscript and study are well structured and provide critical mechanistic updates on the roles of SMAD1/5 in decidualization and preparation of the maternal endometrium for pregnancy.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This paper conducted a GWAS meta-analysis for COVID-19 hospitalization among admixed American populations. The authors identified four genome-wide significant associations, including two novel loci (BAZ2B and DDIAS), and an additional risk locus near CREBBP using cross-ancestry meta-analysis. They utilized multiple strategies to prioritize risk variants and target genes. Finally, they constructed and assessed a polygenic risk score model with 49 variants associated with critical COVID-19 conditions.

      Strengths:<br /> Given that most of the previous studies were done in European ancestries, this study provides unique findings about the genetics of COVID-19 in admixed American populations. The GWAS data would be a valuable resource for the community. The authors conducted comprehensive analyses using multiple different strategies, including Bayesian fine mapping, colocalization, TWAS, etc., to prioritize risk variants and target genes. The polygenic risk score (PGS) result demonstrated the ability of the cross-population PGS model for COVID-19 risk stratification.

      Weaknesses:<br /> 1. One of the major limitations of this study is that the GWAS sample size is relatively small, which limits its power.

      2. The fine mapping section is unclear and there is a lack of information. The authors assumed one causal signal per locus, and only provided credible sets, but did not provide posterior inclusion probabilities (PIP) for the variants to be causal.

      3. Colocalization and TWAS used eQTL data from GTEx data, which are mainly from European ancestries. It is unclear how much impact the ancestry mismatch would have on the result. The readers should be cautious when interpreting the results and designing follow-up studies.

    1. Reviewer #1 (Public Review):

      Germe and colleagues have investigated the mode of action of bacterial DNA gyrase, a tetrameric GyrA2GyrB2 complex that catalyses ATP-dependent DNA supercoiling. The accepted mechanism is that the enzyme passes a DNA segment through a reversible double-stranded DNA break formed by two catalytic Tyr residues-one from each GyrA subunit. The present study (now described in a revised manuscript) sought to understand an intriguing earlier observation that gyrase with a single catalytic tyrosine that cleaves a single strand of DNA, nonetheless has DNA supercoiling activity. This unexpected finding led to the proposal that gyrase acts instead via a nicking closing mechanism. Germe et al used bacterial co-expression to make the wild-type and mutant heterodimeric BA(fused).A complexes with only one catalytic tyrosine. Whether the Tyr mutation was on the A side or BA fusion side, both complexes plus GyrB reconstituted fluoroquinolone-stabilised double-stranded DNA cleavage and DNA supercoiling activity. This indicates that the preparations of these complexes sustain double strand DNA passage as envisaged in the current double-strand break mechanism of gyrase. Of possible explanations for how double-strand cleavage arises, contamination of heterodimeric complexes or GyrB with GyrA dimers was ruled unlikely by the meticulous prior analysis of the proteins on native Page gels, by analytical gel filtration and by mass photometry (although low levels of endogenous GyrA were seen in some preparations). Involvement of an alternative nucleophile on the Tyr-mutated protein was ruled out by analysis of mutagenesis studies focused on the catalytic ArgTyrThr triad of residues. Similarly, analysis of 5'- and 3'- DNA ends generated by cleavage ruled out water as a nucleophile. Instead, results of the present study favour a third explanation wherein double-strand DNA breakage arises as a consequence of subunit (or interface/domain) exchange. The authors showed that although the A subunits in the GyrA dimer were thought to be tightly associated, addition of GyrB to heterodimers with one catalytic tyrosine stimulated DNA cleavage with a time lag consistent with rapid DNA-dependent subunit or interface exchange to generate complexes with two catalytic tyrosines capable of double-stranded DNA breakage. Subunit exchange between heterodimeric complexes was facilitated by DNA bending and wrapping by gyrase, by the ability of both GyrA and GyrB to form higher order aggregates and by dense packing of gyrase complexes on DNA. By addressing a puzzling paradox, this study provides further support for the accepted double strand break (strand passage) mechanism of gyrase (without having to invoke a nicking-closing mechanism) and opens new insights on subunit exchange that may have biological significance in promoting DNA recombination and genome evolution.

      The conclusions of the work are mostly well supported by the experimental data. Moreover, in the revised manuscript, the various concepts, experiments and outcomes are better explained and more accessible to the reader through a reorganised text, clearer figures and an extended Supplementary section.


      The study examines a fundamental biological question, namely the mechanism of DNA gyrase, an essential and ubiquitous enzyme in bacteria, and the target of fluoroquinolone antimicrobial agents.

      The experiments have been carefully done and the analysis of their outcomes is comprehensive, thoughtful and considered.

      The work uses an array of complementary techniques to characterize preparations of GyrA, GyrB and various gyrase complexes. In this regard, mass photometry seems particularly useful. Analysis revealed that purified GyrA and GyrB can each form multimeric complexes and highlights the complexities involved in investigating the gyrase system.

      The various possible explanations for the double-strand DNA breakage by gyrase heterodimers with a single catalytic tyrosine are considered and addressed by appropriate experiments.

      The study highlights the potential biological importance of interactions between gyrase complexes through domain-or subunit-exchange.

    1. Reviewer #1 (Public Review):

      The goal of the current study was to evaluate the effect of neuronal activity on blood-brain barrier permeability in the healthy brain, and to determine whether changes in BBB dynamics play a role in cortical plasticity. The authors used a variety of well-validated approaches to first demonstrate that limb stimulation increases BBB permeability. Using in vivo-electrophysiology and pharmacological approaches, the authors demonstrate that albumin is sufficient to induce cortical potentiation and that BBB transporters are necessary for stimulus-induced potentiation. The authors include a transcriptional analysis and differential expression of genes associated with plasticity, TGF-beta signaling, and extracellular matrix were observed following stimulation. Overall, the results obtained in rodents are compelling and support the authors' conclusions that neuronal activity modulates the BBB in the healthy brain and that mechanisms downstream of BBB permeability changes play a role in stimulus-evoked plasticity. These findings were further supported with fMRI and BBB permeability measurements performed in healthy human subjects performing a simple sensorimotor task. There is literature to suggest that there are sex differences in BBB dysfunction in pathophysiological conditions and the authors have acknowledged the use of only males as a minor limitation of the study that should be addressed in the future. Future studies should also test whether the upregulation of OAT3 plays a role in cortical plasticity observed following stimulation. Overall, this study provides novel insights into how neurovascular coupling, BBB permeability, and plasticity interact in the healthy brain.

    1. Reviewer #1 (Public Review):

      Wang et al., present a paper aiming to identify NALCN and TRPC6 channels as key mechanisms regulating VTA dopaminergic neuron spontaneous firing and investigating whether these mechanisms are disrupted in a chronic unpredictable stress model mouse.

      Major strengths:

      This paper uses multiple approaches to investigate the role of NALCN and TRPC6 channels in VTA dopaminergic neurons.

      Major weaknesses:<br /> In this revision, the authors have addressed the concerns about non-selective pharmacological tools.

      Are the author's claims supported by the data?

      The multimodal approach including shRNA knockdown experiments alleviates much of the concern about the non-specific pharmacological agents. Therefore, the author's claim that NALCN is involved in VTA dopaminergic neuron pacemaking is well-supported.

      The claim that TRPC6 channels in the VTA are involved in the depressive-like symptoms of CMUS is supported.


      It is important to compare pacemaking mechanisms in VTA and SNc neurons and this paper convincingly shows that NALCN contributes to VTA pacemaking, as it is known to contribute to SNc pacemaking. It also shows that TRPC6 channels in VTA dopamine neurons contribute to the depressive-like symptoms associated with CMUS.

      Additional context:

      One of the only demonstrations of the expression and physiological significance of TRPCs in VTA DA neurons was published by (Rasmus et al., 2011; Klipec et al., 2016) which are not cited in this paper. In their study, TRPC4 expression was detected in a uniformly distributed subset of VTA DA neurons, and TRPC4 KO rats showed decreased VTA DA neuron tonic firing and deficits in cocaine reward and social behaviors.

      Update: The authors say they have added a discussion of these papers, but I do not see it in the updated manuscript.

    1. Reviewer #1 (Public Review):

      Midbrain dopamine neurons have attracted attention as a part of the brain's reward system. A different line of research, on the other hand, has shown that these neurons are also involved in higher cognitive functions such as short-term memory. However, these neurons are thought not to encode short-term memory itself because they just exhibit a phasic response in short-term memory tasks, which cannot seem to maintain information during the memory period. To understand the role of dopamine neurons in short-term memory, the present study investigated the electrophysiological property of these neurons in rodents performing a T-maze version of short-term memory task, in which a visual cue indicated which arm (left or right) of the T-maze was associated with a reward. The animal needed to maintain this information while they were located between the cue presentation position and the selection position of the T-maze. The authors found that the activity of some dopamine neurons changed depending on the information while the animals were located in the memory position. This dopamine neuron modulation was unable to explain the motivation or motor component of the task. The authors concluded that this modulation reflected the information stored as short-term memory.

      Comments on revised submission:

      The authors adequately responded to all my concerns in the revised manuscript.

    1. Reviewer #1 (Public Review):

      The paper submitted by Yogesh and Keller explores the role of cholinergic input from the basal forebrain (BF) in the mouse primary visual cortex (V1). The study aims to understand the signals conveyed by BF cholinergic axons in the visual cortex, their impact on neurons in different cortical layers, and their computational significance in cortical visual processing. The authors employed two-photon calcium imaging to directly monitor cholinergic input from BF axons expressing GCaMP6 in mice running through a virtual corridor, revealing a strong correlation between BF axonal activity and locomotion. This persistent activation during locomotion suggests that BF input provides a binary locomotion state signal. To elucidate the impact of cholinergic input on cortical activity, the authors conducted optogenetic and chemogenetic manipulations, with a specific focus on L2/3 and L5 neurons. They found that cholinergic input modulates the responses of L5 neurons to visual stimuli and visuomotor mismatch, while not significantly affecting L2/3 neurons. Moreover, the study demonstrates that BF cholinergic input leads to decorrelation in the activity patterns of L2/3 and L5 neurons.

      This topic has garnered significant attention in the field, drawing the interest of many researchers actively investigating the role of BF cholinergic input in cortical activity and sensory processing. The experiments and analyses were thoughtfully designed and conducted with rigorous standards, leading to convincing results which align well with findings in previous studies. In other words, some of the main findings, such as the correlation between cholinergic input and locomotor activity and the effects of cholinergic input on V1 cortical activity, have been previously demonstrated by other labs (Goard and Dan, 2009; Pinto et al., 2013; Reimer et al., 2016). However, the study by Yogesh and Keller stands out by combining cutting-edge calcium imaging and optogenetics to provide compelling evidence of layer-specific differences in the impact of cholinergic input on neuronal responses to bottom-up (visual stimuli) and top-down inputs (visuomotor mismatch).

    1. Reviewer #1 (Public Review):

      In this manuscript, Yao et al. explored the transcriptomic characteristics of neural stem cells (NSCs) in the human hippocampus and their changes under different conditions using single-nucleus RNA sequencing (snRNA-seq). They generated single-nucleus transcriptomic profiles of human hippocampal cells from neonatal, adult, and aging individuals, as well as from stroke patients. They focused on the cell groups related to neurogenesis, such as neural stem cells and their progeny. They revealed genes enriched in different NSC states and performed trajectory analysis to trace the transitions among NSC states and towards astroglial and neuronal lineages in silico. They also examined how NSCs are affected by aging and injury using their datasets and found differences in NSC numbers and gene expression patterns across age groups and injury conditions. One major issue of the manuscript is questionable cell type identification. For example, more than 50% of the cells in the astroglial lineage clusters are NSCs, which is extremely high and inconsistent with classic histology studies.

      While the authors have made efforts to address previous critics, major concerns have not been adequately addressed, including a very limited sample size and with poor patient information. In addition, some analytical approaches are still questionable and the authors acknowledged that some they cannot address. Therefore, while the topic is interesting, some results are preliminary and some conclusions are not fully supported by the data presented.

    1. Reviewer #1 (Public Review):


      Otarigho et al. presented a convincing study revealing that in C. elegans, the neuropeptide Y receptor GPCR/NPR-15 mediates both molecular and behavioral immune responses to pathogen attack. Previously, three npr genes were found to be involved in worm defense. In this study, the authors screened mutants in the remaining npr genes against P. aeruginosa-mediated killing and found that npr-15 loss-of-function improved worm survival. npr-15 mutants also exhibited enhanced resistance to other pathogenic bacteria but displayed significantly reduced avoidance to S. aureus, independent of aerotaxis, pathogen intake and defecation. The enhanced resistance in npr-15 mutant worms was attributed to upregulation of immune and neuropeptide genes, many of which were controlled by the transcription factors ELT-2 and HLH-30. The authors found that NPR-15 regulates avoidance behavior via the TRPM gene, GON-2, which has a known role in modulating avoidance behavior through the intestine. The authors further showed that both NPR-15-dependent immune and behavioral responses to pathogen attack were mediated by the NPR-15-expressing neurons ASJ. Overall, the authors discovered that the NPR-15/ASJ neural circuit may regulate distinct defense mechanisms against pathogens under different circumstances. This study provides novel and useful information to researchers in the fields of neuroimmunology and C. elegans research.


      1. This study uncovered specific molecules and neuronal cells that regulate both molecular immune defense and behavior defense against pathogen attack and indicate that the same neural circuit may regulate distinct defense mechanisms under different circumstances. This discovery is significant because it not only reveals regulatory mechanisms of different defense strategies but also suggests how C. elegans utilize its limited neural resources to accomplish complex regulatory tasks.

      2. The conclusions in this study are supported by solid evidence, which are often derived from multiple approaches and/or experiments. Multiple pathogenic bacteria were tested to examine the effect of NPR-15 loss-of-function on immunity; the impacts of pharyngeal pumping and defecation on bacterial accumulation were ruled out when evaluating defense; RNA-seq and qPCR were used to measure gene expression; gene inactivation was done in multiple strains to assess gene function.

      3. Gene differential expression, gene ontology and pathway analyses were performed to demonstrate that NPR-15 controls immunity through regulating immune pathways.

      4. Elegant approaches were employed to examine avoidance behavior (partial lawn, full lawn, and lawn occupancy) and the involvement of neurons in regulating immunity and avoidance (the use of a diverse array of mutant strains).

      5. Statistical analyses were appropriate and adequate.

    1. Reviewer #1 (Public Review):

      Summary: The authors have used transcranial magnetic stimulation (TMS) and motor evoked potentials (MEPs) to determine whether the peripheral auditory confound arising from TUS can drive motor inhibition on its own. They gathered data from three international centers in four experiments testing:

      - Experiment 1 (n = 11), two different TUS durations and intensities under sound masking or without.<br /> - Experiment 2 (n = 27) replicates Exp 1 with different intensities and a fixed TUS duration of 500ms.<br /> - Experiment 3 ( n = 16) studies the effect of various auditory stimuli testing different duration and pitches while applying TUS in an active site, on-target or no TUS.<br /> - Experiment 4 (n = 12) uses an inactive control site to reproduce the sound without effective neuromodulation, while manipulating the volume of the auditory confound at different US intensities with and without continuous sound masking.

      Strengths: This study comes from three very strong groups in noninvasive brain stimulation with long experience in neuromodulation, multimodal and electrophysiological recordings. Although complex to understand due to slightly different methodologies across centers, this study provides quantitative evidence relating to the potential auditory confound in online TUS. The results are in line with reductions seen in motor-evoked responses during online 1kHz TUS, and remarkable efforts were made to isolate peripheral confounds from actual neuromodulation factors, highlighting the confounding effect of sound itself.

      Weaknesses: However, there are some points that need attention. In my view, the most important are:

      1. Despite the main conclusion of the authors stating that there is no dose-response effect of TUS on corticospinal inhibition, the point estimates for change in MEP and Ipssa indicate a more complex picture. The present data and analyses cannot rule out that there is a dose-response function which cannot be fully attributed to difference in sound (since the relationship in inversed, lower intracranial Isppa leads to higher MEP decrease). These results suggest that dose-response function needs to be further studied in future studies.

      2. Other methods to test or mask the auditory confound are possible (e.g., smoothed ramped US wave) which could substantially solve part of the sound issue in future studies or experiments in deaf animals etc.

    1. Reviewer #1 (Public Review):

      The propagation of electrical signals within neuronal circuits is tightly regulated by the physical and molecular properties of neurons. Since neurons vary in size across species, the question arises whether propagation speed also varies to compensate for it. The present article compares numerous speed-related properties in human and rat neurons. They found that the larger size of human neurons seems to be compensated by a faster propagation within dendrites but not the axons of these neurons. The faster dendritic signal propagation was found to arise from wider dendritic diameters and greater conductance load in human neurons. In addition, the article provides a careful characterization of human dendrites and axons, as the field has only recently begun to characterize post-operative human cells. There are only a few studies reporting dendritic properties and these are not all consistent, hence there is the added value of reporting these findings, particularly given that the characterization is condensed in a compartmental model.

      Strengths:<br /> The study was performed with great care using standard techniques in slice electrophysiology (pharmacological manipulation with somatic patch-clamp) as well as some challenging ones (axonal and dendritic patch-clamp). Modeling was used to parse out the role of different features in regulating dendritic propagation speed. The finding that propagation speed varies across species is novel as previous studies did not find a large change in membrane time constant or axonal diameters (a significant parameter affecting speed). A number of possible, yet less likely factors were carefully tested (Ih, membrane capacitance). The main features outlined here are well-known to regulate speed in neuronal processes. The modeling was also carefully done to verify that the magnitude of the effects is consistent with the difference in biophysical properties. Hence, the findings appear very solid to me.

      Weaknesses:<br /> The role of diameter in regulating propagation speed is well-known in the axon literature.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This important study investigated the role of the PHOX2B transcription factor in neurons in the key brainstem chemosensory structure, the retrotrapezoid nucleus (RTN), for maintaining proper CO2 chemoreflex responses of breathing in the adult rat in vivo. PHOX2B has an important transcriptional role in neuronal survival and/or function, and mutations of PHOX2B severely impair the development and function of the autonomic nervous system and RTN, resulting in the developmental genetic disease congenital central hypoventilation syndrome (CCHS) in neonates, where the RTN may not form and is functionally impaired. The function of the wild-type PHOX2B protein in adult RTN neurons that continue to express PHOX2B is not fully understood. By utilizing a viral PHOX2B-shRNA approach for knockdown of PHOX2B specifically in RTN neurons, the authors' solid results show impaired ventilatory responses to elevated inspired CO2, measured by whole-body plethysmography in freely behaving adult rats, that develop progressively over a four-week period in vivo, indicating effects on RTN neuron transcriptional activity and associated blunting of the CO2 ventilatory response. The RTN neuronal mRNA expression data presented suggests the impaired hypercapnic ventilatory response is possibly due to the decreased expression of key proton sensors in the RTN. This study will be of interest to neuroscientists studying respiratory neurobiology as well as the neurodevelopmental control of motor behavior.

      Strengths:<br /> 1. The authors used a shRNA viral approach to progressively knock down the PHOX2B protein, specifically in RTN neurons to determine whether PHOX2B is necessary for the survival and/or chemosensory function of adult RTN neurons in vivo.

      2. To determine the extent of PHOX2B knockdown in RTN neurons, the authors combined RNAScope® and immunohistochemistry assays to quantify the subpopulation of RTN neurons expressing PHOX2B and neuromedin B (Nmb), which has been proposed to be key chemosensory neurons in the RTN.

      3. The authors demonstrate that knockdown efficiency is time-dependent, with a progressive decrease in the number of Nmb-expressing RTN neurons that co-express PHOX2B over a four-week period.

      4. Their results convincingly show hypoventilation particularly in 7.2% CO2 only for PHOX2B-shRNA RTN-injected rats after four weeks as compared to naïve and non-PHOX2B-shRNA targeted (NT-shRNA) RTN injected rats, suggesting a specific impairment of chemosensitive properties in RTN neurons with PHOX2B knockdown.

      5. Analysis of the association between PHOX2B knockdown in RTN neurons and the attenuation of the hypercapnic ventilatory response (HCVR), by evaluating the correlation between the number of Nmb+/PHOX2B+ or Nmb+/PHOX2B- cells in the RTN and the resulting HCVR, showed a significant correlation between HCVR and number of Nmb+/PHOX2B+ and Nmb+/PHOX2B- cells, suggesting that the number of PHOX2B-expressing cells in the RTN is a predictor of the chemoreflex response and the reduction of PHOX2B protein impairs the CO2-chemoreflex.

      6. The data presented indicate that PHOX2B knockdown not only causes a reduction in the HCVR but also a reduction in the expression of Gpr4 and Task2 mRNAs, suggesting that PHOX2B knockdown affects RTN neurons transcriptional activity and decreases the CO2 response, possibly by reducing the expression of key proton sensors in the RTN.

      7. Results of this study show that independent of the role of PHOX2B during development, PHOX2B is still required to maintain proper CO2 chemoreflex responses in the adult brain, and its reduction in CCHS may contribute to the respiratory impairment in this disorder.

      Weaknesses:<br /> 1. The authors found a significant decrease in the total number of Nmb+ RTN neurons (i.e., Nmb+/PHOX2B+ plus Nmb+/ PHOX2B-) in NT-shRNA rats at two weeks post viral injection, and also at the four-week period where the impairment of the chemosensory function of the RTN became significant, suggesting some inherent cell death possibly due to off-target toxic effects associated with shRNA procedures that may affect the experimental results.

      2. The tissue sampling procedures for quantifying numbers of cells expressing proteins/mRNAs throughout the extended RTN region bilaterally have not been completely validated to accurately represent the full expression patterns in the RTN under experimental conditions.

      3. The inferences about RTN neuronal expression of NMB, GPR4, or TASK2 are based on changes in mRNA levels, so it remains speculation that the observed reduction in Gpr4 and Task2 mRNA translates to a reduction in the protein levels and associated reduction of RTN neuronal chemosensitive properties.

    1. Reviewer #1 (Public Review):

      Gazula and co-workers presented in this paper a software tool for 3D structural analysis of human brains, using slabs of fixed or fresh brains. This tool will be included in Freesurfer, a well-known neuroimaging processing software. It is possible to reconstruct a 3D surface from photographs of coronal sliced brains, optionally using a surface scan as model. A high-resolution segmentation of 11 brain regions is produced, independent of the thickness of the slices, interpolating information when needed. Using this method, the researcher can use the sliced brain to segment all regions, without the need of ex vivo MRI scanning.

      The software suite is freely available and includes 3 modules. The first accomplishes preprocessing steps, for correction of pixel sizes and perspective. The second module is a registration algorithm that registers a 3D surface scan obtained prior to sectioning (reference) to the multiple 2D slices. It is not mandatory to scan the surface, -a probabilistic atlas can also be used as reference- however the accuracy is lower. The third module uses machine learning to perform the segmentation of 11 brain structures in the 3D reconstructed volume. This module is robust, dealing with different illumination conditions, cameras, lens and camera settings. This algorithm ("Photo-SynthSeg") produces isotropic smooth reconstructions, even in high anisotropic datasets (when the in-plane resolution of the photograph is much higher than the thickness), interpolating the information between slices.

      To verify the accuracy and reliability of the toolbox, the authors reconstructed 3 datasets, using real and synthetic data. Real data of 21 postmortem confirmed Alzheimer's disease cases from the Massachusetts Alzheimer's Disease Research Center (MADRC)and 24 cases from the AD Research at the University of Washington(who were MRI scanned prior to processing)were employed for testing. These cases represent a challenging real-world scenario. Additionally, 500 subjects of the Human Connectome project were used for testing error as a continuous function of slice thickness. The segmentations were performed with the proposed deep-learning new algorithm ("Photo-SynthSeg") and compared against MRI segmentations performed to "SAMSEG" (an MRI segmentation algorithm, computing Dice scores for the segmentations. The methods are sound and statistically showed correlations above 0.8, which is good enough to allow volumetric analysis. The main strengths of the methods are the datasets used (real-world challenging and synthetic) and the statistical treatment, which showed that the pipeline is robust and can facilitate volumetric analysis derived from brain sections and conclude which factors can influence in the accuracy of the method (such as using or not 3D scan and using constant thickness).

      Although very robust and capable of handling several situations, the researcher has to keep in mind that processing has to follow some basic rules in order for this pipeline to work properly. For instance, fiducials and scales need to be included in the photograph, and the slabs should be photographed against a contrasting background. Also, only coronal slices can be used, which can be limiting for certain situations.

      The authors achieved their aims, and the statistical analysis confirms that the machine learning algorithm performs segmentations comparable to the state-of-the-art of automated MRI segmentations.<br /> Those methods will be particularly interesting to researchers who deal with post-mortem tissue analysis and do not have access to ex vivo MRI. Quantitative measurements of specific brain areas can be performed in different pathologies and even in the normal aging process. The method is highly reproducible, and cost-effective since allows the pipeline to be applied by any researcher with small pre-processing steps.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This fundamental study provides compelling neuroanatomical evidence underscoring the sensory function of the trunk in African and Asian elephants. Whereas myelinated tracts are classically appreciated as mediating neuronal connections, the authors speculate that myelinated bundles provide functional separation of trunk folds and display elaboration related to the "finger" projections. The authors avail themselves of many classical neuroanatomical techniques (including cytochrome oxidase stains, Golgi stains, and myelin stains) along with modern synchrotron X-ray tomography. This work will be of interest to evolutionary neurobiologists, comparative neuroscientists, and the general public, with its fascinating exploration of the brainstem of an icon sensory specialist.

      Strengths:<br /> - The authors made excellent use of the precious sample materials from 9 captive elephants.<br /> - The authors adopt a battery of neuroanatomical techniques to comprehensively characterize the structure of the trigeminal subnuclei and properly re-examine the "inferior olive".<br /> - Based on their exceptional histological preparation, the authors reveal broadly segregated patterns of metabolic activity, similar to the classical "barrel" organization related to rodent whiskers.

      Weaknesses:<br /> - As the authors acknowledge, somewhat limited functional description can be provided using histological analysis (compared to more invasive techniques).<br /> - The correlation between myelinated stripes and trunk fold patterns is intriguing, and Figure 4 presents this idea beautifully. I wonder - is the number of stripes consistent with the number of trunk folds? Does this hold for both species?

    1. Reviewer #1 (Public Review):

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

      The authors have addressed most criticisms raised in the initial review and the manuscript has improved significantly. One of the major concerns in the first review was whether PCDHg isoforms, which are expressed at a much lower level than C-type isoforms, have true physiological significance. The authors conducted additional experiments to address this point by using PCDHg cKO and provided convincing data supporting their conclusion. The results from PCDHg C4 overexpression, showing no impact on synaptic connectivity, further clarified the importance of isoforms. The limitation of the paper is that most experiments relied on overexpression of isoforms. Whether the isoform diversity is necessary for the synapse refinement in a physiological condition remains further clarification.

    1. Reviewer #1 (Public Review):

      Hyperactivation of mTOR signaling causes epilepsy. It has long been assumed that this occurs through overactivation of mTORC1, since treatment with the mTORC1 inhibitor rapamycin suppresses seizures in multiple animal models. However, the recent finding that genetic inhibition of mTORC1 via Raptor deletion did not stop seizures while inhibition of mTORC2 did, challenged this view (Chen et al, Nat Med, 2019). In the present study, the authors tested whether mTORC1 or mTORC2 inhibition alone was sufficient to block the disease phenotypes in a model of somatic Pten loss-of-function (a negative regulator of mTOR). They found that inactivation of either mTORC1 or mTORC2 alone normalized brain pathology but did not prevent seizures, whereas dual inactivation of mTORC1 and mTORC2 prevented seizures. As the functions of mTORC1 versus mTORC2 in epilepsy remain unclear, this study provides important insight into the roles of mTORC1 and mTORC2 in epilepsy caused by Pten loss and adds to the emerging body of evidence supporting a role for both complexes in the disease development.

      Strengths:<br /> The animal models and the experimental design employed in this study allow for a direct comparison between the effects of mTORC1, mTORC2, and mTORC1/mTORC2 inactivation (i.e., same animal background, same strategy and timing of gene inactivation, same brain region, etc.). Additionally, the conclusions on brain epileptic activity are supported by analysis of multiple EEG parameters, including seizure frequencies, sharp wave discharges, interictal spiking, and total power analyses.

      Weaknesses:<br /> The original concerns regarding the hippocampal contribution to the seizure phenotypes in this Pten loss-of-function model have been addressed with the inclusion of new data in the revised manuscript.

      The issue of sample sizes being small and do not allow for the assessment of whether mTORC1 or mTORC2 inactivation reduces seizure frequency or incidence remains a limitation of the study. However, the study's main conclusion that spontaneous seizures and epileptiform activity persist following inactivation of mTORC1 or mTORC2 alone while it is rescued following inactivation of both mTORC1 and mTORC2 is supported by the provided data and remains valid.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This study examines a hypothesized link between autism symptomatology and efference copy mechanisms. This is an important question for several reasons. Efference copy is both a critical brain mechanism that is key to rapid sensorimotor behaviors, and one that has important implications for autism given recent empirical and theoretical work implicating atypical prediction mechanisms and atypical reliance on priors in ASD.

      The authors test this relationship in two different experiments, both of which show larger errors/biases in spatial updating for those with heightened autistic traits (as measured by AQ in neurotypical (NT) individuals).

      Strengths:<br /> The empirical results are convincing - effects are strong, sample sizes are sufficient, and the authors also rule out alternative explanations (ruling out differences in motor behavior or perceptual processing per se).

      Weaknesses:<br /> My main concern is that the paper should be more transparent about both (1) that this study does not include individuals with autism, and (2) acknowledging the limitations of the AQ.

      On the first point, and I don't think this is intentional, there are several instances where the line between heightened autistic traits in the NT population and ASD is blurred or absent. For example, in the second sentence of the abstract, the authors state "Here, we examine the idea that sensory overload in ASD may be linked to issues with efference copy mechanisms". I would say this is not correct because the authors did not test individuals with ASD. I don't see a problem with using ASD to motivate and discuss this work, but it should be clear in key places that this was done using AQ in NT individuals.

      For the second issue, the AQ measure itself has some problems. For example, reference 38 in the paper (a key paper on AQ) also shows that those with high AQ skew more male than modern estimates of ASD, suggesting that the AQ may not fully capture the full spectrum of ASD symptomatology. Of course, this does not mean that the AQ is not a useful measure (the present data clearly show that it captures something important about spatial updating during eye movements), but it should not be confused with ASD, and its limitations need to be acknowledged. My recommendation would be to do this in the title as well - e.g. note impaired visuomotor updating in individuals with "heightened autistic traits".

      Suggestions for improvement:<br /> - Figure 5 is really interesting. I think it should be highlighted a bit more, perhaps even with a model that uses the results of both tasks to predict AQ scores.<br /> - Some discussion of the memory demands of the tasks will be helpful. The authors argue that memory is not a factor, but some support for this is needed.<br /> - With 3 sessions for each experiment, the authors also have data to look at learning. Did people with high AQ get better over time, or did the observed errors/biases persist throughout the experiment?

    1. Reviewer #1 (Public Review):


      Walsh and colleagues investigated how cued probabilistic expectations about future stimuli may influence different stages of decision-making as implemented in the human brain. In their study, participants were provided with cues that could correctly (or incorrectly) cue which visual stimulus would be presented. These cues also predicted the motor action that would likely produce a correct judgment for that trial. In addition a 'neutral' cue was included that did not predict any particular stimulus. They report that measures of steady-state visual evoked potentials (SSVEPs, proposed to index the magnitude of visual neural activity in favour of the correct response) were smaller when the cue incorrectly predicted the upcoming image, compared to when an accurate cue or a neutral cue was presented. Their primary finding adds to an ongoing debate in the field of decision-making research about how cued expectations may influence how we make decisions.


      This study uses a carefully-constructed experiment design and decision-making task that allows separation of multiple electroencephalographic (EEG) signals thought to track different stages of decision-making. For example, the steady-state visual evoked potential measures can be cleanly dissociated from more anterior beta-band activity over motor cortex. They also allow evaluation of how cued expectancy effects may unfold over a number of testing sessions. This is important because the most consistent evidence of expectation-related modulations of electrophysiological measures (using EEG, local field potentials or single neuron firing rates) is from studies of non-human primates that involved many days of cue-stimulus contingency learning, and there is a lack of similar work using several testing sessions in humans. Although there were several experimental conditions included in the study, careful trial-balancing was conducted to minimise biases due to incidental differences in the numbers of trials included for analyses across each condition. Performance for each individual was also carefully calibrated to maximise the possibility of identifying subtle changes in task performance by expectation and avoid floor or ceiling effects.


      Although the experiment and analysis methods are cohesive and well-designed, there are some shortcomings that limit the inferences that can be drawn from the presented findings.

      The first relates to the measures of SSVEPs and their relevance for decision-making in the task. In order to eliminate the influence of sporadic pulses of contrast changes that occurred during stimulus presentation, a time window of 680-975 ms post stimulus onset was used to measure the SSVEPs. As shown in the response time quantile plot in Supplementary Figure S1, a substantial portion of response times are earlier than all, or a portion of, the time period included in the SSVEP measurement window. It has also been estimated to require 70-100 ms to execute a motor action (e.g., a keypress response) following the commitment to a decision. This raises some concerns about the proportion of trials in which the contrast-dependent visual responses (indexed by the stimulus-locked SSVEPs) indexed visual input that was actually used to make the decision in a given trial. While response-locked SSVEP plots are provided, statistical analyses testing for differences during the pre-response period were not performed. Standard errors in Figure 4D (depicting differences in SSVEPs for validly and invalidly cued trials) partly overlap with zero during the pre-response time window. There is no strong evidence for clear SSVEP modulations in any specific time windows leading to the response.

      In addition, an argument is made for changes in the evidence accumulation rate (called the drift rate) by stimulus expectancy, corresponding to the observed changes in SSVEP measures and differences in the sensory encoding of the stimulus. As the authors acknowledge, this inference is limited by the fact that evidence accumulation models (such as the Diffusion Decision Model) were not used to test for drift rate changes as could be determined from the behavioural data (by modelling response time distributions). Plots of response quantiles in Supplementary Figure S1 also do not show a typical pattern that indicates changes in the drift rate (i.e., larger differences between validly and invalidly cued trials for relatively slower response time quantiles). There appear to be ample numbers of trials per participant to test for drift rate changes in addition to the starting point bias captured in earlier models. Due to the very high number of trials, models could potentially be evaluated for each single participant, although modelling would be substantively complicated by effects of the pulses of contrast changes, as noted by the authors. This could be done in future work (in experiments without contrast pulses) and would provide more direct evidence for drift rate changes than the findings based on the SSVEPs, particularly due to the issues with the measurement window relating to the response times as mentioned above.

      In addition, there is some uncertainty regarding how to interpret the SSVEP effects in relation to phenomena such as expectation suppression enabled via sharpening or dampening effects. The measure used in this study is marginal SSVEPs, indexing the difference in SSVEP amplitudes between relatively higher- and lower-contrast gratings (termed target and non-target gratings). The observed increase in marginal SSVEPs for validly as compared to invalidly cued trials could arise due to an increase in SSVEP amplitudes for target grating orientations, a decrease for non-target orientations, a combination of these two, or even an increase or decrease for both target and non-target SSVEPs (with a larger increase/decrease for the target or non-target orientation). Some analyses were performed to investigate predictive cueing effects on target as compared to non-target SSVEPs, but these did not provide clear evidence that favoured a specific interpretation. This should be considered when interpreting the SSVEP effects in relation to different variants of expectation suppression that have been proposed in the literature.

    1. Reviewer #1 (Public Review):


      This study investigated behavioural performance on a competing speech task and neural attentional filtering over the course of two years in a group of middle-aged to older adults. Neural attentional filtering was quantified using EEG by comparing neural envelope tracking to an attended vs. an unattended sentence. This dataset was used to examine the stability of the link between behavior and neural filtering over time. They found that neural filtering and behavior were correlated during each measurement, but EEG measures at the first timepoint did not predict behavioural performance two years later. Further, while behavioural measures showed relatively high test-retest reliability, the neural filtering reliability was weak with an r value of 0.21. The authors conclude that neural tracking-based metrics have limited ability to predict longitudinal changes in listening behavior.


      This study is novel in its tracking of behavioural performance and neural envelope tracking over time, and it includes an impressively large dataset of 105 participants. The manuscript is clearly written.


      The weaknesses are minor, primarily concerning how the reviewers interpret their data. Specifically, the envelope tracking measure is often quite low, close to the noise floor, and this may affect test-retest reliability. Furthermore, the trajectories may be affected by accelerated age-related declines that are more apparent in neural tracking than in behaviour.

      Comments on revised version:

      The authors have satisfactorily addressed my previous comments and they present a strong case for the interpretation of their findings.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors aimed to investigate the contribution of antigenic drift in the HA and NA genes of seasonal influenza A(H3N2) virus to their epidemic dynamics. Analyzing 22 influenza seasons before the COVID-19 pandemic, the study explored various antigenic and genetic markers, comparing them against indicators characterizing the epidemiology of annual outbreaks. The central findings highlight the significant influence of genetic distance on A(H3N2) virus epidemiology and emphasize the role of A(H1N1) virus incidence in shaping A(H3N2) epidemics, suggesting subtype interference as a key factor.

      Major Strengths:<br /> The paper is well-organized, written with clarity, and presents a comprehensive analysis. The study design, incorporating a span of 22 seasons, provides a robust foundation for understanding influenza dynamics. The inclusion of diverse antigenic and genetic markers enhances the depth of the investigation, and the exploration of subtype interference adds valuable insights.

      Major Weaknesses:<br /> While the analysis is thorough, some aspects require deeper interpretation, particularly in the discussion of certain results. Clarity and depth could be improved in the presentation of findings. Furthermore, the evolving dynamics of H3N2 predominance post-2009 need better elucidation.

    1. Reviewer #2 (Public Review):

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

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

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

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

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

      Below I have listed some references for the authors to read:

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

    1. Reviewer #1 (Public Review):

      This work describes a new and powerful approach to a central question in ecology: what are the relative contributions of resource utilisation vs interactions between individuals in the shaping of an ecosystem? This approach relies on a very original quantitative experimental set-up whose power lies in its simplicity, allowing an exceptional level of control over ecological parameters and of measurement accuracy.

      In this experimental system, the shared resource corresponds to 10^12 copies of a fixed single stranded target DNA molecule to which 10^15 random single stranded DNA molecules (the individuals populating the ecosystem) can bind. The binding process is cycled, with a 1000x-PCR amplification step between successive binding steps. The composition of the population is monitored via high-throughput DNA sequencing. Sequence data analysis describes the change of population diversity over cycles. The results are interpreted using estimated binding interactions of individuals with the target resource, as well as estimated binding interactions between individuals and also self-interactions (that can all be directly predicted as they correspond to DNA-DNA interactions). A simple model provides a framework to account for ecosystem dynamics over cycles. Finally, the trajectory of some individuals with high frequency in late cycles is traced back to the earliest cycles at which they are detected by sequencing. Their propensities to bind the resource, to form hairpins or to form homodimers suggest how different interaction modes shape the composition of the population over cycles.

      The authors report a shift from selection for binding to the resource to interactions between individuals and self-interactions over the course of cycles as the main driver of their ecosystem. The outcome of the experiment is far from trivial as the individual-resource binding energy initially determines the relative enrichment of individuals, and then seems to saturate. The richness of the population dynamics observed with this simple system is thus comparable to that found in some natural ecosystems. The findings obtained with this new approach will likely guide the exploration of natural ecosystems in which parameters and observables are much less accessible.

      My review focuses mainly on experimental aspects of this work given my own expertise. The introduction exposes very convincingly the scientific context of this work, justifying the need for such an approach to address questions pertaining to ecology. The manuscript describes very clearly and rigorously the experimental set-up. The main strengths of this work are (i) the outstanding originality of the experimental approach and (ii) its simplicity. With this setup, central questions in ecology can be addressed in a quantitative manner, including the possibility to run trajectories in parallel to generalize the findings, as reported here. Technical aspects have been carefully implemented, from the design of random individuals bearing flanking regions for PCR amplification, binding selection and (low error) amplification protocols, and sequencing read-out whose depth is sufficient to capture the relevant dynamics. With this setup one can tune the relative contributions of binding selection vs amplification for instance (to disentangle forces that shape the ecosystem). One can also run cycles with new DNA individuals, designed with arbitrarily chosen resource binding vs self-binding, that are predicted to dominate depending on chosen ecological parameters. These exciting perspectives underlie the strong potential of the new approach described in the current study.

    1. Reviewer #1 (Public Review):

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

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

    1. Reviewer #1 (Public Review):

      Kou and Kang et al. investigated the role of Notch-RBP-J signaling in regulating monocyte homeostasis. Specifically, they examined how a conditional knockout of Rbpj expression in monocytes though a Rbpjfl/fl Lyz2cre/cre mouse affects the homeostasis of Ly6Chi versus Ly6Clo monocytes. They discovered that Rbpj deficiency did not affect the percentage of Ly6Chi monocytes but instead, led to an accumulation of Ly6Clo monocytes in the peripheral blood. Using a comprehensive number of in vivo techniques to investigate the origin of this increase, the authors revealed that the accumulation of Rbpj deficient Ly6Clo monocytes was not due to an increase in bone marrow egress and homing and that this defect was cell intrinsic. However, EdU-labelling and apoptosis assays revealed that this defect was not due to an increase in proliferation nor conversion of Ly6Chi to Ly6Clo monocytes. Interestingly, it was revealed that Rbpj deficient Ly6Clo monocytes had increased expression of CCR2 and ablation of CCR2 expression reversed the accumulation of these cells in the periphery. Lastly, they discovered that Rbpj deficiency also led to downstream effects such as an accumulation of Ly6Clo monocytes in the lung tissue and increased CD16.2+ interstitial macrophages, presumably due to dysregulated monocyte differentiation and function.

      Their findings are interesting and highlight a previously unexplored association between Notch-RBP-J signaling and CCR2 expression in monocyte homeostasis, providing further insight into the distinct pathways that regulate Ly6Chi vs Ly6Clo monocyte subsets individually.

      The strengths of this paper include the use of multiple conditional genetic knock out mouse models to explore their hypothesis and the use of sophisticated in vivo techniques to study the major mechanisms involved in monocyte homeostasis. However, a major weakness of the paper is the exact role of how CCR2 compensates for the increase in Ly6Clo monocytes in the circulation in the RBP-J knockout mice as the authors showed no differences in their conversion, egress or homing back to the bone marrow. The authors were also unable to show that RBP-J knockout mice were functionally different in their response to CCL2 due to technical difficulties, which makes it challenging to conclude how CCR2 compensates for their trafficking patterns. Consequently the link between CCR2 and RBP-J remains correlative based on the data presented in the paper.

      The conclusions of this paper are mostly well substantiated from the experimental data but as mentioned above, the mechanism of how CCR2 relates to the increase in Ly6Clo monocytes in RBP-J knockout mice is still unclear. Nevertheless, this work will be of interest to immunologists and biologists working on Notch-signalling in diseases. In addition, the methods and data would be useful for researchers who are seeking to use the Rbpjfl/fl Lyz2cre/cre mouse model for their studies.

    1. Joint Public Review:

      TGN46 is a prominent TGN protein that cycles to the plasma membrane. It has been used as a TGN marker for many years, but its function has been unknown. This manuscript provides evidence that the luminal domain of TGN46 serves as a cargo receptor for incorporation of the soluble secretory protein PAUF into a class of TGN-derived carriers called CARTS. Interestingly, the luminal domain also plays an important role in the intracellular and intra-Golgi localization of TGN46, and it contains a positive signal for Golgi export in CARTS. They demonstrate that TGN46 loading into CARTS is not dependent on its cytosolic terminus using a deltaCT mutant. A speculative part of the manuscript proposes that the luminal domain of TGN46 might form biomolecular condensates that help to capture cargo proteins for export.

      This is a very nice study that makes a significant contribution to the field. New insights are obtained regarding the function of TGN46 and the role of its various domains. Various potential interpretations of the data are presented in a balanced and constructive way.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The present study addresses how the local abundance of metabolites impacts the biology of the tumor microenvironment. The authors enroll patients harboring kidney tumors and use freshly resected tumor material for metabolic studies. Specifically, the authors separate the adjacent normal kidney tissue from the tumor material and then harvest the interstitial fluid from the normal kidney (KIF) or the tumor (TIF) for quantitative metabolomics. The plasma samples from the patient are used for comparison. Additionally, the authors also compare metabolite levels in the plasma of patients with kidney versus lung cancer (or healthy donors) to address how specific tumor types might contribute to circulating levels of metabolites. Altogether, the authors find that the metabolite levels in the KIF and TIF, although vastly different than plasma, are largely overlapping. These findings indicate that tissue of origin appears to have a stronger role in determining the local metabolic environment of tumors than the genetics or biochemistry of the tumor itself.

      Strengths:<br /> The biggest strength of the current study is the use of human patient-derived samples. The cohort size (~50 patients) is relatively large, which adds to the rigor of the work. The work also relies on a small pool of metabolites that can be quantitatively measured using methods developed by the authors. Focusing on a smaller metabolic pool also likely increases the signal-to-noise ratio and enables the more rigorous determination of any underlying differences. The manuscript is well-written and highlights both the significance of the findings and also acknowledges many of the caveats. The recognition of the metabolic contributions of surrounding normal tissue as the primary driver of local nutrient abundance is a novel finding in the work, which can be leveraged in future studies.

      Weaknesses:<br /> The work has certain caveats, some of which have been already recognized by the authors. These include the use of steady-state metabolites and the possibility of cross-contamination of some TIF into the adjacent KIF. This study is also unable to distinguish the mechanisms driving the metabolic changes in KIF/TIF relative to circulating levels in plasma.

      The relative similarity of KIF and TIF is quite surprising. However, this interpretation is presently based on a sampling of only ~100 polar metabolites and ~200 lipid molecules. It is, perhaps, possible that future technological developments that enable more comprehensive quantitative metabolic profiling might distinguish between KIF and TIF composition.

      In vitro, tissue culture is recognized to suffer from 'non-physiological' nutrient dependencies, which are impacted by the composition of culture media. Thus, in vivo studies remain our current gold-standard in mechanistic studies of tumor metabolism. It is presently unclear whether the findings of this work will be recapitulated in any of the kidney cancer in vivo models and thus be functionally testable.

    1. Graphic designers produce representations of society, and they help create access to information and ideas. But who gets to be represented, and who gets access?

      Beyond this question lies another to me. Who is art for? Do artists/graphic designers have a greater responsibility to the people that engage with their art?

    2. Graphic designers produce representations of society, and they help create access to information and ideas. But who gets to be represented, and who gets access?

      It is essential to question what we see as authority just because it looks official. The internet has opened information and platform access to many more people. What that looks like in the future is literally up to us. We can have a great effect as visual artists and strategic thinkers on how culture grows. What will "inclusive" mean 20 years from now. Will we still be fighting the same fights?