7,004 Matching Annotations
  1. Oct 2023
    1. Reviewer #1 Public Review

      Summary<br /> This paper presents a new, but simple and low-cost technique for multimodal EM imaging that combines the strengths of both volume scanning electron microscopy (SEM) and electron microscopic tomography. The novel ATUM-Tomo approach enables the consecutive inspection of selected areas of interest by correlated serial SEM and TEM, optionally in combination with CLEM, as demonstrated here. The most important feature of ATUM-Tomo, particularly of correlative ATUM-Tomo, is that it can bridge scales, from the cellular to the high-resolution subcellular scale, from micrometer to low nanometer resolution. This is particularly important for ultrastructural analyses of biological regions of interest, which is demonstrated here for focal pathologies or rare cellular and subcellular events. Both imaging modalities are non-destructive, thus allowing re-imaging and hierarchical imaging at the SEM and TEM levels. This is particularly important for precious samples, including human biopsies and samples from complex CLEM experiments. Beyond the demonstrated neuropathology-related application, further use in investigating normal and pathologically altered brains, including human brain tissue samples that require high-resolution SEM and TEM in combination with immunohistochemistry, and virus or tracer injections, would be possible. Thus, ATUM-Tomo provides new possibilities in multimodal volume EM imaging for diverse areas of biological research.

      Strengths<br /> This paper is a very nice piece of work, bringing together modern high-end state-of-the-art technology that will allow us to investigate diverse biological questions in different areas of interest and at different scales. The paper is clear and well-written, although some additions are necessary to the methods section and the scientific results as exemplified by investigations of the blood-brain barrier. The discussion would benefit from an expansion of the part dealing with the scientific results. The paper is accompanied by excellent figures, supplemental information, and colored 3D-reconstructions, which makes it easy for the reader to follow the experimental procedure and the scientific context. The authors may consider moving the supplemental figures into the main body of the paper, which would then still contain 'only' eight figures.

      Weaknesses<br /> There is some imbalance between the description of the state-of-the-art methodology and the scientific context.

    1. Reviewer #1 (Public Review):

      Sun and co-authors have determined the crystal structures of EHEP with/without phlorotannin analog, TNA, and akuBGL. Using the akuBGL apo structure, they also constructed model structures of akuBGL with phlorotannins (inhibitor) and laminarins (substrate) by docking calculation. They clearly showed the effects of TNA on akuBGL activity with/without EHEP and resolubilization of the EHEP-phlorotannin (eckol) precipitate under alkaline conditions (pH >8). Based on this knowledge, they propose the molecular mechanism of the akuBGL-phlorotannin/laminarin-EHEP system at the atomic level. Their proposed mechanism is useful for further understanding of the defensive-offensive association between algae and herbivores.

    1. Reviewer #1 (Public Review):

      Gambelli et al. provide a structural study of the SlaA/SlaB S-layer of the archaeon Sulfolobus acidocaldarius. S-layers form an essential component of most archaeal cell envelopes, where their self-assembling properties and activity as cell envelope support structures have raised substantial interest, both from researchers seeking to understand the fundamental biology of archaea, as well as researchers seeking to exploit the biomaterial properties of S-layers in biotechnological applications. Both interests are hampered by the paucity of structural information on archaeal S-layer assembly, structure, and function to date, in large part due to technical difficulties in their study.

      In this study, Gambelli and coworkers overcome these difficulties and report the high-resolution 3D cryoEM structures of the purified SlaA monomers at three different pH, as well as the medium resolution 3D cryoET structures of the SlaA/SlyB lattices determined from S-layer fragments isolated from the Sulfolobus cells.

      The structural work is generally well executed, although lacks in detail in places to allow a proper review, particularly in the cryoET. A further drawback of the current manuscript is that the structural work remains rather descriptive and speculative, with little validation of the proposed models.

      The authors run a plethora of representation, analyses, prediction, and simulation software on their structures resulting in an abundance of Figures that risk overloading the reader and in several cases bring little new insight beyond unsubstantiated speculation.

      The structural description of the S. acidocaldarius S-layer will be of high general interest and the authors have made a substantial leap forward, but the current manuscript would benefit from a better validation and basic atomic description of the SlaA/SlaB S-layer.

      Specific points.

      - It is not possible to review the quality of the SlaA and SlaA/SlaB models in the cryoET reconstruction. No detailed fits of the map and model are shown, and no correlation statistics are given (the latter is also true for the higher resolution 3D reconstructions at pH4, 7, and 10). To be of use to the community, the S-layer model and cryoET maps should also be deposited in PDB and EMDB, and an autodep report and ideally the cryoET maps should be available.

      - The authors spend a great deal on the MD simulation of the SlaA glycans and the description of the 'glycan shield' and its possible role in subunit electrostatics and intersubunit contacts. This does not result in testable hypotheses, however, and does not bring much more than vague speculation on the role of the glycans or the subunits contacts in S-layer assembly and stability. For the primary description of the SlaA/B S-layer, more important would be a detailed atomic description and validation of the intermolecular contacts in the proposed lattice model. Given the low resolution of the cryoET, this would require MD simulation of the contacts. Lattice stability during MD simulation and/or the confirmation of lattice contacts by cross-linking mass spectrometry would go a great way in validating the proposed lattice model.

      - The discussion of the subunit electrostatics and the role they could play in subunit assembly/disassembly remains superficial and speculative. No real model or hypothesis is put forward, let alone validated.

      - The authors solve the cryoEM structure of SlaA released and purified form S. acidocaldarius S-layers by an alkaline pH shift. When shifted back to acidic pH, does this native material self-assemble in vitro? If not, do the authors have an explanation for this? Are components missing or could the solved structures represent SlaA conformations that are no longer assembly competent?

    1. Reviewer #1 (Public Review):

      The manuscript by Royall et al. builds on previous work in the mouse that indicates that neural progenitor cells (NPCs) undergo asymmetric inheritance of centrosomes and provides evidence that a similar process occurs in human NPCs, which was previously unknown.

      The authors use hESC-derived forebrain organoids and develop a novel recombination tag-induced genetic tool to birthdate and track the segregation of centrosomes in NPCs over multiple divisions. The thoughtful experiments yield data that are concise and well-controlled, and the data support the asymmetric segregation of centrosomes in NPCs. These data indicate that at least apical NPCs in humans undergo asymmetric centrosome inheritance. The authors attempt to disrupt the process and present some data that there may be differences in cell fate, but this conclusion would be better supported by a better assessment of the fate of these different NPCs (e.g. NPCs versus new neurons) and would support the conclusion that younger centriole is inherited by new neurons.

    1. Reviewer #1 (Public Review):

      Myelodysplastic syndrome (MDS) represents as a rather complex and serious hematologic malignancy that affects the production of normal blood cells in the bone marrow. Some types of MDS could stay mild for years and other types of MDS could be more serious and progressed into AML. Tremendous efforts have been made to investigate the pathogenesis and treatment of MDS. For instance, a pile of papers has found that iron chelation therapy could benefit the overall survival in low risk MDS patients. Yet, the risk and benefit of this therapy remain in much debate. The authors demonstrated that erythrocyte precursors could re-gain EPO responsiveness after DFP chelation therapy. In addition, the authors investigated iron trafficking in erythroblasts using the MDS mouse model. The paper is rather interesting as it discussed the biological effects and underlying mechanisms of DFP for the treatment of low-risk MDS. More importantly, the paper adds practical values and theoretical evidences for chelation therapy towards low-risk MDS. The paper is overall well-written.

    1. Reviewer #1 (Public Review):

      In this manuscript, Kim et al. investigate the molecular basis for hindbrain segmentation by performing combined single cell nucleus RNAseq and ATACseq (scMultiome) on zebrafish embryonic hindbrain tissue. Hindbrain segmentation is fundamental to head development in vertebrate species. Decades of research have provided many insights into the gene regulatory cascades that control the progressive subdivision of the hindbrain territory into segments (rhombomeres). These studies have enabled the formulation of gene regulatory network (GRN) models that depict these regulatory interactions. However, many aspects of the GRN need further clarification, including the early steps of pre-rhombomeric patterning, and the factors that respond to axial signaling pathways such as RA and FGF. The dataset in this study provides a comprehensive view of gene expression and chromatin states during hindbrain segmentation, thus it is a valuable resource for characterizing the underlying GRN. The authors demonstrate the utility of this data by comparing the molecular profiles between different rhombomeres and tracing when and how these profiles arise during development.

      Four main findings are presented:

      1. Each rhombomere has a unique molecular profile.<br /> 2. There is no clear molecular signature for odd versus even rhombomeres, nor any overt repeating two-segment molecular identities.<br /> 3. The mature rhombomeres emerge through the subdivision of three mixed-identity 'primary hindbrain progenitor domains' (PHPDs) that correspond to r2/r3, r4, and r5/r6, respectively.<br /> 4. RA and FGF signaling control formation of the primary hindbrain progenitor domains.

      These findings are well supported by the data but in my opinion they mainly confirm what was already known and do not significantly advance our mechanistic understanding of rhombomere formation, which is the aim of the paper.

      Strengths:<br /> This comprehensive dataset will be very valuable to researchers in the field. The authors successfully demonstrate its utility by resolving unique molecular profiles for each rhombomere and identifying some novel markers.

      The authors make excellent use of HCR to validate their findings, such as the co-expression of vgll3 and egr2b in r2/r3 cells at 10hpf, which implies mixed identities of PHPD cells.

      The performance of scMultiome analysis on tissue from DEAB-treated embryos (depleted RA signaling) is exciting and holds much promise for identifying RA-dependent gene regulatory cascades that govern caudal hindbrain patterning. Assessing the contribution of control versus DEAB-treated cells to the various UMAP clusters is a very nice way to identify the altered cell states in the RA-depleted hindbrain. This confirms a complete absence of r5 and r6 in the DEAB-treated embryos at this developmental stage, as was inferred from in-situ approaches in earlier studies.

      Weaknesses:<br /> The major weakness of this work is that it only provides an incremental mechanistic advance to our current understanding of the molecular basis for rhombomere formation. The descriptions of gene expression are useful but for the most part they are rather shallow lines of enquiry that confirm what was already known from previous, less comprehensive studies of gene expression. For example, regarding the identification of PHPDs, it has long been known that r5/r6 share a progenitor domain that is demarcated by mafba expression. Similarly, RA and Fgf signaling have already been shown to be required for anterior-posterior patterning in the pre-rhombomeric hindbrain. The identification of mixed-identity progenitors in PHPDs, and the characterisation of the changes in transcription and chromatin state in response to RA signaling perturbation are really exciting starting points for deeper analysis of the underlying GRN. However, it is a shame that no effort is made to glean mechanistic insights from this dataset by computational GRN inference.

    1. Reviewer #1 (Public Review):

      This manuscript by Proskurin, Manakov, and Karpova, posits a unique role for the anterior cingulate cortex (ACC) in the flexible control of learned sequences of motor actions. The authors marshall evidence from behavioral-electrophysiological analyses in support of two major claims: 1) that action encoding by ACC ensembles tracks 1) the current "context", i.e., which behavioral sequence is rewarded, and 2) the "prevalence", i.e., number of repetitions of one specific sequence. An important aspect of this later point is that the authors propose prevalence encoding is not strictly dependent on trial-by-trial reward receipt.

      In this work, the authors wish to focus on self-initiated behavior when the correct behavioral sequence, out of four or fewer (mostly two it appears), changes across blocks in an unsignaled manner. Rats learn to enter a left and right nose poke in a sequence of three responses, with a required entry into a central port prior to each intra-sequence response, with correct sequence completion reinforced by a sucrose delivery in the relevant side nose poke port. Extracellular spike activity is acquired from well-trained rats performing this task. The authors' analyses of the behavior of well-trained rats show rats adjust their behavior when the block switches to a different one of the sequences in the known 'library'. The rats also perform non-reinforced responses/sequences within a given block which the authors suggest is exploration likely not triggered by changes in reinforcement in contrast to behavior change after a block switch.

      The authors next provide a very rich set of analyses to examine the encoding of responses and sequences by ACC neural activity. Overall, these data provide intriguing support for ACC's integral contributions to flexible behavioral control. However, some of the individual analyses are a bit difficult to follow and could be clarified with greater detail within the results section of the paper, permitting an easier evaluation of the quality of the supporting data. Second, there are some proposals that could be strengthened by fuller analysis, in particular the authors' suggestion that "prevalence" encoding is distinct from reward encoding and/or is not impacted by reward presence or omission. Given the likely rich data set in hand, the authors could do more to demonstrate how "prevalence" encoding interacts with reinforcement parameters or perhaps be more specific in their word choice. More importantly, I was left unclear on how "prevalence" encoding intersects with the decision to repeat the same behavioral sequence on the next trial or not. These issues aside, this work provides further information on the physiology of ACC during flexible behavior and will add importance to this field.

      Below are specific issues:

      1. Some greater attention to the behavioral parameters could be helpful, especially regarding the impact of reward rate on behavior. For example, looking at some of the figures of individual rat behavior, exploratory sequences seemed triggered by reward omission. Is this just a chance for the examples chosen or is there something systematic here? Upon block switch, how exactly does the switch in sequences emitted by the rat track with reinforcement history? The authors mention that reinforcement probability differed across sessions, and one would thus expect switching behavior would as well. Because of the interesting existence of sometimes quite long 'tails' of performance of the original sequence after a block switch, I am wondering how the length of such tails relates to reinforcement rate parameters.

      2. The authors provide strong data indicating that a given L or R response is associated with distinct ACC activity depending on which sequence that response is embedded within, a finding reminiscent of other reports in multiple brain regions. While not a criticism per se, I was interested in the center port responses, also embedded within unique sequences, yet never preceding reward. A key difference in the performance of a given R or L response is that it is sometimes the terminal response, and thus the rat knows a given R or L response to be sometimes reinforced in one of the contexts, but not the other, in each of these comparisons. I wonder if there was an opportunity to cleanly demonstrate the context dependence of a given individual action by comparing center port responses across distinct sequences.

      3. In analyzing neural activity accompanying the behavioral persistence of the dominant sequence after a block change, the authors find that the ACC ensemble firing pattern is closer to the original dominant sequence pattern during reinforcement and less like this pattern during exploration. This makes sense and must be the case, as, in the example shown in the figure, the rat does not "know" the block has switched since no reward has yet been delivered that would signal that switch. (As an aside, it would be interesting to know, given a specific reward schedule in a given session, what would be the maximum number of unrewarded trials within the block, and how might that impact the performance/reward expectation during the tails?)<br /> As time, and trials, progress the rat is approaching the point at which it explores another strategy. The authors find strengthened "prevalence" encoding with increasing sequence repetition, but if this parameter is related to behavioral change/flexibility, this was not clear to me. Might there be something unique about the last trials in a tail "predicting" an upcoming switch? Can the authors please expand?<br /> Relatedly, if the prediction of upcoming behavioral change is not observed in the neural activity from sequence steps 2-6, it is notable that these are the steps 'within' the sequence, that leaves out the initiation (first center poke) and termination (reward/reward omission). Thus one could imagine this information is "missed" in the current analysis given that both the reward period and the initiation of a trial at the center are not analyzed. This does lead me to suggest a softening of some claims made of identifying "unifying principles" of ACC function, as the authors state, based on the analyses included in the current report, since the neural activity related to the full unit of behavior is not considered. (I appreciate the motivation behind this focus on within-sequence behavior - the wish to compare time periods with similar movement parameters .)

      4. The variance in neural activity explained by the prevalence models is on average quite low. However, the authors find that the variance explained differs quite dramatically by anatomical coordinate within ACC. Would it make sense to focus the control analyses (vigor, reward history, and so on) on those sessions/ensembles with greater variance explained, ie, perhaps there might be greater sensitivity to detecting interactions among variables within ensembles recorded more rostrally?

      5. A very intriguing aspect of this work is the position that (from the abstract): "Prevalence encoding in the ACC is ...independent of reward delivery." This is a novel aspect of the current work. However, I am wondering if the authors can refine and expand upon this. I find it difficult to disentangle prevalence encoding and impacts of reward in the way the data and interpretation are presented in some areas of the text. While neural encoding may not reflect trial-by-trial reward receipt, clearly the rat's decision to repeat a given sequence or initiate a new sequence is impacted by reinforcement parameters and reward expectation. Thus being very exact in the interpretation would be helpful.

    1. Reviewer #1 (Public Review):

      Summary:

      Fox, Dan, and Loewenstein investigated how people explored six maze-like environments. They show that roughly one-third of their participants make choices now that increase the potential for future information gain and also temporally discount potential information gain based on how far in the future potential gains might be. The authors argue that rather than valuing exploration in its own right, participant behavior is most consistent with using exploration as a way to reduce uncertainty. They then propose a reinforcement learning (RL) model in which agents estimate an "exploration value" (the expected cumulative information gained by taking a given action in a given state) using standard RL techniques for estimating value (expected cumulative reward). They find that this model exhibits several qualitative similarities with human behavior and that it best captures the temporal dynamics of human exploration when propagating information through the entire history of a behavioral episode (as opposed to merely propagating it in a single step as some of the simplest RL models do).

      While the core insight and basic method of the paper are compelling, the way in which both the behavioral experiment and computational modeling were conducted raise concerns that mean that, in their present form, the results do not fully justify the conclusions. After resolving these issues, the work would demonstrate how human exploration is sensitive to long-range dependencies in information gain, as well as valuable insights about how best to characterize this behavior computationally. I am not particularly well-versed in the literature on exploration so cannot comment on novelty here.

      Strengths:<br /> The entire paper is logically well-motivated. It builds on a valuable basic insight, namely that while bandit tasks are an ideally minimal platform for testing certain questions about decision-making and exploration, richer paradigms are needed to capture the long-range informational dependencies distinguishing between various approaches to exploration.

      Even so, the maze navigation paradigm explored here remains simple. Participants navigate a maze with two main branches which are identical save for minimal, theoretically motivated differences. Moreover, the tested differences are designed to clearly and explicitly test well-identified questions. The task, and really the entire paper, is clearly organized, and each component is logically connected to a larger argument.

      The proposed model is also simple, clearly presented, and a clever way of applying ideas typically used to reason about reward-motivated behavior to reason here about information-motivated behavior.

      One other strength of this work is that it combines behavioral experiments with computational modeling. This approach pairs a detailed and objectively specified theory (i.e. the model) with novel data specifically designed to test that theory and thus in principle presents a particularly strong test of the authors' hypotheses.

      Weaknesses:<br /> Despite many strengths in the underlying logic of the paper, the presented evidence does not provide compelling support for the conclusions. In particular:

      - The main claims are based on the behavior of 452 participants classed as good explorers, out of 1,052 participants included in the analyses and 1,336 participants who completed the study. That is, the authors' broad claims about human exploration are based on a third of their total sample; the other two-thirds displayed very different behavior, including 20% who performed at or below chance levels. That is, while a significant sub-population may demonstrate the claimed abilities, it is far from clear that they are universal.

      - While the experimental manipulations are elegant, the behavioral study seems underpowered. In each of the primary manipulations, key theoretical predictions are not statistically validated. For example, in Experiment 1, the preference for the right door increases from the 4:3 condition to the 5:2 condition, but not when moving from the 5:2 condition to the 6:1 condition, as predicted (Figure 1c). Similar results can be seen for other analyses in Figures 3b and 4b. Relatedly, the experiments comprised just 20 episodes, and it is unclear whether that was sufficiently long for participants to demonstrate asymptotic behavior (e.g. Figure 5b). Either more participants or greater differences between conditions (e.g. testing 9:8, 12:5, and 15:2 conditions in a revised Experiment 1), as well as running a greater number of total episodes, would be needed to resolve this concern.

      - The model is presented after the behavioral results, giving the impression that it was perhaps constructed to fit the data. No attempt is made to fit the model to a subset of the data and then validate the rest or give any clear indication as to how the model parameters were set. Moreover, as noted, even where the model is successful, it only explains the behavior of a minority of the total participants. No modeling work is done to explain the behavior of the other two-thirds of the participants.

      - The authors helpfully discuss several meaningful alternative models of exploration, such as visit-counting and incorporating an objective function sensitive to information gain. They do not, however, compare their model against these or any other meaningful baselines. Moreover, the comparison between model and human participants is qualitative rather than quantitative. These issues could be resolved by introducing a more rigorous analysis quantitatively comparing a variety of theoretically relevant models as quantitative explanations of the human data.

    1. Reviewer #1 (Public Review):

      In this study, the authors utilise different chemical inhibitors and celular markers to examine the roles of macropinocytosis in WNT signalling activation in development (Xenopus), cell culture (3T3 cells) and cancer (CRC sections). Furthermore, they investigate the effect of the inflammation inducer Phorbol-12-myristate-13-acetate (PMA) in WNT signalling activation through macropinocytosis. The authors show 1) that PMA induces macropinocytosis-dependent WNT signalling activation, and 2) that CRC development correlates with increased levels and co-localisation of macropinocytosis components and b-catenin.

      I found the analyses and conclusions compelling. Additional epistatic analyses could be done in the future to further disentangle the roles of macropinocytosis during WNT signalling activation, especially upon oncogenic alterations (e.g. in APC). The studies on CRC samples open interesting questions for specialists in tumour progression.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Ngoune et al. present compelling evidence that Slender cells are challenged to infect tsetse flies. They explore the experimental context of a recent important paper in the field, Schuster et al., that presents evidence suggesting the proliferative Slender bloodstream T.brucei can infect juvenile tsetse flies. Schuster et al. were disruptive to the widely accepted paradigm that the Stumpy bloodstream-form is solely responsible for tsetse infection and T.brucei transmission potential.

      Evidence presented here shows that in all cases, Stumpy form parasites are exponentially more capable of infecting tsetse flies. They further show that Slender cells do not infect mature flies.

      However, they raise questions of immature tsetse immunological potential and field transmission potential that their experiments do not address. Specifically, they do not show that teneral tsetse flies are immunocompromised, that tsetse flies must be immunocompromised for Slender infection nor that younger teneral tsetse infection is not pertinent to field transmission.

      Strengths:<br /> Experimental Design is precise and elegant, outcomes are convincing. Discussion is compelling and important to the field. This is a timely piece that adds important data to a critical discussion of host: parasite interactions, of relevance to all parasite transmission.

      Weaknesses:<br /> As above, the authors dispute the biological relevance of teneral tsetse infection in the wild, without offering evidence to the contrary. Statements need to be softened for claims regarding immunological competence or relevance to field transmission.

    1. Reviewer #1 (Public Review):

      DeKraker et al. propose a new method for hippocampal registration using a novel surface-based approach that preserves the topology of the curvature of the hippocampus and boundaries of hippocampal subfields. The surface-based registration method proved to be more precise and resulted in better alignment compared to traditional volumetric-based registration. Moreover, the authors demonstrated that this method can be performed across image modalities by testing the method with seven different histological samples. This work has the potential to be a powerful new registration technique that can enable precise hippocampal registration and alignment across subjects, datasets, and image modalities.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Van der Heijden et al perform an ambitious analysis of single-unit activity in the interposed nuclei of multiple mouse models of cerebellar dysfunction. Based on these recordings, they develop a classifier to predict the behavioral phenotype (ataxic, dystonic, or tremor) of each model, suggesting that highly regular spiking is associated with ataxia, irregular spiking is associated with dystonia and rhythmic spiking is associated with tremor. After developing this classifier, they show that activating Purkinje neurons in different patterns that evoke interposed nuclear activity similar to their "ataxic", "dystonic", and "tremor" firing patterns induce similar behaviors in healthy mice. These results show convincingly that specific patterns of cerebellar output are sufficient to cause specific movement abnormalities. The extent to which cerebellar nuclear firing patterns are solely responsible for phenotypes in human disease remains to be established, however.

      Strengths:<br /> Major strengths are the recordings across multiple phenotypic models including genetic and pharmacologic manipulations, and the robust phenotypes elicited by Purkinje neuron stimulation.

      Weaknesses:<br /> The number of units recorded was small for each model (on the order of 20), limiting the conclusions that can be drawn from the recording/classifier experiments.

    1. Joint Public Review:

      Summary<br /> This is a very meticulous and precise anatomical description of the external sensory organs (sensillia) in Drosophila larvae. Extending on their previous study (Rist and Thum 2017) that analyzed the anatomy of the terminal organ, a major external taste organ of fruit fly larva, the authors examined the anatomy of the remaining head sensory organs - the dorsal organ, the ventral organ, and the labial organ-also described the sensory organs of the thoracic and abdominal segments. Improved serial electron microscopy and digital modeling are used to the fullest to provide a definitive and clear picture of the sensory organs, the sensillia, and adjacent ganglia, providing an integral and accurate map, which is dearly needed in the field. 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 and construct a complete structural and neuronal map of the external larval sensilla.

      Strengths<br /> 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 use it as a reference paper. In addition to the classification and nomenclature of the different types of sensilla throughout the larval body, the wealth of data presented here will be valuable to the scientific community. It will allow for investigating sensory processing in depth. Serial electron microscopy and digital modeling are used to the fullest to provide a comprehensive, definitive, and clear picture of the sensory organs. The discussion places the anatomical data into a functional and developmental frame. The study offers fundamental anatomical insights, which will be helpful for future functional studies and to understand the sensory strategies of Drosophila larvae in response to the external environment. By analyzing different larval stages (L1 and L3), this work offers some insights into the developmental aspects of the larval sense organs and their corresponding sensory cells.

      Weaknesses<br /> There are no apparent weaknesses, although it is not a complete novel anatomical study. It revisits many data that already existed, adding new information. However, the repetitiveness of some data and prior studies may be avoided for easy readability.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors aimed to infer the trajectories of long range and local neuronal synchrony across the Alzheimer's disease continuum, relative to neurodegeneration and cognitive decline. The trajectories are inferred using event-based models, which infer a set of data-driven disease stages from a given dataset. The authors develop an adapted event-based modelling approach, in which they characterise each stage as a particular biomarker increasing by a particular z-score deviation from controls. Fitting infers the optimal set of z-scores to use for each biomarker and the order in which each biomarker reaches each z-score. The authors apply this approach to data from 148 individuals (70 cognitively unimpaired older adults and 78 individual with mild cognitive impairment or Alzheimer's disease), identifying trajectories in which long-range (amplitude-envolope correlation) and local (regional spectral power) neuronal synchrony in the alpha and beta bands becomes abnormal prior to neurodegeneration (measured as the volume of the parahippocampal gyrus) and cognitive decline (measured using the mini-mental state examination).

      Strengths:<br /> - The main strength is that the authors assess two models. In the first they derive a staging system based only on the volume of the parahippocampal gyrus and mini-mental state examination score. They then investigate how neuronal synchrony metrics change compared to this staging system. In the second they derive a staging system that also includes an average (combined long-range and local) neuronal synchrony metric and investigate how long-range and local synchrony metrics change relative to this staging system. This is a strength as the first model provides confidence that there is not overfitting to the neuronal synchrony data, and the second provides more detailed insights into the dynamics of the early neuronal synchrony changes.<br /> - Another strength is that the authors automatically infer the optimal z-scores to choose, rather than having to pre-select them manually, as in previous approaches.

      Weaknesses:<br /> - The dataset is small and no external validation is performed.<br /> - A high proportion of the data is from controls (nearly 50%) with no biomarker evidence of Alzheimer's disease, and so the changes may be driven by aging or other non-Alzheimer's effects.<br /> - Inferring the optimal z-scores is a strength, however as different sets of z-scores are allowed per biomarker, there is a concern that the changes reflected are mainly driven by the choice of z-score, rather than the markers themselves (e.g. if lower z-scores are selected for one marker than another, then changes in that marker will appear to be detected earlier, even if both markers change at the same time).<br /> - In equation 2 it is unclear why the gaussian is measured based on a sum over I. The more obvious choice would be to use a multivariate gaussian with no covariance, which would mean taking the product rather than the sum over I.<br /> - In the original event-based model, k is a hidden variable. Presumably that is also the case here, however the notation k=stage(j) makes it seem like each subject is assigned a stage during the sequence optimisation.<br /> - Typically for event-based modelling, positional variance diagrams are created from the markov chain monte carlo samples of the event sequence, enabling visualisation of the uncertainty in the sequence, but these are not included in the study.<br /> - Many of the figures in the manuscript (e.g. Figure 1E/G, Figure 2A/B, Figure 3A/B/E/F/I/J, Figure 4 A/B/E/F/I/J) are based on averages in both the x and the y axis. In the x dimension, individuals have a weighted contribution to the value on the y axis, depending on their stage probability. In the y dimension, the values are averages across those individuals, and the error bars represent the standard error rather than the standard deviation. Whilst the trajectories themselves are interesting, they may not be discriminative at the individual level and may be more heterogeneous than it appears.<br /> - The bootstrapped statistical analyses comparing metrics between the stages do not consider the variability in the sequence.

    1. Reviewer #1 (Public Review):

      In this manuscript, Lee et al. compared encoding of odor identity and value by calcium signaling from neurons in the ventral pallidum (VP) in comparison to D1 and D2 neurons in the olfactory tubercle (OT).

      Strengths: They utilize a strong comparative approach, which allows the comparison of signals in two directly connected regions. First, they demonstrate that both D1 and D2 OT neurons project strongly to the VP, but not the VTA or other examined regions, in contrast to accumbal D1 neurons which project strongly to the VTA as well as the VP. They examine single unit calcium activity in a robust olfactory cue conditioning paradigm that allows them to differentiate encoding of olfactory identity versus value, by incorporating two different sucrose, neutral and air puff cues with different chemical characteristics. They then use multiple analytical approaches to demonstrate strong, low-dimensional encoding of cue value in the VP, and more robust, high-dimensional encoding of odor identity by both D1 and D2 OT neurons, though D1 OT neurons are still somewhat modulated by reward contingency/value. Finally, they utilize a modified conditioning paradigm that dissociates reward probability and lick vigor to demonstrate that VP encoding of cue value is not dependent on encoding of lick vigor during sucrose cues, and that separable populations of VP neurons encode cue value/sucrose probability and lick vigor.

      Weaknesses: The conclusions of the data are mostly well supported by the analyses, but the statistical analysis is somewhat limited and needs to be clarified and extended.

      1) The manuscript includes limited direct statistical comparison of the neural populations, and many of the comparisons between the subregions are descriptive, including descriptions of the percentage of neurons having specific response types, or differences in effect sizes or differing "levels" of significance. An additional direct comparison of data from each subpopulation would help to confirm whether the differences reported are statistically meaningful.

      2) When hypothesis tests are conducted between the neural populations, it is not clear whether the authors have accounted for the random effect of the subject, or whether individual units were treated as fully independent. For instance, pairwise differences are reported in Figures 4I, 5G/I/L, and others, but the statistical methods are unclear. Assessment of the statistics is further limited by the lack of reporting of degrees of freedom.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Ever-improving techniques allow the detailed capture of brain morphology and function to the point where individual brain anatomy becomes an important factor. This study investigated detailed sulcal morphology in the parieto-occipital junction. Using cutting-edge methods, it provides important insights into local anatomy, individual variability, and local brain function. The presented work advances the field and will stimulate future research into this important area.

      Strengths:<br /> Detailed, very thorough methodology. Multiple raters mapped detailed sulci in a large cohort. The identified sulcal features and their functional and behavioural relevance are then studied using various complementary methods. The results provide compelling evidence for the importance of the described sulcal features and their proposed relationship to cortical brain function.

      Weaknesses:<br /> A detailed description/depiction of the various sulcal patterns is missing. A possible relationship between sucal morphology and individual demographics might provide more insight into anatomical variability. The unique dataset offers to opportunity to provide insights into laterality effects that should be explored.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors provide very compelling evidence that the lateral septum (LS) engages in theta cycle skipping.

      Strengths:<br /> The data and analysis are highly compelling regarding the existence of cycle skipping.

      Weaknesses:<br /> The manuscript falls short on describing the behavioral or physiological importance of the witnessed theta cycle skipping, and there is a lack of attention to detail with some of the findings and figures:

      More/any description is needed in the article text to explain the switching task and the behavioral paradigm generally. This should be moved from only being in methods as it is essential for understanding the study.

      An explanation is needed as to how a cell can be theta skipping if it is not theta rhythmic.

      The most interesting result, in my opinion, is the last paragraph of the entire results section, where there is more switching in the alternation task, but the reader is kind of left hanging as to how this relates to other findings. How does this relate to differences in decoding of relative arms (the correct or incorrect arm) during those theta cycles or to the animal's actual choice? Similarly, how does it relate to the animal's actual choice? Is this phenomenon actually behaviorally or physiologically meaningful at all? Does it contribute at all to any sort of planning or decision-making?

      The authors state that there is more cycle skipping in the alternation task than in the switching task, and that this switching occurs in the lead-up to the choice point. Then they say there is a higher peak at ~125 in the alternation task, which is consistent. However, in the final sentence, the authors note that "This result indicates that the representations of the goal arms alternate more strongly ahead of the choice point when animals performed a task in which either goal arm potentially leads to reward." Doesn't either arm potentially lead to a reward (but different amounts) in the switching task, not the alternation task? Yet switching is stronger in the alternation task, which is not constant and contradicts this last sentence.

      Additionally, regarding the same sentence - "representations of the goal arms alternate more strongly ahead of the choice point when the animals performed a task in which either goal arm potentially leads to reward." - is this actually what is going on? Is there any reason at all to think this has anything to do with reward versus just a navigational choice?

      Similarly, the authors mention several times that the LS links the HPC to 'reward' regions in the brain, and it has been found that the LS represents rewarded locations comparatively more than the hippocampus. How does this relate to their finding?

    1. Reviewer #1 (Public Review):

      Summary:

      This manuscript describes the development of an oral THC consumption model in mice where THC is added to a chocolate flavored gelatin. The authors compared the effects of THC consumed in this highly palatable gelatin (termed E-gel) to THC dissolved in a less palatable gelatin (CTR-gel), and to i.p. injections of multiple doses of THC, on the classic triad of CB1R dependent behaviors (hypolocomotion, antinociception, and body temperature).

      The authors found that they could achieve consumption of higher concentrations of THC in the E-gel than the CTR-gel, and that this led to larger total dose exposure and decreases in locomotor activity, antinociception, and body temperature reductions similar to 3-4 mg/kg THC when tested after 2 hour consumption and roughly 10 mg/kg if tested immediately after 1 hour consumption. The majority of THC E-gel consumption was found to occur in the first hour on the first exposure day. THC E-gel consumption was lower than VEH E-gel consumption and this persisted on a subsequent consumption day, suggesting that the animals may form a taste aversion and that THC at the dose consumed likely has aversive properties, consistent with the literature on i.p. dosing. The authors also report the pharmacokinetics in brain and plasma of THC and metabolites after 1 or 2 hour consumption, finding high levels of THC in the brain that begins to dissipate at 2.5 hours is gone 24 hours later. Finally, the authors tested THC effects on the acoustic startle response and found an inverted dose response that was more pronounced in males than females after i.p. dosing and a greater startle response in males after E-gel dosing.

      Overall, the authors find that voluntary oral consumption of THC can achieve levels of intake that are consistent with the present and prior reported literature on i.p. dosing.

      Strengths:

      The strengths of the article include a direct comparison of voluntary oral THC consumption to noncontingent i.p. administration, the use of multiple THC doses and oral THC formulations, the inclusion of multiple assays of cannabinoid agonist effects, and the inclusion of males and females. Additional strengths include monitoring intake over 10 minute intervals and validating that effects are CB1R dependent via antagonist studies.

      Weaknesses:

      1. The abstract does not discuss the reduction of E-gel consumption that occurs after multiple days of exposure to the THC formulation, but rather implies that a new model for chronic oral self-administration has been developed. Given that only two days of consumption was assessed, it is not clear if the model will be useful to determine THC effects beyond the acute measures presented here. The abstract should clarify that there was evidence of reduced consumption/aversive effects with repeated exposures.<br /> 2. In the results section, the authors sometimes describe effects in terms of the concentration of gel as opposed to the dose consumed in mg/kg, which can make interpretation difficult. For example, the text describing Figure 1i states that significant effects on body temperature were achieved at 4 mg CTR-gel and 5 mg THC-gel, but were essentially equivalent doses consumed? It would be helpful to describe what average dose of THC produced effects given that consumption varied within each group of mice assigned to a particular concentration.<br /> 3. The description of the PK data in Figure 3 did not specify if sex differences were examined. Prior studies have found that males and females can exhibit stark differences in brain and plasma levels of THC and metabolites, even when behavioral effects are similar. However, this does depend on species, route, timing of tissue collection. It would be helpful to describe the PK profile of males and females separately.<br /> 4. In Figure 5, it is unclear how the predicted i.p. THC dose could be 30 mg/kg when 30 mg/kg was not tested by the i.p. route according to the figure, and if it had been it would have likely been almost zero acoustic startle, not the increased startle that was observed in the 2 hr gel group. It seems more likely that it would be equivalent to 3 mg/kg i.p. Could there be an error in the modeling, or was it based on the model used for the triad effects? This should be clarified.

    1. Reviewer #1 (Public Review):

      Prior research demonstrated that vocal learning is sexually dimorphic in zebra finches; female song nuclei atrophy and fail to develop, but can be rescued with exogenous 17-𝛃-estradiol (E2) treatment. In previous research, the authors treated both male and female birds with exogenous E2. They laser-captured dissected tissue samples from the E2-treated individuals as well as untreated controls. They then extracted RNA and used RNA-seq to characterize the transcriptomes within and adjacent to four major song nuclei (HVC, LMAN, RA, Area X) in these birds. In this study, Davenport et al. remapped this massive amount of transcriptome data (n=3 birds per sex/treatment group) to fully resolve the genomic location of differentially expressed genes, which they assigned to several modules based on co-expression. Adequate read mapping to all chromosomes was previously impossible with zebra finch genome assemblies lacking W chromosome data. Using the high-quality zebra finch genome assembly with Z and W chromosomes (bTaeGut2.pat.W.v2), the authors were able to demonstrate the enrichment of certain modules on certain chromosomes; most interestingly, Z chromosome gene expression was increased in E2-treated females. This research greatly improves our understanding of the ontology and location of genes involved in song development in E2-treated females, providing insight into the development of vocal learning in the zebra finch.

      The authors' main conclusions on the importance of certain gene modules in the vocal learning process are well warranted by their excellent data and thorough analyses, but should not be too broadly interpreted as necessarily applying to the gene expression involved in vocal learning in other species. While the data here further supports convergent evolution in vocal learning genes in humans and zebra finches, vocal learning is unusually sexually dimorphic in zebra finches compared to most other vocal learners.

      The authors note the possibility of female haploinsufficiency of Z-linked genes such as the growth hormone receptor (GHR) and also imply there are potential effects of the fission of the ancestral chromosome into passerine chromosomes 1 and 1A impacting the typical development of male zebra finch song and the lack thereof in females. These thoughts are intriguing and should prompt further transcriptomic research in avian species with the same genomic features (ZW females, split 1 and 1A chromosomes) where females also learn song, i.e. female-singing passerine species. Currently, it is impossible to say if female-singing species are, as is likely with the E2-treated zebra finch females, using estrogen signaling pathways to regulate an increase in dosage of these genes. Alternately, these female-singing birds may be using different gene modules, which is also worthy of investigation. This research excels at elucidating the genomic underpinnings of vocal learning in a model organism; further research will demonstrate how broadly applicable these authors' findings are across other species.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This paper presents an innovative decoding approach for brain-computer interfaces (BCIs), introducing a new method named MINT. The authors develop a trajectory-centric approach to decode behaviors across several different datasets, including eight empirical datasets from the Neural Latents Benchmark. Overall, the paper is well written and their method shows impressive performance compared to more traditional decoding approaches that use a simpler approach. While there are some concerns (see below), the paper's strengths, particularly its emphasis on a trajectory-centric approach and the simplicity of MINT, provide a compelling contribution to the field.

      Strengths:<br /> The adoption of a trajectory-centric approach that utilizes statistical constraints presents a substantial shift in methodology, potentially revolutionizing the way BCIs interpret and predict neural behaviour. This is one of the strongest aspects of the paper.

      The thorough evaluation of the method across various datasets serves as an assurance that the superior performance of MINT is not a result of overfitting. The comparative simplicity of the method in contrast to many neural network approaches is refreshing and should facilitate broader applicability.

      Weaknesses:<br /> Scope: Despite the impressive performance of MINT across multiple datasets, it seems predominantly applicable to M1/S1 data. Only one of the eight empirical datasets comes from an area outside the motor/somatosensory cortex. It would be beneficial if the authors could expand further on how the method might perform with other brain regions that do not exhibit low tangling or do not have a clear trial structure (e.g. decoding of position or head direction from hippocampus)

      When comparing methods, the neural trajectories of MINT are based on averaged trials, while the comparison methods are trained on single trials. An additional analysis might help in disentangling the effect of the trial averaging. For this, the authors could average the input across trials for all decoders, establishing a baseline for averaged trials. Note that inference should still be done on single trials. Performance can then be visualized across different values of N, which denotes the number of averaged trials used for training.

    1. Reviewer #1 (Public Review):

      This EEG study probes the prediction of a mechanistic account of P300 generation through the presence of underlying (alpha) oscillations with a non-zero mean. In this model, the P300 can be explained by a baseline shift mechanism. That is, the non-zero mean alpha oscillations induce asymmetries in the trial-averaged amplitudes of the EEG signal, and the associated baseline shifts can lead to apparent positive (or negative) deflections as alpha becomes desynchronized at around P300 latency. The present paper examines the predictions of this model in a substantial data set (using the typical P300-generating oddball paradigm and careful analyses). The results show that all predictions are fulfilled: the two electrophysiological events (P300, alpha desynchronization) share a common time-course, anatomical sources (from inverse solutions), and covariations with behaviour; plus relate (negatively) in amplitude, while the direction of this relationship is determined by the non-zero-mean deviation of alpha oscillations pre-stimulus (baseline shift index, BSI). This is indictive of a link of the P300 with underlying alpha oscillations through a baseline shift account, and hence that the P300 can be explained, at least in parts, by non-zero mean brain oscillations as they undergo post-stimulus changes.

    1. Reviewer #1 (Public Review):

      This paper aims to study the effects of choice history on action-selective beta band signals in human MEG data during a sensory evidence accumulation task. It does so by placing participants in three different stochastic environments, where the outcome of each trial is either random, likely to repeat, or likely to alternate across trials. The authors provide good behavioural evidence that subjects have learnt these statistics (even though they are not explicitly told about them) and that they influence their decision-making, especially on the most difficult trials (low motion coherence). They then show that the primary effect of choice history on lateralised beta-band activity, which is well-established to be linked to evidence accumulation processes in decision-making, is on the slope of evidence accumulation rather than on the baseline level of lateralised beta.

      The strengths of the paper are that it is: (i) very well analysed, with compelling evidence in support of its primary conclusions; (ii) a well-designed study, allowing the authors to investigate the effects of choice history in different stochastic environments.

      There are no major weaknesses to the study. On the other hand, investigating the effects of choice/outcome history on evidence integration is a fairly well-established problem in the field. As such, I think that this provides a valuable contribution to the field, rather than being a landmark study that will transform our understanding of the problem.

      The authors have achieved their primary aims and I think that the results support their main conclusions. One outstanding question in the analysis is the extent to which the source-reconstructed patches in Figure 2 are truly independent of one another (as often there is 'leakage' from one source location into another, and many of the different ROIs have quite similar overall patterns of synchronisation/desynchronisation.). A possible way to investigate this further would be to explore the correlation structure of the LCMV beamformer weights for these different patches, to ask how similar/dissimilar the spatial filters are for the different reconstructed patches.

      The revised paper now states explicitly how source-reconstructed patches are indeed affected by leakage, but also why the focus of the authors on differences (rather than similarities) between patches leaves their findings and conclusions essentially unaffected by this intrinsic limitation of cortical source reconstruction from surface MEG data.

    1. Reviewer #1 (Public Review):

      The Eph receptor tyrosine kinase family plays a critical function in multiple physiological and pathophysiological processes. Hence, understating the regulation of these receptors is highly important question. Through extensive experiments in cell lines and cultured neurons Chang et.al show that the signaling hub protein, MYCBP2 positively regulates the overall stability of a specific member of the family, EPHB2, and by that the cellular response to ephrinBs.<br /> Overall, this work sheds light on the divergent in the regulatory mechanisms of the Eph receptors family. Although the physiological importance of this new regularly mechanism in mammals awaits to be discovered, the authors provide genetic evidence using C.elegans that it is evolutionarily conserved.

    1. Reviewer #1 (Public Review):

      Colin et al demonstrated that condensin is a key factor for the disjunction of sister-telomeres during mitosis and proposed that it is due to that condensin restrains the telomere association of cohesin. The authors first showed that condensin binds telomeres in mitosis evidenced by ChIP-qPCR and calibrated ChIP-seq. They further demonstrated that compromising condensin's activity leads to a failure in the disjunction of telomeres, with convincing cytological and HI-seq evidence. Two telomeric proteins Taz1 and Mit1 were identified to specifically regulate the telomere association of cohesin. Deletion of these genes decreased/increased condensin's telomere association and exacerbated/remedied the defected telomere disjunction in a condensin mutant, echoing the role of condensin in telomere disjunction. They proposed that the underlying mechanism is that condensin inhibits cohesin's accumulation at telomeres. However, the evidence for this claim might need to be further strengthened. Nevertheless, this study uncovered a novel role of condensin in the separation of telomeres of sister chromosomes and open a question of how condensin regulates the structure of chromosomal ends.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this paper the authors present genome-wide association analyses of 11 different cancers including time-to-event analyses. The authors use two recently published Bayesian methods, one of which is constructed to handle time-to-event data. The authors demonstrate that polygenic risk scores trained on these models give nominally better predictions than standard polygenic risk scores. Further they show that performing 11 GWASs in UKB while adjusting for the polygenic effects estimated by their improved predictor, they find seven novel loci are implicated by one or both of these methods of which the authors find that three replicate in Estonian Biobank.

      Strengths:<br /> A clear strength is that the authors evaluate the performance of the model in a completely different dataset (Estonian Biobank) than the one it is trained in.

      Weaknesses:<br /> The 11 phenotypes that the authors chose have the challenge that they are rare, particularly in healthy biobank participants, which means that (i) the benefit of modeling it as a time-to-event analysis is expected to be smaller and (ii) that models have to be stable under imbalanced case/control fractions. In GWAS analyses authors handle this second problem by using a recently published association test, which is robust to imbalanced data, which likely means that they avoid inflated test statistics, but also that they do not leverage the actual time-to-event information to its full potential.

      The authors chose not to use the recently published methods BayesRR-RC and BayesW, but instead they run these models and then add an extra step where they run a logistic regression with an offset term set to the LOCO genomic values as estimated by GRMR-BayesW and GRMR-BayesRR-RC respectively. They write that this was because of the imbalanced case/control proportion, but not how the problem was detected. If the authors have insight about when the standard GRMR-BayesW and GRMR-BayesRR-RC become unreliable, I think it would be helpful to share in this paper. Further, if the associations implicated by standard GRMR-BayesW and GRMR-BayesRR-RC are not reliable, I think we need some justification that the variance components reported in Figure 1 are still reliable.

      The authors chose to compare the two new GWAS methods, GMRM-BayesW-adjusted and GMRM-BayesRR-RC-adjusted, to REGENIE, so an obvious first question in my opinion is if GMRM-BayesW-adjusted and GMRM-BayesRR-RC-adjusted find more signal than REGENIE.<br /> a. We see that 7 loci where found by GMRM-BayesW but not by REGENIE, but how many were found by REGENIE but not by GMRM-BayesW?<br /> b. Figure S5 as I understand it is showing that the mean -log(p-value) is lower in GMRM-BayesW than REGENIE for variants that have a p-value in GMRM-BayesW that is lower than 5e-8. I don't think this is a valid way to check if GMRM-BayesW has more power. I have a feeling that there could be a winner's curse-like phenomenon here. I think a more principled comparison could be provided.

      The title of the paper ("Novel discoveries and enhanced genomic prediction from modelling genetic risk of cancer age-at-onset") seems to imply that the age of onset informed model (GMRM-BayesW) does better. But I think the foundation for that statement could be strengthened.<br /> Figure S6 shows that 261 previously reported loci were replicated by GMRM-BayesW-adjusted whereas 256 were replicated by GMRM-BayesRR-RC. How were previously reported loci defined? did they include UKB data? and how many where there in total?<br /> In the PRS analyses presented in Figure 3a GMRM-BayesW does better than GMRM-BayesRR-RC in 8/11 phenotypes, which does not itself appear significant to me. And with overlapping confidence intervals the significance of the improvement is hard to see.

      In Table 1 it says that rs35763415, rs117972357 and rs7902587 replicated in the Estonian Biobank but Figure 3b it says that rs35763415, rs117972357 and rs1015362 replicated in the Estonian Biobank. What is the difference between these two analyses? In the methods it says that you checked your findings for replication in FinnGen, but I don't see any results from FinnGen anywhere?

    1. Reviewer #1 (Public Review):

      Summary<br /> The authors investigated the antigenic diversity of recent (2009- 2017) A/H3N2 influenza neuraminidases (NAs), the second major antigenic protein after haemagglutinin. They used 27 viruses and 43 ferret sera and performed NA inhibition. This work was supported by a subset of mouse sera. Clustering analysis determined 4 antigenic clusters, mostly in concordance with the genetic groupings. Association analysis was used to estimate important amino acid positions, which were shown to be more likely close to the catalytic site. Antigenic distances were calculated and a random forest model was used to determine potential important sites.

      This has the potential to be a very interesting piece of work. At present, there are inconsistencies in the methods, results and presentation that limit its impact. In particular, there are weaknesses in some of the computational work.

      Strengths<br /> 1. The data cover recent NA evolution and a substantial number (43) of ferret (and mouse) sera were generated and titrated against 27 viruses. This is laborious experimental work and is the largest publicly available neuraminidase inhibition dataset that I am aware of. As such, it will prove a useful resource for the influenza community.

      2. A variety of computational methods were used to analyse the data, which give a rounded picture of the antigenic and genetic relationships and link between sequence, structure and phenotype.

      Weaknesses<br /> 1. Inconsistency in experimental methods<br /> Two ferret sera were boosted with H1N2, while recombinant NA protein for the others. This, and the underlying reason, are clearly explained in the manuscript. The authors note that boosting with live virus did not increase titres. Nevertheless, these results are included in the analysis when it would be better to exclude them (Figure 2 shows much lower titres to their own group than other sera).

      2. Inconsistency in experimental results<br /> Clustering of the NA inhibition results identifies three viruses which do not cluster with their phylogenetic group. Again this is clearly pointed out in the paper. Further investigation of this inconsistency is required to determine whether this has a genetic basis or is an experimental issue. It is difficult to trust the remaining data while this issue is unresolved.

      3. Inconsistency in group labelling<br /> A/Hatay/4990/2016 & A/New Caledonia/23/2016 are in phylogenetic group 1 in Figure 2 and phylogenetic group 1 in Figure 5 - figure supplement 1 panel a.<br /> A/Kansas/14/2017 is selected as a representative of antigenic group 2, when in Figure 2 it is labelled as AC1 (although Figure 2 - supplement 4 which the text is referring to shows data for A/Singapore/Infimh-16-0019/2016 as the representative of AC2). A/Kansas/14/2017 is coloured and labelled as AC2 in Figure 2 - supplement 5.<br /> The colouring is changed for Figure 3a at the bottom. A/Heilongjiang-Xiangyang/1134/2011 is coloured the same as AC4 viruses when it is AC1 in Figure 2.<br /> This lack of consistency makes the figures misleading.

      4. Data not presented, without explanation<br /> The paper states that 44 sera and 27 H6N2 viruses were used (line 158). However, the results for the Kansas/14/2017 sera do not appear to be presented in any of the figures (e.g. Figure 2 phylogenetic tree, Figure 5 - figure supplement 1). It is not obvious why these data were not presented. The exclusion of this serum could affect the results as often the homologous titre is the highest and several heatmaps show the fold down from the highest titre.

      5. The cMDS plot does not have sufficient quality assurance<br /> A cMDS plot is shown in Figure 5 - figure supplement 1, generated using classical MDS. The following support for the appropriateness of this visualisation is not given.<br /> a. Goodness of fit of the cMDS projection, including per point and per titre.<br /> b. Testing of the appropriate number of dimensions (the two sera from phylogenetic group 3 are clustered with phylogenetic group 2; additional dimensions might separate these groups).<br /> c. A measure of uncertainty in positioning, e.g. bootstrapping.<br /> d. A sensitivity analysis of the assumption about titres below the level of detection (i.e. that <20 = 10).<br /> Without this information, it is difficult to judge if the projection is reliable.

      6. Choice of antigenic distance measure<br /> The measure of antigenic distance used here is the average difference between titres for two sera. This is dependent on which viruses have been included in the analysis and will be biased by the unbalanced number of viruses in the different clusters (12, 8, 2, 5).

      7. Association analysis does not account for correlations<br /> For each H6N2 virus and position, significance was calculated by comparing the titres between sera that did or did not have a change at that position. This does not take into account the correlations between positions. For haemagglutinin, it can be impossible to determine the true antigenic effects of such correlated substitutions with mutagenesis studies.

      8. Random forest method<br /> 25 features are used to classify 43 sera, which seems high (p/3 is typical for classification). By only considering mismatches, rather than the specific amino acid changes, some signals may be lost (for example, at a given position, one amino acid change might be neutral while another has a large antigenic effect). Features may be highly, or perfectly correlated, which will give them a lower reported importance and skew the results.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In the present study, the authors examined the possibility of using phosphatidyl-inositol kinase 3-kinase alpha (PI3Ka) inhibitors for heterotopic ossification (HO) in fibrodysplasia ossificans progressiva (FOP). Administration of BYL719, a chemical inhibitor of PI3Ka, prevented HO in a mouse model of FOP that expressed a mutated ACVR1 receptor. Genetic ablation of PI3Ka (p110a) also suppressed HO in mice. BYL719 blocked osteochondroprogenitor specification and reduced inflammatory responses, such as pro-inflammatory cytokine expression and migration/proliferation of immune cells. The authors claimed that inhibition of PI3Ka is a safe and effective therapeutic strategy for HO.

      Strengths:<br /> This manuscript reports an interesting finding that BYL719 inhibits HO in a mouse model of FOP.

      Weaknesses:<br /> The molecular mechanisms of BYL719 were still unclear because BYL719 affected multiple events and many types of cells. Additional experimental data would be needed to show more clearly how PI3Ka regulates HO.

    1. Reviewer #1 (Public Review):

      This study compares visuospatial working (VWM) memory performance between patients with MS and healthy controls, assessed using analog report tasks that provide continuous measures of recall error. The aim is to advance on previous studies of VWM in MS that have used binary (correct/incorrect) measures of recall, such as from change detection tasks, that are not sensitive to the resolution with which features can be recalled, and to use mixture modelling to disentangle different contributions to overall performance. The results identify a specific decrease in the precision of VWM recall in MS, although the possibility that visual and/or motor impairments contribute to performance decrements on the memory task cannot be ruled out.

    1. Reviewer #1 (Public Review):

      The manuscript aims to provide mechanistic insight into the activation of PI3Kbeta by its known regulators tyrosine phosphorylated peptides, GTP-loaded Rac1 and G-protein beta-gamma subunits. To achieve this the authors have used supported lipid bilayers, engineered recombinant peptides and proteins (often tagged with fluorophores) and TIRF microscopy to enable bulk (averages of many molecules) and single molecule quantitation. The great strength of this approach is the precision and clarity of mechanistic insight. Although the study does not use "in transfecto" or in vivo models the experiments are performed using "physiologically-based" conditions and provide a powerful insight into core regulatory principles that will be relevant in vivo.

      The results are beautiful, high quality, well controlled and internally consistent (and with other published work that overlaps on some points) and as a result are compelling. The primary conclusion is that the primary regulator of PI3Kbeta are tyrosine phosphorylated peptides (and by inference tyrosine phsophorylated receptors/adaptors) and that the other activators can synergise with that input but have relatively weak impacts on their own.

      Although the methodology is not easily imported, for reasons of both cost and the experience needed to execute them well, the results have broad importance for the field and reverse an impression that had built in large parts of the broader signalling and PI3K communities that all of the inputs to PI3Kbeta were relatively equivalent, however, these conclusions were based on "in cell" or in vivo studies that were very difficult to interpret clearly.

    1. Reviewer #1 (Public Review):

      Warming and precipitation regime change significantly influences both above-ground and below-ground processes across Earth's ecosystems. Soil microbial communities, which underpin the biogeochemical processes that often shape ecosystem function, are no exception to this, and although research shows they can adapt to this warming, population dynamics and ecophysiological responses to these disturbances are not currently known. The Qinghai-Tibet Plateau, the Third Pole of the Earth, is considered among the most sensitive ecosystems to climate change. The manuscript described an integrated, trait-based understanding of these dynamics with the qSIP data. The experimental design and methods appear to be of sufficient quality. The data and analyses are of great value to the larger microbial ecological community and may help advance our understanding of how microbial systems will respond to global change. There are very few studies in which the growth rates of bacterial populations from multifactorial manipulation experiments on the Qinghai-Tibet Plateau have been investigated via qSIP, and the large quantity of data that comprises the study described in this manuscript, will substantially advance our knowledge of bacterial responses to warming and precipitation manipulations.

    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.

      Strengths:<br /> 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. It would be beneficial for the dataset to be made available to other researchers using a public data repository.

      Weaknesses:<br /> There are several primary weaknesses, described below:

      Sex was not considered in the analyses<br /> The number of experimental animals of each sex are not clearly represented in the paper, but are buried in supplemental tables, and the Ns for the RNAseq are unclear. No analyses were done to examine sex differences in male/female DS or WT animals with or without treatment. Body measurements will greatly vary by sex, but this was not taken into consideration during assessments. As such, there is a high amount of variability within each cohort measured for body assessments (tibia, body weight, skeletal development etc.). Supplemental table 14 had the list of each animal, but not collated by sex, genotype or treatment, making it difficult to assess the strength of each measurement.

      Key results are not clearly depicted in the main figures<br /> Rigorous assessment of each figure and the clarity of the figure to convey the results of the analysis needs to be performed. Many of the figures do not clearly represent the findings, with authors heavily relying on supplemental figures to present details to explain results. Figure legends do not adequately describe figures; rather, they are limited to describing how the analysis is performed. For example, LDA plots in Figure 4 do not clearly convey the results of metabolite analysis.<br /> Overall, the amount of data presented here is overwhelming, making it difficult to interpret the findings. Some assessments that do not add to the overall paper need to be removed. Clarifying the text, figures and trimming the supplement to represent the data in a manner that is easily understood will improve the readability of the paper. For example, perhaps measures which are not strongly impacted by genotype could be moved to the supplement, because they are not directly relevant to the question of whether GTE-EGCG reverses the impact of trisomy on the measures.

      Lack of clarity in the behavioral analyses<br /> Behavioral assessments are not clearly written in the methods. For example, for the novel object recognition task, it isn't clear how preference was calculated. Is this simply the percent of time spent with the novel object, or is this a relative measure (novel:familiar ratio)? This matters because if it is simply the percent of time, the relevant measure is to compare each group to 50% (the absence of a preference). The key measures for each test need to be readily distinguished from the control measures.<br /> There are also many dependent behavioral measures. For example, speed and distance are directly related to each other, but these are typically reported as control measures to help interpret the key measure, which is the anxiety-like behavior. Similarly, some behavioral tests were used to represent multiple behavioral dimensions, such as anxiety and arousal. In general, the measurements of arousal seem atypical (speed and distance are typically reported as control measures, not measures of arousal). Similarly, measures of latency during training would not typically be used as a measure of long-term memory but instead reported as a control measure to show learning occurred. LDA analysis requires independence of the measures, as well as normality. It does not appear that all of the measures fed into this analysis would have met these assumptions, but the methods also do not clearly describe which measures were actually used in the LDA.

      Unclear value of RNAseq<br /> RNAseq was performed in cerebellum, a relatively spared region in DS pathology at an early time point in disease. Further, the expression of 125 genes triplicated in DS was shown in a PCA plot to highly overlap with WT, indicating that there are minimal differences in gene expression in these genes. If these genes are not critical for cerebellar function, perhaps this could account for the lack of differences between WT and Ts65Dn mice. If the authors are interested in performing RNAseq, it would have made more sense to perform this in hippocampus (to compare with metabolites) and to perform more stringent bioinformatic analysis than assessment by PCA of a limited subset of genes. Supplementary Table S14, which shows the differentially expressed genes, appears to be missing from the manuscript and cannot be evaluated. Additionally, the methods of the RNAseq are not sufficiently described and lack critical details. For example, what was the normalization performed, and which groups were compared to identify differentially expressed genes? It would also be worthwhile to describe how animals were identified for RNAseq-were those animals representative of their groups across other measures?

    1. Reviewer #1 (Public Review):

      Wheeler et al. have discovered a new RNA circuit that regulates T-cell function. They found that the long non-coding RNA Malat1 sponges miR-15/16, which controls many genes related to T cell activation, survival, and memory. This suggests that Malat1 indirectly regulates T-cell function. They used CRISPR to mutate the miR-15/16 binding site in Malat1 and observed that this disrupted the RNA circuit and impaired cytotoxic T-cell responses. While this study presents a novel molecular mechanism of T-cell regulation by Malat1-miR-15/16, the effects of Malat1 are weaker compared to miR-15/16. This could be due to several reasons, including higher levels of miR-15/16 compared to Malat1 or Malat1 expression being mostly restricted to the nucleus. Although the role of miR15/16 in T-cell activation has been previously published, if the authors can demonstrate that miR15/16 and/or Malat1 affect the clearance of Listeria or LCMV, this will significantly add to the current findings and provide physiological context to the study.

    1. Joint Public Review:

      The authors of this manuscript studied cell-cell interaction between fibroblast and cancer cells as an intermediary model of tumor cell migration/invasion. The work focused on the mesenchymal cadherin-11 (CDH11) which is expressed in the later stages of the epithelial mesenchymal transition (EMT) in tumor cellular models, and whose expression is correlated with tumor progression in vivo. The authors employed 3-D matrix and live cell imaging to visualize the nutrient-dependent co-migration of fibroblast and cancer cells. By siRNA-based suppression of CDH11 expression in tumor cell line and/or fibroblast cells, the authors observed decreased co-movement and attenuated growth of mixed xenograft. Accordingly, the authors conclude that post-EMT cancer cells are capable of migrating/invading through CDH11-mediated cell-cell contact.

      While the data point to the involvement of CDH11 in fibroblast mediated co-invasion, as it stands it is difficult to fully contextualize these observations within the broader context of the molecular mechanisms underlying metastasis, and in particular do not firmly establish a primary role for CDH11 at this time. The reviewers were specifically concerned about indirect effects of CDH11 manipulation on the physiology and cell biology of the tumor cells, and the possibility that several of the results could be consequences of these changes rather than due specifically to CDH11 mediated interactions.

      The reviewers acknowledge the difficulty in fully controlling for these phenomena, and believe this work will be of interest to the large number of researchers investigating the molecular basis for metastasis and specifically of trans cell-type interactions. However until experiments establishing the specific formation and CDH11-mediated interactions in co-invasion are carried out, the author's conclusions about the prominent role of CDH11 should be treated as intriguing, but speculative.

    1. Reviewer #1 (Public Review):

      The overall tone of the rebuttal and lack of responses on several questions was surprising. Clearly, the authors took umbrage at the phrase 'no smoking gun' and provided a lengthy repetition of the fair argument about 'ticking boxes' on the classic list of criteria. They also make repeated historical references that descriptions of neurotransmitters include many papers, typically over decades, e.g. in the case of ACh and its discovery by Sir Henry Dale. While I empathize with the authors' apparent frustration (I quote: '...accept the reality that Rome was not built in a single day and that no transmitter was proven by a one single paper') I am a bit surprised at the complete brushing away of the argument, and in fact the discussion. In the original paper, the notion of a receptor was mentioned only in a single sentence and all three reviewers brought up this rather obvious question. The historical comparisons are difficult: Of course many papers contribute to the identification of a neurotransmitter, but there is a much higher burden of proof in 2023 compared to the work by Otto Loewi and Sir Henry Dale: most, if not all, currently accepted neurotransmitter have a clear biological function at the level of the brain and animal behavior or function - and were in fact first proposed to exist based on a functional biological experiment (e.g. Loewi's heart rate change). This, and the isolation of the chemical that does the job, were clear, unquestionable 'smoking guns' a hundred years ago. Fast forward 2023: Creatine has been carefully studied by the authors to tick many of the boxes for neurotransmitters, but there is no clear role for its function in an animal. The authors show convincing effects upon K+ stimulation and electrophysiological recordings that show altered neuronal activity using the slc6a8 and agat mutants as well as Cr application - but, as has been pointed out by other reviewers, these effects are not a clear-cut demonstration of a chemical transmitter function, however many boxes are ticked. The identification of a role of a neurotransmitter for brain function and animal behavior has reasonably more advanced possibilities in 2023 than a hundred years ago - and e.g. a discussion of approaches for possible receptor candidates should be possible.

      Again, I reviewed this positively and agree that a lot of cumulative data are great to be put out there and allow the discovery to be more broadly discussed and tested. But I have to note, that the authors simply respond with the 'Rome was not built in a single day' statement to my suggestions on at least 'have some lead' how to approach the question of a receptor e.g. through agonists or antagonists (while clearly stating 'I do not think the publication of this manuscript should not be made dependent' on this). Similarly, in response to reviewer 2's concerns about a missing receptor, the authors' only (may I say snarky) response is ' We have deleted this sentence, though what could mediate postsynaptic responses other than receptors?' The bullet point by reviewer 3 ' • No candidate receptor for creatine has been identified postsynaptically.' is the one point by that reviewer that is simply ignored by the authors completely. Finally, I note that my reivew question on the K stimulation issues (e.g. 35 neurons that simply did not respond at all) was: ' Response: To avoid the disadvantage of K stimulation, we also performed optogenetic experiments recently and obtained encouraging preliminary results.' No details, not data - no response really.

      In sum, I find this all a bit strange and the rebuttal surprising - all three reviewers were supportive and have carefully listed points of discussion that I found all valid and thoughtful. In response, the authors selectively responded scientifically to some experimental questions, but otherwise simply rather non-scientifically dismissed questions with 'Rome was not built in a day'-type answers, or less. I my view, the authors have disregarded the review process and the effort of three supportive reviewers, which should be part of the permanent record of this paper.

    1. We first examined whether it is possible to evoke auditory-visual synesthesia in non-synesthetes undergoing short-term sensorydeprivation and parameterized the features that maximize the strength of these experiences (Klüver, 1966).
    1. Reviewer #1 (Public Review):

      Summary:

      HP1 plays a pivotal role in orchestrating chromatin packaging through the creation of biomolecular condensates. The existence of distinct homologs offers an intriguing avenue for investigating the interplay between genetic sequence and condensate formation. In this study, the authors conducted extensive coarse-grained simulations to delve into the phase separation behavior of HP1 paralogs. Additionally, the researchers delved into the captivating possibility of various HP1 paralogs co-localizing within assemblies composed of multiple components. Importantly, the study also delved into the critical role of DNA in finely tuning this complex process.

      Strengths:

      I applaud the authors for their methodical approach in conducting simulations aimed at dissecting the contributions of hinges, CTE, NTE, and folded regions. The comprehensive insights unveiled in Figure 3 compellingly substantiate the significance of these protein components in facilitating the process of phase separation.

      This systematic exploration has yielded several innovative revelations. Notably, the authors uncovered a nuanced interplay between the folded and disordered domains. Although disordered regions have traditionally been linked to driving phase separation through their capacity for forming multivalent interactions, the authors have demonstrated that the contribution of the CD cannot be overlooked, as it significantly impacts the saturation concentration.

      The outcomes of this study serve to elucidate the intricate mechanisms and regulatory aspects governing HP1 LLPS.

      Weaknesses:

      The authors do not provide an assessment of the quantitative precision of their model. To illustrate, HP1a is anticipated to undergo phase separation primarily under low salt concentrations. Does the model effectively capture this sensitivity to salt conditions? Regrettably, the specific salt conditions employed in the simulations are not explicitly stated. While I anticipate that numerous findings in the manuscript remain valid, it could be beneficial to acknowledge potential limitations tied to the simulations. For instance, might the absence of quantitative precision impact certain predictions, such as the CD's influence on phase separation?

    1. Reviewer #1 (Public Review):

      The authors have generated a set of yeast S. cerevisiae strains containing different numbers of chromosomes. 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 Type I or Type II survivors.

      Strengths: The authors examined whether the subtelomeric sequences X and Y' promote HR-mediated telomere maintenance using the strain SY12 carrying three chromosomes.

      Weaknesses:<br /> It is not determined how atypical survivors or Type X survivors are generated in telomerase-deficient SY12 cells.<br /> Survivor generation of each type (Type I, Type II, Type X or atypical and circularization) is not quantitated.

    1. Joint Public Review:

      Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

      This manuscript is the third in a recent series of reports of cryo-EM structures of Sirt6:nucleosome complexes. The main conclusions of the three studies are similar, but this manuscript from Smirnova et al. includes additional molecular dynamics analysis of the histone tails. These studies suggest that part of the specificity for sites on the H3 tail is the result of only this tail having significant access to the active site. The results are partially validated by showing that H3-K27Ac is sometimes found near the active site in the simulations, and is a weak substrate for the deacetylase in vitro. All of the structures show Sirt6 contacting the acidic patch of H2A-H2B, partial displacement of the H2A C-terminal tail, and displacement of the DNA at the entry-exit site to "unclamp" the H3 N-terminal tail. This manuscript provides additional support for the conclusions drawn in the first two published structures, adds molecular dynamics simulations that provide further insight and includes a biochemical assay that helps to resolve an apparent conflict regarding the deacetylation of H3-K27Ac from the other two papers.

    1. Reviewer #1 (Public Review):

      In this study, Satake and colleagues endeavored to explore the rates and patterns of somatic mutations in wild plants, with a focus on their relationship to longevity. The researchers examined slow- and fast-growing tropical tree species, demonstrating that slow-growing species exhibited five times more mutations than their fast-growing counterparts. The number of somatic mutations was found to increase linearly with branch length. Interestingly, the somatic mutation rate per meter was higher in slow-growing species, but the rate per year remained consistent across both species. A closer inspection revealed a prevalence of clock-like spontaneous mutations, specifically cytosine-to-thymine substitutions at CpG sites. The author suggested that somatic mutations were identified as neutral within an individual, but subject to purifying selection when transmitted to subsequent generations. The authors developed a model to assess the influence of cell division on mutational processes, suggesting that cell-division independent mutagenesis is the primary mechanism.

      The authors have gathered valuable data on somatic mutations, particularly regarding differences in growth rates among trees. Their meticulous computational analysis led to fascinating conclusions, primarily that most somatic mutations accumulate in a cell-division independent manner. The discovery of a molecular clock in somatic mutations significantly advances our comprehension of mutational processes that may generate genetic diversity in tropical ecosystems. The interpretation of the data appears to be based on the assumption that somatic mutations can be effectively transmitted to the next generation unless negative selection intervenes. However, accumulating evidence suggests that plants may also possess "effective germlines," which could render the somatic mutations detected in this study non-transmittable to progeny. Incorporating additional analyses/discussion in the context of plant developmental biology, particularly recent studies on cell lineage, could further enhance this study.

      Specifically, several recent studies address the topics of effective germline in plants. For instance, Robert Lanfear published an article in PLoS Biology exploring the fundamental question, "Do plants have a segregated germline?" A study in PNAS posited that "germline replications and somatic mutation accumulation are independent of vegetative life span in Arabidopsis." A phylogenetic-based analysis titled "Rates of Molecular Evolution Are Linked to Life History in Flowering Plants" discovered that "rates of molecular evolution are consistently low in trees and shrubs, with relatively long generation times, as compared with related herbaceous plants, which generally have shorter generation times." Another compelling study, "The architecture of intra-organism mutation rate variation in plants," published in PLoS Biology, detected somatic mutations in peach trees and strawberries. Although some of these studies are cited in the current work, a deeper examination of the findings in relation to the existing literature would strengthen the interpretation of the data.

    1. Reviewer #1 (Public Review):

      The authors have employed a digital twin approach to show that depending on the underlying disease mechanism, a digital replica constructed from human data can both recapitulate clinical findings, but also provide important insights into the fundamental disease state by revealing underlying contributing mechanisms. Moreover, the authors are able to show that a disease state caused by two different underlying genetic anomalies exhibit different electrical and morphological profiles.

      This is important information as it allows for potential stratification of treatment approaches in future cases based on underlying phenotype by linking it to specific genotype properties. One of the most innovative aspects of the study is the mismatch switching between personalized structure, remodeling and genotype specific electrophysiological properties. The approach is elegant and allows for further exposure of the key mechanisms that contribute to the development of ventricular tachycardia circuits. One addition that could add more insight is to predict the effect of structural remodeling alone well, considering only normal electrophysiological models. Another interesting approach would be a sensitivity analysis, to determine how sensitive the VT circuits are to the specific geometry of the patient and remodeling that occurs during the disease, such an approach could also be used to determine how sensitive the outputs are to electrophysiological model inputs.

    1. Reviewer #1 (Public Review):

      The research titled "Spatial and temporal distribution of ribosomes in single cells reveals aging differences between old and new daughters of Escherichia coli" by Lin Chao, Chun Kuen Chen, Chao Shi, and Camilla U. Rang addresses the asymmetric distribution of ribosomes in single E. coli cells during aging by time-lapse microscopy, as well as its correlation to protein misfolding. The presented research is an important contribution to the field of protein biosynthesis pathways and their link to aging, especially in regard to the thorough analysis of variation in cell elongation rate in old and new daughter cells derived from old and new mother cells. However, the imaging results, analysis, and methodologies require substantial elaboration, as in its current form several key characteristics remain unanswered. Furthermore, the results should be compared and discussed in regard to several other reports, which analyzed ribosome asymmetric distribution and inheritance in E.coli, see detailed comments below.

      Major comments:<br /> *It is not clear from the results or the material and methods sections how the authors define and detect old vs. new mother cells up to 128 cells division, which is the limit the manuscript describes in line 574: "To avoid effects of crowding within the micro-colonies, movies were ended when micro-colonies exceeded 128 cells". The results described only refer to 3 cell divisions (Fig.1 for example). As this is the key issue the manuscript addresses this requires elaboration.

      * The authors should present several representative images of the results described, including: "New daughters at birth from old mothers have more ribosomes" - this should include clear quantification, of normalized fluorescence intensity vs. normalized cell length, as well as for "Ribosomal asymmetry between daughters are spatially in place in mothers before division"(line 218) for example. This should include annotation of the exact time points in minutes. The quantification can be done and presented as in their previous work, which provides the basis for this study: (Figure 2b, for example) "Allocation of gene products to daughter cells is determined by the age of the mother in single Escherichia coli cells" Chao Shi, Lin Chao, Audrey Menegaz Proenca, Andrew Qiu, Jasper Chao and Camilla U. Rang, May 2020, https://doi.org/10.1098/rspb.2020.0569.

      * Quantification of variations over generations time during the time lapse is required: the change in cell-length (y-axis, the length of full-grown cell normalized to 1) vs. ribosomes number (y-axis) relative to the generation time analysis should be presented, based on the time-lapse images. The mean from ~10 independent cells should be presented, as in many similar research, for example: "Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence" Qian Chai, Bhupender Singh, Kristin Peisker, Nicole Metzendorf, Xueliang Ge, Santanu Dasgupta, Suparna Sanyal, 2014, JBC (Figure 3b).

      * The distribution of ribosomes should be compared to the nucleoid distribution, as this is a major factor in RNA and translation distribution in bacterial cells (for example Gray, W. T., Govers, S. K., Xiang, Y., Parry, B. R., Campos, M., Kim, S., & Jacobs-Wagner, C. (2019). Nucleoid size scaling and intracellular organization of translation across bacteria. Cell, 177(6), 1632-1648.e20. https://doi.org/10.1016/j.cell.2019.05.017 , as reviewed in RNA localization in prokaryotes: Where, when,how, and why, Mikel Irastortza-Olaziregi, Orna Amster-Choder, 2020). The authors should add and discuss this, or elaborate on the reasons to omit this analysis.

      * The results should be compared and discussed in regard to several other reports, which analyzed ribosome asymmetric distribution and inheritance in E.coli by tagging different ribosomal proteins, as well as different methodologies, including:

      Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence" Qian Chai, Bhupender Singh, Kristin Peisker, Nicole Metzendorf, Xueliang Ge, Santanu Dasgupta, Suparna Sanyal, 2014, JBC

      Gray, W. T., Govers, S. K., Xiang, Y., Parry, B. R., Campos, M., Kim, S., & Jacobs-Wagner, C. (2019). Nucleoid size scaling and intracellular organization of translation across bacteria. Cell, 177(6), 1632-1648.e20. https://doi.org/10.1016/j.cell.2019.05.017

      Spatiotemporal Organization of the E. coli Transcriptome: Translation Independence and Engagement in Regulation Graphical Abstract Highlights d RNAs in E. coli exhibit asymmetric distribution on a transcriptome-wide scale, Shanmugapriya Kannaiah, Jonathan Livny, Orna Amster-Choder, 2019

      Several of the findings reported, including asymmetric ribosome distribution and inheritance levels seem different than the ones reported here. This should be discussed in regard to the different methodologies.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Here the authors have tethered a Pgp substrate to strategically place cysteine residues in the protein. Notably, the cysteine-linked substrate (ANC-DNPT)- stimulates ATP hydrolyse and so is able to undergo IF to OF transitions. The authors then determined cryo-EM structures of these complexes and MD simulations of bound states. By capturing unforeseen OF conformations with substate they propose that TM1 undergoes local conformational changes that are sufficient to translocate substrates, rather than large bundle movements.

      Strengths:<br /> This paper provides the first substrate (ANC-DNPT)- bound conformations of PgP and a new mechanistic model of how substrates are translocated.

      Weaknesses:<br /> Although the cross-links stimulate ATP hydrolysis, further controls are needed to convince me that the TM1 conformations observed in the structures are physiologically relevant, since they have been trapped by "large" substrates covalently-tethered by cross-links.

    1. Reviewer #1 (Public Review):

      Summary. In this investigation Kapustin et al. demonstrate that vascular smooth muscle cells (VSMCs) exposed to the extracellular matrix fibronectin stimulates the release of small extracellular vesicles (sEVs). The authors provide experimental evidence that stimulation of the actin cytoskeleton boosts sEV secretion and posit that sEVs harbor both fibronectin and collagen IV protein themselves which also, in turn, alter cell migration parameters. It is well established that fibronectin is associated with increased cell migration and adherence; therefore, this association with VSMCs is not novel. The authors purport that sEV are largely born of filopodia origin; however, this data is not well executed and seems generally at odds with the presented data. Similarly, the effect of sEVs on parameters of cell migration has almost no magnitude of effect, making mechanism exploration somewhat nebulous. Lastly, the proposed mechanism of VSMCs responding to, and depositing, ECM proteins via sEVs was not rigorously executed; again, making the conclusions challenging for the reader to interpret.

      Strengths. The authors provide a comprehensive battery of cytoskeletal experiments to test how fibronectin and sEVs impact both sEV release and vascular smooth muscle cell migratory activation.

      Weaknesses. Unfortunately, this article suffers from many weaknesses. First, the rigor of the experimental approach is low, which calls into question the merit of the conclusions. In this vein, there is a lack of proper controls or inclusion of experiments addressing alternative explanations for the phenotype or lack thereof.

    1. Reviewer #1 (Public Review):

      In their article, "Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings," Tendolkar and colleagues explore Ubx regulation in butterflies. The authors investigated how Ubx expression is restricted to the hindwing in butterflies through a series of genomic analyses and genetic perturbations. The authors provide evidence that a Topoologiacally Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin around Ubx, largely through an apparent boundary element. CRISPR mutations of this boundary element led to ectopic Ubx expression in forewings, resulting in homeotic transformation in the wings. The authors also explore the results of the mutation in two non-coding RNA regions as well as a possible enhancer module. Each of these induces homeotic phenotypes. Finally, the authors describe a number of homeotic phenotypes in butterflies, which they relate to their work.

      Together, this was an interesting paper with compelling initial data. That said, I have several items that I feel would warrant further discussion, presentation, or data.

      First, I would not state, "Little is known about how Hox genes are regulated outside of flies." They should add "in insects" since so much in known in vertebrates

      For Figure 1, it would aid the readers if the authors could show the number of RNAseq reads across the locus. This would allow the readership to evaluate the frequency of the lncRNAs, splice variants, etc.

      How common are boundary elements within introns? Typically, boundary elements are outside gene bodies, so this could be explored further. This seems like an interesting bit of biology which, following from the above point, it would be interesting to, at a minimum, discuss, but also relate to how transcription occurs through a possible boundary element (are there splice variants, for example?).

      The CRISPR experiments led to compelling phenotypes. However, as a Drosophila biologist, I found it hard to interpret the data from mosaic experiments. For example, in control experiments, how often do butterflies die? Are there offsite effects? It's striking that single-guide RNAs led to such strong effects. Is this common outside of this system? Is it possible to explore the function effects at the boundary element - are these generating large deletions (for example, like Mazo-Vargas et al., 2022)?

      For the mosaic experiments, how frequent are these effects in nature or captive stocks? Would it be possible to resequence these types of effects? At the moment, this data, while compelling, was hard to put into the context of the experiments above without understanding how common the effects are. Ideally, there would be resequencing of these tissues, which could be targeted, but it was not clear to me the general rates of these variants.

      In sum, I enjoyed the extensive mosaic perturbations. However, I feel that more molecular descriptions would elevate the work and make a larger impact on the field.

    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 impact 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. 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. Joint Public Review:

      Strengths:

      Gain-of-function mutations and amplifications of PPM1D are found across several human cancers and are associated with advanced tumor stage, worse prognosis, and increased lymph node metastasis. In this study, Zhang and colleagues investigate the synthetic-lethal dependencies of PPM1D (protein phosphatase, Mg2+/Mn2+ dependent 1D) in leukemia cells using CRISPR/Cas9 screening. They identified that SOD1 (superoxide dismutase-1) as the top hit, whose loss reduces cellular growth in PPM1D-mutant cells, but not wild-type (WT) cells. Consistently, the authors demonstrate that PPM1D-mutant cells are more sensitive to SOD1 inhibitor treatment. By performing different in vitro studies, they show that PPM1D-mutant leukemia cells have an elevated level of reactive oxygen species (ROS), decreased basal respiration, increased genomic instability, and impaired non-homologous end-joining repair. The data strongly support that PPMD1 mutant cells have high levels of total peroxides and elevated DNA breaks and that genetic depletion of SOD1 decreases cell growth in two AML cell lines. These data highlight the potential of SOD1 inhibition as a strategy to achieve therapeutic synergism for PPM1D-mutant leukemia; and demonstrate the redox landscape of PPM1D-mutant cells.

      Weaknesses:

      It is not explained how superoxide radical (which is not damaging by itself) induces damage, the on-target effects of the SOD1 inhibitors at the concentrations are not clear, the increase in total hydroperoxides is not supported by loss of SOD1, the changes in mitochondrial function are small, and there is no assessment of how the mitochondrial SOD2 expression or function, which dismutates mitochondrial superoxide, is altered. Overall these studies do not distinguish between signal vs. damaging aspects of ROS in their models and do not rule out an alternate hypothesis that loss of SOD1 increases superoxide production by cytosolic NADPH activity which would significantly alter ROS-driven regulation of kinase/phosphatase signal modulation, affecting cell growth and proliferation as well as DNA repair. Additionally, with the exception of growth defects demonstrated with sgSOD1, the majority of data are acquired using two chemical inhibitors, LCS1 and ATN-224, without supporting evidence that these inhibitors are acting in an on-target manner.

      Overall, the authors address an important problem by seeking targetable vulnerabilities in PPM1D mutant AML cells. It is clear that SOD1 deletion induces strong growth defects in the AML cell lines tested, that most of the approaches are appropriate for the outcomes being evaluated, and that the data are technically solid and well-presented. The major weakness lies in which redox pathways and ROS species are evaluated, how the resulting data are interpreted, and gaps in the follow-up experiments. Due to these omissions, as currently presented, the broader impact of these findings is unclear.

    1. Reviewer #1 (Public Review):

      Summary: Hansen et al. dissect the molecular mechanisms of bacterial ice nucleating proteins mutating the protein systematically. They assay the ice nucleating ability for variants changing the R-coils as well as the coil capping motifs. The ice nucleation mechanism depends on the integrity of the R-coils, without which the multimerization and formation of fibrils are disrupted.

      Strengths: The effects of mutations are really dramatic, so there is no doubt about the effect. The variants tested are logical and progressively advance the story. The authors identify an underlying mechanism involving multimerization, which is plausible and compatible with EM data. The model is further shown to work in cells by tomography.

      Weaknesses: The theoretical model presented for how the proteins assemble into fibrils is simple, but not supported by much data.

    1. Reviewer #1 (Public Review):

      Owen D et al. investigated the protein partners and molecular functions of ZMYM2, a transcriptional repressor with key roles in cell identity and mutated in several human diseases, in human U2OS cells using mass spectrometry, siRNA knockdown, ChIP-seq and RNA-seq. They tried to identify chromatin bound complexes containing ZMYM2 and identified known and novel protein partners, including ADNP and the newly described partner TRIM28. Focusing mainly on these two proteins, they show that ZMYM2 physically interacts with ADNP or TRIM28, and co-occupies an overlapping set of genomic regions with ADNP and TRIM28. By generating a large set of knockdown and RNA-seq experiments, they show that ZMYM2 co-regulates a large number of genes with ADNP and TRIM28 in U2OS cells. Interestingly, ZMYM2-TRIM28 do not appear to repress genes directly at promoters, but the authors find that ZMYM2/TRIM28 repress LTR elements and suggest that this leads to gene deregulation at distance by affecting the chromatin environment within TADs.

      A strength of the study is that, compared to previous studies of ZMYM2 protein partners, it investigates binding partners of ZMYM2 using the RIME method on chromatin. The RIME method makes it possible to identify low-affinity protein-protein interactions and proteins interactions occurring at chromatin, therefore revealing partners most relevant for gene regulation at chromatin. This allowed the identification of novel ZMYM2 partners not identified before, such as TRIM28.

      The authors present solid interaction data with appropriate controls and generated an impressive amount of datasets (ChIP-seq for TRIM28 and ADNP, RNA-seq in ZMYM2, ADNP and TRIM28 knockdown cells) that are important to understand the molecular functions of ZMYM2. These datasets were generated with replicates and will be very useful for the scientific community. This study provides important novel insights into the molecular roles of ZMYM2 in human U2OS cells.

    1. Reviewer #1 (Public Review):

      In the present study, the authors carefully evaluated the metabolic effects of intermittent fasting on normal chow and HFD fed mice and reported that intermittent fasting induces beiging of subcutaneous white adipose tissue. By employing complementary mouse models, the authors provided compelling evidence to support a mechanism through ILC3/IL-22/IL22R pathway. They further performed comprehensive single-cell sequencing analyses of intestinal immune cells from lean, obese, obese undergone intermittent fasting mice and revealed altered interactome in intestinal myeloid cells and ILC3s by intermittent fasting via activating AhR. Overall, this is a very interesting and timely study uncovering a novel connection between intestine and adipose tissue in the context of executing metabolic benefits of intermittent fasting.

    1. Reviewer #1 (Public Review):

      The authors propose a hypothesis for ovarian carcinogenesis based on epidemiological data, and more specifically they suggest that the latter relates to ascending genital tract "infection" or "dysbiosis", the resulting fallopian tube inflammation ultimately predisposing to ovarian cancer.

      While this hypothesis would ideally be addressed in a longitudinal set-up with repeated female genital tract sampling, such an approach is obviously hard to realize. Rather, the authors present this hypothesis as a rationale for a cross-sectional study involving 81 patients with ovarian cancer (most with the most common subtype of high grade serous ovarian carcinoma, though other subtypes were also included), as well as 106 control patients with various non-infectious conditions including endometriosis and benign ovarian cysts. In all patients was there a comprehensive microbiome sampling of ovarian surface/fallopian tube, cervix and peritoneal cavity as well sampling of a number of potential sources of contamination, including surgery sites, ambient environment, consumables used in the DNA extraction and sequencing pipeline, etc. In line with the hypothesis presented at the outset, species with a threshold of at least 100 reads in both at least one cervical and at least one fallopian tube sample, while absent from environmental swabs, were considered relevant to the postulated pathway.

      Remarkably, fallopian tube microbiota in ovarian cancer patients tended to cluster more closely to those retrieved from the paracolic gutter, than fallopian tube microbiota in non-cancer controls, which showed more relative similarity to vaginal/genital tract microbiota.

      Although not really addressed by the authors, there also seem to be quite a few differences, at least in terms of abundance, in cervical microbiota between ovarian cancer patients and controls as well, which is an interesting finding, even when accounting for differences in age distribution between ovarian cancer patients and included control patients.

      Overall, very few data are available thus far on the upper genital tract/fallopian tube microbiome, while also invariably controversial, as it has proven extremely difficult to obtain pelvic samples in a valid, "sterile" manner, i.e. without affecting a resident low-biomass microbiome to be analyzed. The authors took a number of measures to counter so, and in this respect, this is likely the largest and most valid study on the subject, even though biases and contamination can never be completely excluded in this context.

      As such, I believe the strength of this study and paper primarily relates to the rigour of the methodology, thereby giving us a valuable insight in the presumed fallopian tube/ovarian surface microbiome, which may definitely serve as an impetus and a reference to future translational ovarian cancer research, or ovarian microbiome research for that matter.

      I believe that the authors should acknowledge in more detail, that the data obtained from their cross-sectional study, valid as these are, do not provide any direct support to the hypothesis - albeit also plausible - set forth, a discussion that I somehow missed to a certain extent. It is important to realize in this and related contexts that neoplasia may well induce microbiome alterations through a variety of mechanisms, hence microbiome alterations not per se being causative. Conclusions should therefore be more reserved. Along the same lines, potential biases introduced through the selection of control patients (some detail here would be insightful) also deserves some discussion, as it is not known, whether other conditions such as benign ovarian cysts or endometriosis have some relationship with the human microbiome, be it causative or 'reversely causative', see for instance very recent work in Science Translational Medicine.

    1. Joint Public Review:

      The enteroviruses comprise a medically important genus in the large and diverse picornavirus family, and are known to be released without lysis from infected cells in large vesicles containing numerous RNA genome-containing capsids - a feature allowing for en bloc transmission of multiple viral genomes to newly infected cells that engulf these vesicles. SIRT-1 is an NAD-dependent protein deacetylase that has numerous and wide ranging effects on cellular physiology and homeostasis, and it is known to be engaged in cellular responses to stress and autophagy.

      Jassey et al. show that RNAi depletion of SIRT-1 impairs the release of enterovirus D-68 (EV-D68) in EVs recovered from the supernatant fluids of infected cells using a commercial exosome isolation kit. The many functions attributed to SIRT-1 in the literature reflect its capacity to deacetylate various cell proteins engaged in transcription, DNA repair, and regulation of metabolism, apoptosis and autophagy. However, Jassey et al. make the surprising claim that the proviral role of SIRT-1 in promoting enterovirus release is not dependent on its deacetylase activity. Fig. S1C is crucial to this suggestion but it is less than completely convincing. It shows that both wild-type and mutant SIRT-1are massively over-expressed in the rescue experiment compared to the normal endogenous level of SIRT-1 expression. Moreover, the blots are heavily saturated, making it difficult to assess the relative expression of wild-type vs. mutant. In addition, Fig. S1B and Fig. 4C convincingly show that EX527, a small molecule inhibitor of the deacetylase activity of SIRT-1, inhibits extracellular release of the virus. This suggests that the deacetylase activity of SIRT-1 may in fact be required for the proviral effect of SIRT-1. This is a fundamentally important question that requires more investigation.

      Fig. 6 shows how SIRT-1 knockdown impacts the release of enterovirus D68 in EVs recovered from cell culture supernatant using a commercial 'Total Exosome Isolation Kit'. The authors are appropriately cautious in describing the vesicles they presume to be isolated by the kit as simply 'extracellular vesicles', since there are multiple types of EVs with very different mechanisms of biogenesis, of which 'exosomes' are but one specific type. It would have been more elegant had the authors shown that SIRT-1 is required for EV-D68 release in detergent-sensitive vesicles with low buoyant density in isopycnic gradients, and to characterize the size and number of viral capsids in these vesicles by electron microscopy.

      The authors claim that "reduction of SIRT-1 attenuates the release of virus-loaded CD63-positive EVs" but they never actually show that the vesicles containing EV-D68 are in fact CD63-positive. Can a CD63 pulldown immunoprecipitate EV-D68 capsid proteins or viral RNA? This is important since CD63 is strongly associated with exosomes released from cells through the multi-vesicular body pathway, which are distinct from the LC3-positive EVs released by secretory autophagy that have previously been associated with enteroviruses.

      The authors claim "that most EV-D68 is released non-lytically in an enveloped form" but they show data from only from early time points following infection (5 or 6 hrs post-infection) - prior to cell lysis. It would have been interesting to see a more complete temporal analysis, and to know the overall proportion of virus released in EVs versus lytic release of nonenveloped virus.

      Fig. 1D indicates that a small fraction of SIRT-1 leaks from the nucleus in EV-D68 infected cells. The authors suggest this is due to targeted nuclear export, rather than simply leaky nuclear pores which are well known to exist in enterovirus-infected cells, but the evidence for this is questionable. The authors present similar fluorescent microscopy data showing inhibition of TFEB export in leptomycin-B treated cells in Fig. S2A in support of their claim that there is specific SIRT-1 export, but there is equivalent residual TFEB and SIRT-1 in the cytoplasm of the treated cells. Quantitative immunoblots of cytoplasmic and nuclear cell fractions might prove more compelling.

    1. Joint Public Review:

      This study investigated the mechanisms and biological processes associated with eccDNA generation in germline cells. They enriched eccDNA from cells at each step of spermatogenesis in mouse as well as human sperm using a commonly-used method to enrich small eccDNA: column purification, exonuclease digestion, rolling circle amplification, followed by short-read Illumina sequencing. From the fragment size analyses, dominant sizes were shown to be those protected by mono- or di-nucleosomes. The authors developed a computational pipeline to investigate eccDNA breakpoints in detail from split reads and reported a prevalence of a microhomology-mediated mechanism. Features of small germline eccDNA closely matched with small eccDNA generated by apoptosis, suggesting apoptotic germline cells as a major source.

      Combined with analyses of publically available data from mouse tissues, the study established a strong link between small eccDNA and DNA fragments protected by mono-or di-nucleosomes. The rigorous investigation of microhomologies revealed that eccDNA sizes correlated with the lengths of microhomology in spermatogonial cells. Small eccDNA tends to originate from euchromatic regions, while longer eccDNA is derived from heterochromatic regions. These are novel findings.

      The authors repeatedly stated the rare association between eccDNA and recombination hotspots. The argument was backed by (1) the abundance of eccDNA coming from dead cells, and (2) the small number of eccDNA from SPA cells undergoing miosis. The argument seems to have a point; however, the observation that the authors recovered hundreds of eccDNA at recombination hotspots may indicate that miotic recombination is a significant source of eccDNA. Because of the bulk isolation of eccDNA, those eccDNAs were outnumbered by the abundant eccDNA coming from apoptotic cell death. Indeed, eccDNAs from recombination hotspots are slightly more than random in all cell types (Fig. 4A).

      Related to this issue, the dominance of both 180- and 360-bp fragments in most mouse tissues put the single 180-bp peaks of SPA, RST, and EST eccDNA in a peculiar position. Fewer numbers of these cells were used for eccDNA isolation than sperm cells, resulting in fewer eccDNA in these cell types, despite the same amount (10ng) of DNA input for rolling circle amplification. There might be a technical issue behind the peculiar observation, which is understandable given the challenging nature of isolating pure cell populations.

    1. Reviewer #1 (Public Review):

      Here, in this revised manuscript, the authors describe the transition between the summer form and the winter form of the pear psyllid pest, Cacopsylla chinensis (hemiptera). While the authors explore many components of this transition, the central hypotheses they seek to test are (i) that a protein they deem CcTRPM is a cold-sensitive Transient Receptor Potential Melastatin (TRPM) channel, and (ii) that this channel is involved in the summer-to-winter transition, in response to cold.<br /> The authors demonstrate that: both cold and menthol can initiate the summer-to-winter transition; that the protein of interest is required for the summer-to-winter transition (in vivo); that the protein of interest is involved in menthol- and cold-dependent Ca2+ transients (in vitro); that miR-252 expression is temperature-dependent, modulates the seasonal transition, and affects the expression of the transcript of interest; and finally, somewhat separately, that the chitin biosynthesis pathway is linked to the summer-to-winter transition.

      However, I note three weaknesses, which are largely inherited from the original manuscript.

      Firstly, the identification of the TRPM gene seems to be partially couched in the ab initio structural identification of "conserved ankyrin repeats." The methodology used to identify these so-called ankyrin repeats is not sufficiently described, and their conserved status is not sufficiently demonstrated nor cited (to my knowledge, this would be the first description of ankyrin repeats in TRPM, whereas previous studies have not detected them). There is also no discussion of previously identified structural components of TRPMs (see: Yin et al 2018, DOI: 10.1126/science.aan4325)

      Secondly, the phylogenetic analysis still appears to be incomplete. The authors claim that "insects TRPM and mammals TRPM belong to different branches in evolution." While this is not a paper centered on the evolutionary analysis of this gene/protein family, the phylogenetic analysis here is insufficient for justifying this claim, especially since this claim is counter to previous studies (many in the literature over the past 10 years).

      Thirdly, the methods lack sufficient detail to completely reproduce the phylogenetics and the cold-induced Ca2+ imaging.

      Despite these weaknesses, I find the organismal/molecular component of this manuscript to be clear and convincing.

    1. Reviewer #1 (Public Review):

      Summary:

      Developing vaccination capable of inducing persistent antibody responses capable of broadly neutralizing HIV strains is of high importance. However, our ability to design vaccines to achieve this is limited by our relative lack of understanding of the role of T-follicular helper (Tfh) subtypes in the responses. In this report Verma et al investigate the effects of different prime and boost vaccination strategies to induce skewed Tfh responses and its relationship to antibody levels. They initially find that live-attenuated measles vaccine, known to be effective at inducing prolonged antibody responses has a significant minority of germinal center Tfh (GC-Tfh) with a Th1 phenotype (GC-Tfh1) and then explore whether a prime and boost vaccination strategy designed to induce GC-Tfh1 is effective in the context of anti-HIV vaccination. They conclude that a vaccine formulation referred to as MPLA before concluding that this is the case.

      Strengths:

      While there is a lot of literature on Tfh subtypes in blood, how this relates to the germinal centers is not always clear. The strength of this paper is that they use a relevant model to allow some longitudinal insight into the detailed events of the germinal center Tfh (GC-Tfh) compartment across time and how this related to antibody production.

      Weaknesses:

      The authors focus strongly on the numbers of GC-Tfh1 as a proportion of memory cells and their comparison to GC-Tfh17. There seems to be little consideration of the large proportion of GC-Tfh which express neither CCR6 and CXCR3 and currently no clear reasoning for excluding the majority of GC-Tfh from most analysis. There seems to be an assumption that since the MPLA vaccine has a higher number of GC-Tfh1 that this explains the higher levels of antibodies. There is not sufficient information to make it clear if the primary difference in vaccine efficacy is due to a greater proportion of GC-Tfh1 or an overall increase in GC-Tfh of which the percentage of GC-Tfh1 is relatively fixed.

    1. Reviewer #1 (Public Review):

      Previous work by this group has established that cholinergic projections from the forebrain to the basolateral amygdala (BLA) contribute to the acquisition of auditory-cued fear memories (Jiang et al., 2016). Here, the authors continue these studies, using a combination of techniques including genetic access to cFos expressing neurons, in-vivo optogenetics, and optical detection of acetylcholine (ACh) in the BLA. The main findings are that ACh is not only released during footshock presentation (the unconditioned stimulus, US, used in the fear learning) but that in addition, ACh is released upon CS presentation after fear learning. This implies that cholinergic neurons in the basal forebrain (BF) "learn" the response to tones and that they are recruited into a memory engram in the brain. The authors then follow up these ideas by showing with genetic, activity-dependent cFos labeling that BF ChAT+ neurons which are activated during the training session, are also re-activated by tone recall (Figure 2). Moreover, hM4Di- mediated block of the activity of those ChAT neurons activated during the training session strongly suppresses tone(CS) - driven freezing behavior during recall (Figure 3), again suggesting that re-activation of ChAT neurons in the BF is an important element for the retrieval of fear memory (or else, for the expression of a fear memory). Overall, I think the paper convincingly shows that learning of a tone response occurs in a neuromodulatory system and that neuromodulatory neurons are recruited to a fear memory engram. This adds a new dimension to the circuit- and neuromodulatory mechanisms that underlie learning and memory.

      The paper, as it stands, has weaknesses in data presentation, data analysis, and statistical reporting. For most experiments, significantly more raw data should be shown (e.g. raw example traces for GRAB-ACh3.0), and also brain section images for almost all experiments (specific examples below). Raw data should also be shown in the Main Figures.

      Major point<br /> 1) The authors use hM4Di to "silence" Fos-tagged neurons in the basal forebrain, but they have not validated the efficiency or the possible various effects of this reagent.<br /> It is possible that hM4Di actually has a relatively small effect on suppressing the AP activity of neurons. Nevertheless, hM4Di might still be an effective manipulation, because it was shown to additionally reduce transmitter release at the nerve terminal (see e.g. Stachniak et al. (Sternson) 2014, Neuron). Thus, the authors should evaluate in control experiments whether hM4Di expression plus CNO actually electrically silences the AP-firing of ChAT neurons in the BF as they seem to suggest, and/or if it reduces ACh release at the terminals. For example, one experiment to test the latter would be to perfuse CNO locally in the BLA; after expressing hM4Di in the cholinergic neurons of the BF. At the very least, the assumed action of hM4Di, and the possible caveats in the interpretation of these results should be discussed in the paper.

      Further specific points.<br /> 1) The names of brain areas like "NBM/SIp" and "VP-SIa" need to be better introduced, and somehow contextualized (in the Introduction, and also at first reading in the Results).

      2) Figure 3C: Application of CNO on the memory recall day leads to a strong reduction in CS-driven freezing. However, in this experiment, and also in Fig. S7, the pre-tone value of freezing is also strongly reduced. This would indicate that the activity of NBM/SIp cells (or else, ACh-release from these cells - see also Major point 1), also influences contextual learning. The authors should, first, statistically, test these effects (I am not sure this was done). If these differences are significant, a possible role of ACh in contextual fear learning should be discussed. Has it been shown before whether ACh is involved in contextual fear learning? Does this indicate the involvement of another target area of ACh neurons (e.g., the hippocampus?).

      3) The discussion could be improved by better comparing what they found, to the wider literature. For example, previous papers studying other neuromodulatory systems found evidence for a modulation of neuromodulator release after learning; e.g. see Martins and Froemke 2015 Nat. Neuroscience for the noradrenergic system, Tang et al. (Schneggenburger lab) 2020 J. Neuroscience for the dopaminergic system and fear learning; and Uematsu et al., 2017, Nat. Neuroscience for the noradrenergic system and fear learning. Maybe the authors could include these and similar references when revising their discussion to take into account a broader view of previous findings related to other neuromodulatory systems.

    1. Reviewer #1 (Public Review):

      The manuscript "Vibrio cholerae´s ToxRS bile sensing system" by Gubensäk et al. reports the crystal structure of a periplasmic, hetero-dimeric bile-sensing protein complex ToxRSp. The authors show that the intrinsically disordered C-terminus of ToxRp folds upon binding to ToxSp, thus completing the defective bile-binding interface of ToxSp. Using NMR experiments they find that bile acid binds to the ToxRSp hetero-dimer but not to ToxSp or ToxRp alone. Results from NMR and microfluidic modulation spectroscopy indicate additional, weak binding sites in the ToxRSp complex and local conformational changes associated with binding. The authors apply AlphaFold to predict ToxRSp structures from various Vibrio strains, showing gross structural conservation with greater variability in ToxRp compared to ToxSp. The authors conclude to have shown that ToxS is a main sensor in Vibrio strains and requires ToxR for binding bile, forming part of a regulation mechanism for survival and virulence after infection.

      Cholera is a severe and often lethal disease affecting a high number of people in the developing world. It is caused by the bacterium Vibrio cholerae, which rapidly adapts to hostile conditions in the stomach where it produces toxins. The pathogen uses sensory proteins, like the ToxR-ToxS system, that facilitate bile resistance and virulence. The present studies by Gubensäk et al. reveal an intriguing molecular mechanism by which V. cholerae creates a sensor for bile, transducing the signal through the cellular membrane of the bacterium. Their crystal structure of ToxRSp and complementary biophysical experiments conclusively show a split binding interface for bile formed by the individual periplasmic domains ToxRp and ToxSp. The folding of an intrinsically disordered segment of ToxRp upon binding to ToxSp adds a missing beta-strand to a defective beta-barrel, thus creating the intact interface for the ligand. The mechanism provides new molecular level insights into bile resistance of V. cholerae. Experiments are carefully conducted and analysed. The manuscript is well written.

      However, there are some ambiguities in the proposed stoichiometry of the ToxRSp/bile interaction inferred from SEC-MALS experiments and MD simulation. Results may contain additional information on the order of events in formation of the ternary complex. Moreover, the quality of the manuscript could be improved by expanding analyses and discussion on the apparent necessity of a split protein binding interface in mechanisms of resistance and virulence.

    1. Reviewer #1 (Public Review):

      The proposed study provides an innovative framework for the identification of muscle synergies taking into account their task relevance. State-of-the-art techniques for extracting muscle interactions use unsupervised machine-learning algorithms applied to the envelopes of the electromyographic signals without taking into account the information related to the task being performed. In this work, the authors suggest including the task parameters in extracting muscle synergies using a network information framework previously proposed. This allows the identification of muscle interactions that are relevant, irrelevant, or redundant to the parameters of the task executed.

      The proposed framework is a powerful tool to understand and identify muscle interactions for specific task parameters and it may be used to improve man-machine interfaces for the control of prostheses and robotic exoskeletons.

      With respect to the network information framework recently published, this work added an important part to estimate the relevance of specific muscle interactions to the parameters of the task executed. However, the authors should better explain what is the added value of this contribution with respect to the previous one, also in terms of computational methods.

      It is not clear how the well-known phenomenon of cross-talk during the recording of electromyographic muscle activity may affect the performance of the proposed technique and how it may bias the overall outcomes of the framework.

    1. Reviewer #1 (Public Review):

      Picard et al. report a novel neural signature of facial expressions of pain. In other words, they provide evidence that a specific set of brain activations, as measured by means of functional magnetic resonance imaging (fMRI), can tell us when someone is expressing pain via a concerted activation of distinctive facial muscles. They demonstrate that this signature provides a better characterization of this pain behaviour when compared with other signatures of pain reported by past research. The Facial Expression of Pain Signature (FEPS) thus enriches this collection and, if further validated, may allow scientists to identify the neural structures subserving important non-verbal pain behaviour. I have, however, some reservations about the strength of the evidence, relating to insufficient characterization of the underlying processes involved.

      Strengths:<br /> The study relies on a robust machine-learning approach, able to capitalise on the multivariate nature of the fMRI data, an approach pioneered in the field of pain by one of the authors (Dr. Tor Wager). This paper extends Wager's and other colleagues' work attempting to identify specific combinations of brain structures subserving different aspects of the pain experience while examining the extent of similarity/dissimilarity with the other signatures. In doing so, the study provides further methodological insight into fine-grained network characterization that may inspire future work beyond this specific field.

      Weaknesses:<br /> The main weakness concerns the lack of a targeted experimental design aimed to dissect the shared variance explained by activations both specific to facial expressions and to pain reports. In particular, I believe that two elements would have significantly increased the robustness of the findings:<br /> 1) Control conditions for both the facial expressions and the sensory input. An efficient signature should not be predictive of neutral and emotional facial expressions (e.g., disgust) other than pain expressions, as well as it should not be predictive of sensations originating from innocuous warm stimulation or other unpleasant but non-painful stimulation.<br /> 2) Graded intensity of the sensory stimulation: different intensities of the thermal stimulation would have caused a graded facial expression (from neutral to pain) and graded verbal reports (from no pain to strong pain), thus offering a sensitive characterisation of the signal associated with this condition (and the warm control condition).<br /> However, these conditions are missing from the current design, and therefore we cannot make a strong conclusion about the generalisability of the signature (regardless of whether it can predict better than other signatures - which may/may not suffer from similar or other methodological issues - another potential interesting scientific question!). The authors seem to work on the assumption that the trials where warm stimulation was delivered are of no use. I beg to disagree. As per my previous comment, warm trials (and associated neutral expressions) could be incorporated into the statistical model to increase the classification sensitivity and precision of the FEPS decoding.

    1. Reviewer #1 (Public Review):

      Activity has effects on the development of neural circuitry during almost any step of differentiation. In particular during specific time periods of circuit development, so-called critical periods (CP), altered neural activity can induce permanent changes in network excitability. In complex neural networks, it is often difficult to pinpoint the specific network components that are permanently altered by activity, and it often remains unclear how activity is integrated during the CP to set mature network excitability. This study combines electrophysiology with pharmacological and optogenetic manipulation in the Drosophila genetic model system to pinpoint the neural substrate that is influenced by altered activity during a critical period (CP) of larval locomotor circuit development. Moreover, it is then tested whether and how different manipulations of synaptic input are integrated during the CP to tune network excitability.

      Strengths:<br /> Based on previous work, during the CP, network activity is increased by feeding the GABA-AR antagonist PTX. This results in permanent network activity changes, as highly convincingly assayed by a prolonged recovery period following induced seizure and by altered intersegmental locomotor network coordination. This is then used to provide two important findings: First, compelling electro- and optophysiological experiments track the site of network change down to the level of single neurons and pre- versus postsynaptic specializations. In short, increased activity during the CP increases both the magnitude of excitatory and inhibitory synaptic transmission to the aCC motoneuron, but excitation is affected more strongly. This results in altered excitation inhibition ratios. Fine electrophysiology shows that excitatory synapse strengthening occurs postsynaptically. High-quality anatomy shows that dendrite size and numbers of synaptic contacts remain unaltered. It is a major accomplishment to track the tuning of network excitability during the CP down to the physiology of specific synapses to identified neurons.

      Second, additional experiments with single neuron resolution demonstrate that during the CP different forms of activity manipulation are integrated so that opposing manipulations can rescue altered setpoints. This provides novel insight into how developing neural network excitability is tuned, and it indicates that during the CP, training can rescue the effects of hyperactivity.

      Weaknesses:<br /> There are no major weaknesses to the findings presented, but the molecular cause that underlies increased motoneuron postsynaptic responsiveness as well as the mechanism that integrates different forms of activity during the CP remain unknown. It is clear that addressing these experimentally is beyond the scope of this study, but some discussion about different candidates would be helpful.

    1. Reviewer #1 (Public Review):

      Summary:<br /> As adult-born granule neurons have been shown to play diverse roles, both positive and negative, to modulate hippocampal circuitry and function in epilepsy, understanding the mechanisms by which altered neurogenesis contributes to seizures is important for future therapeutic strategies. The work by Jain et al. demonstrates that increasing adult neurogenesis before status epilepticus (SE) leads to a suppression of chronic seizures in the pilocarpine model of temporal lobe epilepsy. This work is potentially interesting because previous studies showed suppressing neurogenesis led to reduced chronic seizures.

      To increase neurogenesis, the authors conditionally delete the pro-apoptotic gene Bax using a tamoxifen-inducible Nestin-CreERT2 which has been previously published to increase proliferation and survival of adult-born neurons by Sahay et al. After 6 weeks of tamoxifen injection, the authors subjected male and female mice to pilocarpine-induced SE. In the first study, at 2 hours after pilocarpine, the authors examine latency to the first seizure, severity and total number of acute seizures, and power during SE. In the second study in a separate group of mice, at 3 weeks after pilocarpine, the authors examine chronic seizure number and frequency, seizure duration, postictal depression, and seizure distribution/cluster seizures. Overall, the study concludes that increasing adult neurogenesis in the normal adult brain can reduce epilepsy in females specifically. However, important BrdU birthdating experiments in both male and female mice need to be included to support the conclusions made by the authors. Furthermore, speculative mechanisms lacking direct evidence reduce enthusiasm for the findings.

      Strengths:<br /> 1. The study is sex-matched and reveals differences in response to increasing adult neurogenesis in chronic seizures between males and females.

      2. The EEG recording parameters are stringent, and the analysis of chronic seizures is comprehensive. In two separate experiments, the electrodes were implanted to record EEG from the cortex as well as the hippocampus. The recording was done for 10 hours post pilocarpine to analyze acute seizures, and for 3 weeks continuous video EEG recording was done to analyze chronic seizures.

      Weaknesses:<br /> 1. Cells generated during acute seizures have different properties to cells generated in chronic seizures. In this study, the authors employ two bouts of neurogenesis stimuli (Bax deletion dependent and SE dependent), with two phases of epilepsy (acute and chronic). There are multiple confounding variables to effectively conclude that conditionally deleting Bax in Nestin-Cre+ cells leads to increased neurogenesis and hilar ectopic granule cells, thereby reducing chronic seizures.

      2. Related to this is the degree of neurogenesis between Cre+ and Cre- mice and the nature of the sex differences. It is crucial to know the rate/fold change of increased neurogenesis before pilocarpine treatment and whether it is different between male and female mice.

      3. The authors observe more hilar Prox1 cells in Cre+ mice compared to Cre- mice. The authors should confirm the source of the hilar Prox1+ cells.

      4. The biggest weakness is the lack of mechanism. The authors postulate a hypothetical mechanism to reconcile how increasing and decreasing adult-born neurons in GCL and hilus and loss of hilar mossy and SOM cells would lead to opposite effects - more or fewer seizures. The authors suggest the reason could be due to rewiring or no rewiring of hilar ectopic GCs, respectively, but do not provide clear-cut evidence.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This study examined the role of statistical learning in pain perception, suggesting that individuals' expectations about a sequence of events influence their perception of pain intensity. They incorporated the components of volatility and stochasticity into their experimental design and asked participants (n = 27) to rate the pain intensity, their prediction, and their confidence level. They compared two different inference strategies - optimal Bayesian inference vs. heuristic-employing Kalman filters and model-free reinforcement learning and showed that the expectation-weighted Kalman filter best explained the temporal pattern of participants' ratings. These results provide evidence for a Bayesian inference perspective on pain, supported by a computational model that elucidates the underlying process.

      Strengths:<br /> Their experimental design included a wide range of input intensities and the levels of volatility and stochasticity.

      Weaknesses:<br /> - Selection of candidate computational models: While the paper juxtaposes the simple model-free RL model against a Kalman Filter model in the context of pain perception, the rationale behind this choice remains ambiguous. It prompts the question: could other RL-based models, such as model-based RL or hierarchical RL, offer additional insights? A more detailed explanation of their computational model selection would provide greater clarity and depth to the study.

      - Effects of varying levels of volatility and stochasticity: The study commendably integrates varying levels of volatility and stochasticity into its experimental design. However, the depth of analysis concerning the effects of these variables on model fit appears shallow. A looming concern is whether the superior performance of the expectation-weighted Kalman Filter model might be a natural outcome of the experimental design. While the non-significant difference between eKF and eRL for the high stochasticity condition somewhat alleviates this concern, it raises another query: Would a more granular analysis of volatility and stochasticity effects reveal fine-grained model fit patterns?

      - Rating instruction: According to Fig. 1A, participants were prompted to rate their responses to the question, "How much pain DID you just feel?" and to specify their confidence level regarding their pain. It is difficult for me to understand the meaning of confidence in this context, given that they were asked to report their *subjective* feelings. It might have been better to query participants about perceived stimulus intensity levels. This perspective is seemingly echoed in lines 100-101, "the primary aim of the experiment was to determine whether the expectations participants hold about the sequence inform their perceptual beliefs about the intensity of the stimuli."

      - Relevance to clinical pain: While the authors underscore the relevance of their findings to chronic pain, they did not include data pertaining to clinical pain. Notably, their initial preprint seemed to encompass data from a clinical sample (https://www.medrxiv.org/content/10.1101/2023.03.23.23287656v1), which, for reasons unexplained, has been omitted in the current version. Clarification on this discrepancy would be instrumental in discerning the true relevance of the study's findings to clinical pain scenarios.

      - Paper organization: The paper's organization appears a little bit weird, possibly due to the removal of significant content from their initial preprint. Sections 2.1-2.2 and 2.4 seem more suitable for the Methods section, while 2.3 and 2.4.1 are the only parts that present results. In addition, enhancing clarity through graphical diagrams, especially for the experimental design and computational models, would be quite beneficial. A reference point could be Fig. 1 and Fig. 5 from Jepma et al. (2018), which similarly explored RL and KF models.

    1. Reviewer #1 (Public Review):

      Summary:

      In the current manuscript, the authors find distinct roles for the calcium sensors Syt7 and Doc2alpha in the regulation of asynchronous release and calcium-dependent synaptic vesicle docking in hippocampal neurons. The authors' data indicate that Doc2 functions in activating a component of asynchronous release beginning with the initial stimulus, while Syt7 does not appear to have a role at this early stage. A role for Syt7 in supporting both synchronous and asynchronous release appears during stimulation trains, where Syt7 is proposed to promote synaptic vesicle docking or capture during stimulation. Doc2 mutants show facilitation initially during a train and display higher levels of synchronous release initially, before reaching a similar plateau to controls later in the train. The authors contribute the increased synchronous release in Doc2 mutants to Syt1 having access to more SVs that can fuse synchronously. In contrast, Syt7 mutants show depression during a train, and continue to decline during stimulation. The authors contribute this to a role for Syt7 in promoting calcium-dependent SV docking and capture that feeds SVs to both synchronous and asynchronous fusion pathways. Importantly, phenotypes of a double Doc2/Syt7 mutant collapse onto the Doc2 phenotype, suggesting the two proteins are not additive in their role in supporting distinct aspects of SV release. Rapid freeze EM after stimulation provides support for a role for Syt7 in SV docking/capture at release sites, as they display less docked SVs after stimulation. In the case of Doc2, EM reveals fewer SVs fusion pits later during a stimulation, consistent with fewer asynchronous fusion events. The authors also provide modeling that supports aspects of their conclusions from the experimental data. I cannot evaluate the modeling data or the specific experimental subtleties of the GluSnFR quantification approach, as these are outside of my reviewer expertise.

      Strengths:

      The use of multiple approaches (optical imaging, physiology, rapid freeze EM, modeling, double mutant analysis) provides compelling support for the distinct roles of the two proteins in regulating SV release.

      Weaknesses:

      Some of the phenotypes for both Doc2 and Syt7 mutants have been reported in the authors' prior publications. It is not clear how well the GluSnFR approach is for accurately separating synchronous versus asynchronous release kinetics. The authors also tend to overstate the significance of the two proteins for asynchronous release in general, as a significant fraction of this release component is still intact in the double mutant, indicating these two proteins are only part of the asynchronous release mechanism.

    1. Reviewer #1 (Public Review):

      The research addresses a key problem in life sciences: While there are millions of commercially available antibodies to human proteins, researchers often find that the reagents do not perform in the assays they are specified for. The consequence is wasted time and research funding, and the publication of misleading results.

      Manufacturers' catalogues often contain images of western blots. Researchers are likely to select antibodies that stain a single band at the position expected from the mass of the intended target. However, the good results shown in catalogues are often not reproduced when researchers use the antibody in their own laboratories. A single band is also weak evidence since many proteins have similar mass and because assessment of mass by WB is at best approximate. In addition, results obtained by WB may not predict performance in applications where the antibody is used to recognize folded proteins. Examples include immunoprecipitation (IP) of native proteins in cell lysates and staining of viable or formalin-fixed/permeablized cells for flow cytometry or immunofluorescence microscopy (IF).

      The authors of this manuscript are from the Canadian, public interest open-science company YCharos. The company webpage (ycharos.com) explains that they have partnered with many leading manufacturers of research antibodies and that their mission is to characterize commercially available antibody reagents for every human protein.

      The authors have developed a standardized pipeline where antibodies are used in WB, IP of native proteins from cell lysates (WB readout) and IF (staining of cell lines that have been fixed with paraformaldehyde and permeabilized with Triton x100). A key component is the use of knockout cell lines as negative controls in WB and IF. Eight cell lines were selected as positive controls on the basis of mRNA expression data that are publicly available in the Expression 22Q1 database.

      Reports for antibodies to each protein are made available online at https://ZENODO.org/communities/ycharos/ as images of western blots, and immunofluorescence staining. In addition, reports for each target are available at https://ycharos.com/data/ .

      MANUSCRIPT:<br /> The manuscript describes validation criteria and results obtained with 614 commercially available antibodies to 65 proteins relevant for neuroscience A major achievement is the identification of successful renewable antibodies for 50/56 (77%) proteins in WB, 49/65 (75%) for IP and (54%) for IF. There can be little doubt that the approach represents a gold standard in antibody validation. The manuscript therefore represents a guide to a very valuable resource that should be of considerable interest to the scientific community.

      While the results are convincing, they could be more accessible. In the current format, researchers have to download reports for each target and look through all images to identify the most useful antibodies from the images. The reports I reviewed did not draw conclusions on performance. A searchable database that returns validated antibodies for each application seems necessary.

      It is worth noting that 95% of the tested antibodies were specified by the manufacturer for use in WB. This supports the view that manufacturers use WB as a first-pass test (Nat Methods. 2017 Feb 28;14(3):215) and that most commercial antibodies are developed to recognize epitopes that are exposed in unfolded proteins. Important exceptions are those used for ELISA or staining of viable cells for flow cytometry. 44% of antibodies specified for WB were classified as "successful" meaning a single band that was absent in the negative control (knockout/KO lysate). Another 35% detected the intended target but showed additional bands that were present also in the KO lysate. A key question is to what extent off-target binding was predictable from the WBs provided by the manufacturers. Thus, how often did the authors find multiple bands when the catalogue image showed a single band and vice versa?

      The authors correctly point out that manufacturers rarely test their reagents in IP. Thus, there is little information about antibodies capable of binding folded proteins. It is encouraging that as many as 37% of those not specified for IP were able to enrich their targets from cell lysates. Yet it is important to explain that a test that involves readout by WB provides information about on-target binding only. Cross-reactive proteins will generally not be detected when blots are stained with an antibody reactive with a different epitope than the one used for IP. Possible solutions to overcome this limitation such as the use of mass spectrometry as readout should be discussed (Nature Methods volume 12, pages 725-731 (2015).

      Performance in immunofluorescence microscopy was performed on cells that were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X100. It seems reasonable to assume that this treatment mainly yields folded proteins wherein some epitopes are masked due to cross-linking. The expectation is therefore that results from IP are more predictive for on-target binding in IF than are WB results (Nature Methods volume 12, pages725-731 (2015). It is therefore surprising that IP and WB were found to have similar predictive value for performance in IF (supplemental Fig. 3). It would be useful to know if failure in IF was defined as lack of signal, lack of specificity (i.e. off-target binding) or both. Again, it is important to note the IP/western protocol used here does not test for specificity.

      The authors report that recombinant antibodies perform better than standard monoclonals/mAbs or polyclonal antibodies. Again, a key question is to what extent this was predictable from the validation data provided by the manufacturers. It seems possible that the recombinant antibodies submitted by the manufacturers had undergone more extensive validation than standard mAbs and polyclonals.

      Overall, the manuscript describes a landmark effort for systematic validation of research antibodies. The results are of great importance for the very large number of researchers who use antibodies in their research. The main limitations are the high cost and low throughput. While thorough testing of 614 antibodies is impressive and important, the feasibility of testing hundreds of thousands of antibodies on the market should be discussed in more detail.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The findings in this manuscript are important in the gene editing in human-derived hematopoietic stem and progenitor cells. By optimizing the delivery tool, adding DNA-PK inhibitor and including spacer-breaking silent mutations, the editing efficiency is significantly increased, and the heterozygosity could be tuned. The editing is even across the hematopoietic hierarchy.

      Strengths:<br /> The precise gene editing is important in gene therapy in vitro and in vivo. The manuscript provides solid evidence showing the efficacy and uniqueness of their gene editing approach.

      Weaknesses:<br /> There are several extended and unique points shown in this paper but in a specific cell population.

    1. Reviewer #1 (Public Review):

      Summary: The manuscript by Heyndrickx et al describes protein crystal formation and function that bears similarity to Charcot-Leyden crystals made of galectin 10, found in humans under similar conditions. Therefore, the authors set out to investigate CLP crystal formation and their immunological effects in the lung. The authors reveal the crystal structure of both Ym1 and Ym2 and show that Ym1 crystals trigger innate immunity, activated dendritic cells in the lymph node, enhancing antigen uptake and migration to the lung, ultimately leading to induction of type 2 immunity.

      Strengths:<br /> We know a lot about expression levels of CLPs in various settings in the mouse but still know very little about the functions of these proteins, especially in light of their ability to form crystal structures. As such data presented in this paper is a major advance to the field.

      Resolving the crystal structure of Ym2 and the comparison between native and recombinant CLP crystals is a strength of this manuscript that will be a very powerful tool for further evaluation and understanding of receptor, binding partner studies including the ability to aid mutant protein generation.

      The ability to recombinantly generate CLP crystals and study their function in vivo and ex vivo has provided a robust dataset whereby CLPs can activate innate immune responses, aid activation and trafficking of antigen presenting cells from the lymph node to the lung and further enhances type 2 immunity. By demonstrating these effects the authors directly address the aims for the study. A key point of this study is the generation of a model in which crystal formation/function an important feature of human eosinophilic diseases, can be studied utilising mouse models. Excitingly, using crystal structures combined with understanding the biochemistry of these proteins will provide a potential avenue whereby inhibitors could be used to dissolve or prevent crystal formation in vivo.

      The data presented flows logically and formulates a well constructed overall picture of exactly what CLP crystals could be doing in an inflammatory setting in vivo. This leaves open a clear and exciting future avenue (currently beyond the scope of this work) for determining whether targeting crystal formation in vivo could limit pathology.

      Weaknesses:<br /> Although resolving the crystal structure of Ym2 in particular is a strength of the authors work, the weaknesses are that further work or even discussion of Ym2 versus Ym1 has not been directly demonstrated. The authors suggest Ym2 crystals will likely function the same as Ym1, but there is insufficient discussion (or data) beyond sequence similarity as to why this is the case. If Ym1 and Ym2 crystals function the same way, from an evolutionary point, why do mice express two very similar proteins that are expressed under similar conditions that can both crystalise and as the authors suggest act in a similar way. Some discussion around these points would add further value.

      Additionally, the crystal structure for Ym1 has been previously resolved (Tsai et al 2004, PMID 15522777) and it is unclear whether the data from the authors represents an advance in the 3D structure from what is previously known.

      Whilst also generating a model to understand charcot-leyden crystals (CLCs), the authors fail to discuss whether crystal shape may be an important feature of crystal function. CLCs are typically needle like, and previous publications have shown using histology and TEM that Ym1 crystals are also needle like. However, the crystals presented in this paper show only formation of plate like structures. It is unclear whether these differences represent different methodologies (ie histology is 2D slides), or differences in CLP crystals that are intracellular versus extracellular. These findings highlight a key question over whether crystal shape could be important for function and has not been addressed by the authors.

      Ym1/Ym2 crystals are often observed in conditions where strong eosinophilic inflammation is present. However, soluble Ym1 delivery in naïve mice shows crystal formation in the absence of a strong immune response. There is no clear discussion as to the conditions in which crystal formation occurs in vivo and how results presented in the paper in terms of priming or exacerbating an immune responses align with what is known about situations where Ym1 and Ym2 crystals have been observed.

    1. Reviewer #1 (Public Review):

      Summary:

      Otarigho et al. presented a solid 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.

      Strengths:

      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. Most 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.

      Weaknesses:

      1. The authors identified NPR-15 and ASJ neurons that are involved in both molecular and behavioral responses to pathogen attack. This finding, by itself, is significant. However, how the NPR-15/ASJ circuit regulates the interplay between the two defense strategies was not explored. Therefore, emphasizing the interplay in the title and the abstract is misleading.

      2. Although the discovery of a single GPCR regulating both immunity and avoidance behavior is significant and novel, NPR-15 is not the first GPCR identified with these functions. Previously, the same lab reported that the GPCR OCTR-1 also regulates immunity and avoidance behavior through ASH and ASI neurons respectively (PMID: 29117551). This point was not mentioned in the current manuscript.

      3. The authors discovered that NPR-15 regulates avoidance behavior via the TRPM gene, GON-2. Only two factors (GON-2 and GTL-2) were examined in this study, and GON-2 happens to function through the intestine. It is possible that NPR-15 may broadly regulate multiple effectors in multiple tissues. Confining the regulation to the amphid sensory neuron-intestinal axis, as stated in the title and elsewhere in the manuscript, is not accurate.

      4. The C. elegans nervous system is simple, and hermaphrodites only have 302 neurons. Individual neurons possessing multiple regulatory functions is expected. Whether this is conserved in mammals and other vertebrates is unknown, because in higher animals, neurons and neuronal circuits could be more specialized.

      5. A key question, that is, why would NPR-15 suppress immunity (which is bad for defense) but enhance avoidance behavior (which is good for defense), is not addressed or explained. This could be due to temporal regulation, for example, upon pathogen exposure, NPR-15 could regulate behavior to avoid the pathogen, but after infection, NPR-15 could suppress excessive immune responses or quench the responses for the resolution of infection.

      6. The discussion appears timid in scope and contains some repetitive statements. Point 5 can be addressed in the Discussion.

      Overall, the authors presented an impactful study that identified specific molecules and neuronal cells that regulate both molecular and behavioral immune responses to pathogen attack. Most conclusions are supported by solid evidence. However, some statements are overreaching, for example, regulation of the interplay between molecular and behavioral immune responses was emphasized but not explored. Nonetheless, this study reported a significant and novel discovery and has laid a foundation for investigating such an interplay in the future.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The author uses CHAT GPT in the assessment linguistic characteristics of peer reviews published from August 2022 to February 2023 in Nature Communications in neuroscience field. The author analysed over 500 reviews, which greatly varied in terms of author characteristics, peer review length, subfield, number of reviews and writing style. Chat GPT analysed reviews and gave the scores regarding the language characteristics related to sentiment score and politeness.

      Strengths:<br /> The innovative method is the biggest strength of this article. Moreover, the method can be implemented across fields and disciplines. I myself would like to see this method implemented in a grander scale. The author invested a lot of effort in data collection and I especially commend the that the chat GPT assessed the reviews twice, to ensure greater objectivity.

      Weaknesses:<br /> The weaknesses listed in my Public Review of the previous version have been addressed in this revised version.

    1. Reviewer #1 (Public Review):

      Summary:

      This important manuscript investigates a subpopulation of glutamatergic neurons in the suprammamillary nucleus that projects to the pre-optic hypothalamus area (SuM-VGLUT2+::POA). First, they define the neural circuitry of these neurons, which make contact with many stress/threat-associated brain regions. Then they employ fibre photometry to measure the activity of these neurons during various threatening tasks and find the responses correlate well with threat stimuli. Finally, they stimulate these neurons and find multiple lines of evidence that mice find this aversive and will act to avoid receiving this stimulation. In sum, they provide compelling evidence that this neuronal population represents a new node in stress response circuitry that allows the animal to produce flexible behaviours in response to stress, which will be of interest to neuroscientists across several sub-fields.

      Strengths:

      Overall I found a lot to like about this manuscript and very little to dislike. It is very novel and interesting, and the evidence given to support the conclusions is compelling.

      Specific strengths:

      • The topic is highly novel.<br /> • The manuscript follows a logical structure and neatly moves through the central story. I found myself quite convinced of the evidence given for the conclusions that were made and many potential alternate interpretations are well-controlled for.<br /> • The manuscript employs an array of different tasks to provide converging evidence for their conclusions.<br /> • The authors provide excellent evidence of the specificity of the function of this neuronal population, both from anatomical studies and from behavioural studies (e.g. demonstrating that activity of gabaergic neurons in the same region does not correlate with behaviours in the same way).<br /> • The study is well-powered (sample sizes are good) and the effects are convincing.

      Weaknesses:

      • Despite the manuscript being generally well-written and easy to follow, there are several grammatical errors throughout that need to be addressed.<br /> • Only p values are given in the text to support statistical differences. This is not sufficient. F and/or t values should be given as well. Moreover, the fibre photometry data does not appear to have any statistical analyses reported - only confidence intervals represented in the figures without any mention of whether the null hypothesis that the elevations in activity observed are different from the baseline. This is particularly important where there is ambiguity, such as in Figure 3K, where the spontaneous activity of the animal appears to correlate with a spike in activity but the text mentions that there is no such difference. Without statistics, this is difficult to judge.<br /> • The use of photostimulation only is unfortunate, it would have been really nice to see some inactivation of these neurons as well. This is because of the well-documented issues with being able to determine whether photostimulation is occurring in a physiological manner, and therefore makes certain data difficult to interpret. For instance, with regards to the 'active coping' behaviours - is this really the correct characterisation of what's going on? I wonder if the mice simply had developed immobile responding as a coping strategy but when they experience stimulation of these neurons that they find aversive, immobility is not sufficient to deal with the summative effects of the aversion from the swimming task as well as from the neuronal activation? An inactivation study would be more convincing.<br /> • Nose poke is only nominally instrumental as it cannot be shown to have a unique relationship with the outcome that is independent of the stimuli-outcome relationships (in the same way that a lever press can, for example). Moreover, there is nothing here to show that the behaviours are goal-directed.

    1. Reviewer #1 (Public Review):

      Sun and colleagues outline structural and mechanistic studies of the bacterial adhesin PrgB, an atypical microbial cell surface-anchored polypeptide that binds DNA. The manuscript includes a crystal structure of the Ig-like domains of PrgB, cryo-EM structures of the majority of the intact polypeptide in DNA-bound and free forms, and an assessment of the phenotypes of E. faecalis strains expressing various PrgB mutants. Generally, the study has been conducted with a good level of rigor, and there is consistency in the findings. Initial concerns about inferences initially made from low-resolution Cryo-EM structures have been addressed experimentally and the manuscript correspondingly updated.

    1. Reviewer #1 (Public Review):

      Bacteria can adapt to extremely diverse environments via extensive gene reprogramming at transcriptional and post-transcriptional levels. Small RNAs are key regulators of gene expression that participate in this adaptive response in bacteria, and often act as post-transcriptional regulators via pairing to multiple mRNA-targets.

      In this study, Melamed et al. identify four E. coli small RNAs whose expression is dependent on sigma 28 (FliA), involved in the regulation of flagellar gene expression. Even though they are all under the control of FliA, expression of these 4 sRNAs peaks under slightly different growth conditions and each has different effects on flagella synthesis/number and motility. Combining RILseq data, structural probing, northern-blots and reporter assays, the authors show that 3 of these sRNAs control fliC expression (negatively for FliX, positively for MotR and UhpU) and two of them regulate r-protein genes from the S10 operon (again positively for MotR, and negatively for FliX). UhpU also directly represses synthesis of the LrhA transcriptional regulator, that in turn regulates flhDC (at the top of flagella regulation cascade). Based on RILseq data, the fourth sRNA (FlgO) has very few targets and may act via a mechanism other than base-pairing.

      As r-protein S10 is also implicated in anti-termination via the NusB-S10 complex, the authors further hypothesize that the up-regulation of S10 gene expression by MotR promotes expression of the long flagellar operons through anti-termination. Consistent with this possible connection between ribosome and flagella synthesis, they show that MotR overexpression leads to an increase in flagella number and in the mRNA levels of two long flagellar operons, and that both effects are dependent on the NusB protein. Lastly, they provide data supporting a more general activating and repressing role for MotR and FliX, respectively, in flagellar genes expression and motility, via a still unclear detailed mechanism.

      This study brings a lot of new information on the regulation of flagellar genes, from the identification of novel sigma 28-dependent sRNAs to their effects on flagella production and motility. It represents a considerable amount of work; the experimental data are clear and solid and support the conclusions of the paper. Even though mechanistic details underlying the observed regulations by MotR or FliX sRNAs are lacking, the effect of these sRNAs on fliC, several rps/rpl genes, and flagellar genes and motility is convincing.<br /> The connection between r-protein genes regulation and flagellar operons is exciting, and so is the general effect of pMotR or pFliX on the expression of multiple middle and late flagellar genes.

    1. Reviewer #1 (Public Review):

      Lim W et al. investigated the mechanisms underlying doxorubicin resistance in triple negative breast cancer cells (TNBC). They use a new multifluidic cell culture chamber to grow MB-231 TNBC cells in the presence of doxorubicin and identify a cell population of large, resistant MB-231 cells they term L-DOXR cells. These cells maintain resistance when grown as a xenograft model, and patient tissues also display evidence for having cells with large nuclei and extra genomic content. RNA-seq analysis comparing L-DOXR cells to WT MB-231 cells revealed upregulation of NUPR1. Inhibition or knockdown of NUPR1 resulted in increased sensitivity to doxorubicin. NUPR1 expression was determined to be regulated via HDAC11 via promoter acetylation. The data presented could be used as a platform to understand resistance mechanisms to a variety of cancer therapeutics.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, the authors used machine learning algorithm to analyze published exosome datasets to find biomarkers to differentiate exosomes of different origin.

      Strengths:

      The performance of the algorithm are generally of good quality.

      Weaknesses:

      The source datasets are heterogeneous as described in Figure 1 and Figure 2, or Line 72-75; and therefore questionable.

    1. Reviewer #1 (Public Review):

      The manuscript investigates how humans store temporal sequences of tones in working memory. The authors mainly focus on a theory named "Language of thought" (LoT). Here the structure of a stimulus sequence can be stored in a tree structure that integrates the dependencies of a stimulus stored in working memory. To investigate the LoT hypothesis, participants listened to multiple stimulus sequences that varied in complexity (e.g., alternating tones vs. nearly random sequence). Simultaneously, the authors collected fMRI or MEG data to investigate the neuronal correlates of LoT complexity in working memory. Critical analysis was based on a deviant tone that violated the stored sequence structure. Deviant detection behavior and a bracketing task allowed a behavioral analysis.

      Results showed accurate bracketing and fast/correct responses when LoT complexity is low. fMRI data showed that LoT complexity correlated with the activation of 14 clusters. MEG data showed that LoT complexity correlated mainly with activation from 100-200 ms after stimulus onset. These and other analyses presented in the manuscript lead the authors to conclude that such tone sequences are represented in human memory using LoT in contrast to alternative representations that rely on distinct memory slot representations.

      Strengths

      The study provides a concise and easily accessible introduction. The task and stimuli are well described and allow a good understanding of what participants experience while their brain activation is recorded. Results are extensive as they include multiple behavioral investigations and brain activation data from two different measurement modalities. The presentation of the behavioral results is intuitive. The analysis provided a direct comparison of the LoT with an alternative model based on estimating a transition-probability measure of surprise.

      For the fMRI data, the whole brain analysis was accompanied by detailed region of interest analyses, including time course analysis, for the activation clusters correlated with LoT complexity. In addition, the activation clusters have been set in relation (overlap and region of interest analyses) to a math and a language localizer. For the MEG data, the authors investigated the LoT complexity effect based on linear regression, including an analysis that also included transitional probabilities and multivariate decoding analysis. The discussion of the results focused on comparing the activation patterns of the task with the localizer tasks. Overall, the authors have provided considerable new data in multiple modalities on a well-designed experiment investigating how humans represent sequences in auditory working memory.

      Weaknesses

      The primary issue of the manuscript is the missing formal description of the LoT model and alternatives, inconsistencies in the model comparisons, and no clear argumentation that would allow the reader to understand the selection of the alternative model. Similar to a recent paper by similar authors (Planton et al., 2021 PLOS Computational Biology), an explicit model comparison analysis would allow a much stronger conclusion. Also, these analyses would provide a more extensive evidence base for the favored LoT model. Needed would be a clear argumentation for why the transitional probabilities were identified as the most optimal alternative model for a critical test. A clear description of the models (e.g., how many free parameters) and a description of the simulation procedure (e.g., are they trained, etc.) Here it would be strongly advised to provide the scripts that allow others to reproduce the simulations.

      Furthermore, the manuscript needs a clear motivation for the type of sequences and some methodological decisions. Central here is the quadratic trend selectively used for the fMRI analysis but not for the other datasets. Also, the description of the linear mixed models is missing (e.g., the random effect structure, e.g., see Bates, D., Kliegl, R., Vasishth, S., & Baayen, H. (2015). Parsimonious mixed models. arXiv preprint arXiv:1506.04967.). Moreover, sample sizes have not been justified by a power analysis.

    1. Reviewer #1 (Public Review):

      The current manuscript by Liu et al entitled "Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma" reports the identification of a novel CRM1 inhibitor and shows its efficiency against extranodal natural killer/T cell lymphoma cells (ENKTL).

      This is a very timely and very original study with potential impact in a variety of pathologies not only in ENKTL. However, the main conclusions of the work are not supported by experimental evidence. The study claims that LFS-1107 reversibly inhibits the nuclear export receptor CRM1 but the authors only show that the compound binds to CRM1 and that the CRM1 substrate IκBα accumulates in the cell nucleus upon LFS-1107 treatment. The evidence is indirect and alternative scenarios are certainly possible. On the other hand, the manuscript is not always well-written and insufficiently referenced. The quality of the images is poor. The figure legends are incomplete. The nuclear translocation in figure 2G is not convincing. The western blot in figure 2G shows that LFS-1107 treatment induces IκBα expression, and both cytoplasmic and nuclear amounts increase in a dose-dependent manner. Together, these data do not support nuclear IκBα accumulation upon LFS-1107 treatment.

    1. Reviewer #1 (Public Review):

      This paper describes the neural activity, measured by intrinsic optical imaging in reach-to-grasp, and reach-only conditions in relation to the Intra-cortical micro stimulation maps. The paper mostly describes a relatively unique and potentially useful data set. However, in the current version, no real hypotheses about the organization of M1 and PMd are tested convincingly. For example, the claim of "clustered neural activity" is not tested against any quantifiable alternative hypothesis of non-clustered activity, and support for this idea is therefore incomplete.

      The combination of intrinsic optical imaging and intra-cortical micro-stimulation of the motor system of two macaque monkeys promised to be a unique and highly interesting dataset. The experiments are carefully conducted. In the analysis and interpretation of the results, however, the paper was disappointing to me. The two main weaknesses in my mind were:

      a) The alternative hypotheses depicted in Figure 1B are not subjected to any quantifiable test. When is an activity considered to be clustered and when is it distributed? The fact that the observed actions only activate a small portion of the forelimb area (Figure 5G, H) is utterly unconvincing, as this analysis is highly threshold-dependent. Furthermore, it could be the case that the non-activated regions simply do not give a good intrinsic signal, as they are close to microvasculature (something that you actually seem to argue in Figure 6b). Until the authors can show that the other parts of the forelimb area are clearly activated for other forelimb actions (as you suggest on line 625), I believe the claim of cluster neural activity stands unsupported.

      b) The most interesting part of the study (which cannot be easily replicated with human fMRI studies) is the correspondence between the evoked activity and intra-cortical stimulation maps. However, this is impeded by the subjective and low-dimensional description of the evoked movement during stimulation (mainly classifying the moving body part), and the relatively low-dimensional nature (4 conditions) of the evoked activity.

      c) Many details about the statistical analysis remain unclear and seem not well motivated.

    1. Reviewer #1 (Public Review):

      This paper evaluates the effect of knocking out CST7(Cystatin 5) on the APPNL-G-F Alzheimer's disease mouse model. They found sexually dimorphic outcomes, with differential transcriptional responses, increased phagocytosis (but interestingly a higher plaque burden) in females and suppressed inflammatory microglial activation in males (but interestingly no change in plaque burden). This study offers new insight into the functional role of CST7 that is upregulated in a subset of disease- associated microglia in AD models and human brain. Despite the discovery of disease-associated microglia several years ago, there has been little effort in understanding the function of the different genes that make up this profile, making this paper especially timely. Overall, the experiments are well-controlled and the data support the main conclusions and the manuscript could be strengthened by addressing the below comments and clarifying questions that could impact the interpretation of their data/ findings.

      1. In the first section discussing CST7 expression levels in AD models, it would be good to involve a discussion of levels of CST7 change in human AD samples. There are sufficient available datasets to look at this, and it would help us understand how comparable the animal models are to human patients. For example, while in mice CST7 is highly enriched in microglia/macrophages, in human datasets it seems like it is not quite so specific to microglia - it is equally expressed in endothelial cells. This might have a significant impact on the interpretation of the data, and it would be good to introduce and assess the findings in mice through the human subjects lens. There is a discussion of the human data in the discussion section, but it would be more appropriately assessed in the same way as the mouse data and comparatively presented in the results section. The authors could also include the data from Gerrits et al. 2021 in their first figure.<br /> 2. The differential RNAseq data is perhaps one of the most striking results of this paper; however it is difficult to see exactly how similar the male v female APPNL-G-F profiles are, in addition to the genes shared or not between the KO condition. Venn diagrams, in addition to statistical tests, would enhance this part of the paper and add more clarity.<br /> 3. A major argument in the paper is a continuation of Sala-Frigiero 2019 which says that the female phenotype is an acceleration of the male phenotype. Does this mean that if males were assessed at later timepoints, they would be more similar to the females? Or are there intrinsic differences that never resolve? It would be helpful to see a later timepoint for males to get at the difference between these two options<br /> 4. If the central argument is that CST7 in females decreases phagocytosis and in males increases microglia activation, are there changes in amyloid plaque burden or structure in the APPNL-G-F /CST 7 KO mice compared to APPNL-G-F/CST7 WT that reflect these changes? Please address. If not, how does this affect the functional interpretation of differential expression observed in phagocytic/reactive microglia genes? Pieces of this are discussed but it could be clearer<br /> 5. It is confusing that increased phagocytosis in the APPNL-G-F/CST7 KO females leads to greater plaque burden, considering proteolysis is not affected. What might explain this observation? Additionally, it is interesting that suppression of microglial activation doesn't lead to an increase in plaques in the male APPNL-G-F/CST7 KO mice. How does the profile of phagocytic microglia in the male APPNL-G-F/CST7 KO mice differ from the APPNL-G-F males?<br /> 6. Seems that the authors have potentially discovered an unusual mechanism for how CST7 could regulate cell autonomous function without impacting its canonical protease target. The authors deal with this extensively in the discussion but an ELISA or ICC to localize CST7 to microglia in vitro or in vitro would help address this point.<br /> 7. The authors focus on plaques in their final figure, however dysregulated microglial phagocytosis could impact many other aspects of brain health. Simple immunohistochemistry for synapses and myelin/oligodendrocytes (especially given the results of the in vitro phagocytosis assay) could provide more insight here.

    1. Reviewer #1 (Public Review):

      The CFTR ion channel belongs to the family of ABC transporters, alternating between inward-facing (IF) and outward-facing (OF) conformations driven by binding and hydrolysis of ATP. ABC transporters are involved in a wide variety of physiologically essential transport processes. In contrast to all other ABC transporters, the OF conformation of CFTR includes an anion-conducting transmembrane pore, which has enabled investigators to use single-channel patch clamp electrophysiology to characterize the energetics of most of the relevant transitions that can be observed during a gating cycle. A transition that had remained elusive to quantify is the non-hydrolytic closing rate in which the channel switches back to an IF conformation even though both nucleotide-binding domain sites are occupied by non-hydrolyzed ATP molecules. The reason for this is that the rate of closure due to ATP hydrolysis occurs much faster, such that the non-hydrolytic closing rate cannot be quantified in WT channel recordings. Further, channels with mutations that are expected to disrupt ATP hydrolysis exhibit high variability between mutants in the non-hydrolytic closing rates, precluding an extrapolation for this rate onto the WT channel. It is presently unclear whether the large spread in the rates is caused by distinct degrees of remnant hydrolytic activity in each of the mutant channels. Regardless of this uncertainty, several of these mutations have been successfully employed in structural studies to stabilize the channel in an OF conformation with two ATP molecules bound. In the present manuscript, Márton A. Simon and collaborators use patch-clamp electrophysiology to measure the rates of non-hydrolytic channel closure in human CFTR channels containing single and double mutations expected to disrupt ATP hydrolysis. First, they find that the E1371Q but not the E1371S mutation significantly stabilizes the OF state and slows channel closure in the human but not the zebrafish channel. Looking at the structures of both human and zebrafish E1371Q mutant channels, a non-native hydrogen bond is identified between the side-chain of E1371Q and the main chain at position G576 that is observed only in the human structure and would be expected to stabilize the OF state. Double mutant cycle analysis is then utilized to compare the effect of removal of either of the hydrogen-bonding partners in the human CFTR, and found to be consistent with a large energy of interaction between the two sites that is interpreted to occur selectively in the OF state. Notably, none of these perturbations altered the intra-burst activity of the CFTR, indicating that those closures do not involve major changes in the NBD. The rates of closure for other mutants are then investigated, and it is found that combining two hydrolysis-disrupting mutations that retain relatively fast closure rates does not slow closure any further, suggesting that their fast closure rates are not due to residual ATP hydrolytic activity. Further, this observation also suggests that these mutations do not affect the intrinsic non-hydrolytic closing rate, allowing their rates to be used as a measure of what occurs in WT channels. The experiments presented are high-quality, the conclusions are well supported by the data and the findings clarify a series of questions that had remained in the field, in addition to finding and precisely quantitating the role of a hydrogen bond in stabilizing a conformation state of this channel. The results could have implications for other members of the ABC family that are harder to study because they do not produce ionic currents. Some methodological details could be explained better, such as the voltage at which each of the recordings was performed, and how data was normalized, which is presently unclear. Additional testing of the hypothesis could have been carried out through double mutant cycle analysis with E1371Q + G576Δ in the zebrafish receptor or the other non-hydrolytic mutants.

    1. Reviewer #1 (Public Review):

      This paper presents an interesting data set from historic Western Eurasia and North Africa. Overall, I commend the authors for presenting a comprehensive paper that focuses the data analysis of a large project on the major points, and that is easy to follow and well-written. Thus, I have no major comments on how the data was generated, or is presented. Paradoxically, historical periods are undersampled for ancient DNA, and so I think this data will be useful. The presentation is clever in that it focuses on a few interesting cases that highlight the breadth of the data.

      The analysis is likewise innovative, with a focus on detecting "outliers" that are atypical for the genetic context where they were found. This is mainly achieved by using PCA and qpAdm, established tools, in a novel way. Here I do have some concerns about technical aspects, where I think some additional work could greatly strengthen the major claims made, and lay out if and how the analysis framework presented here could be applied in other work.

      ## clustering analysis<br /> I have trouble following what exactly is going on here (particularly since the cited Fernandes et al. paper is also very ambiguous about what exactly is done, and doesn't provide a validation of this method). My understanding is the following: the goal is to test whether a pair of individuals (lets call them I1 and I2) are indistinguishable from each other, when we compare them to a set of reference populations. Formally, this is done by testing whether all statistics of the form F4(Ref_i, Ref_j; I1, I2) = 0, i.e. the difference between I1 and I2 is orthogonal to the space of reference populations, or that you test whether I1 and I2 project to the same point in the space of reference populations (which should be a subset of the PCA-space). Is this true? If so, I think it could be very helpful if you added a technical description of what precisely is done, and some validation on how well this framework works.

      An independent concern is the transformation from p-values to distances. I am in particular worried about i) biases due to potentially different numbers of SNPs in different samples and ii) whether the resulting matrix is actually a sensible distance matrix (e.g. additive and satisfies the triangle inequality). To me, a summary that doesn't depend on data quality, like the F2-distance in the reference space (i.e. the sum of all F4-statistics, or an orthogonalized version thereof) would be easier to interpret. At the very least, it would be nice to show some intermediate results of this clustering step on at least a subset of the data, so that the reader can verify that the qpWave-statistics and their resulting p-values make sense.

      The methodological concerns lead me to some questions about the data analysis. For example, in Fig2, Supp 2, very commonly outliers lie right on top of a projected cluster. To my understanding, apart from using a different reference set, the approach using qpWave is equivalent to using a PCA-based clustering and so I would expect very high concordance between the approaches. One possibility could be that the differences are only visible on higher PCs, but since that data is not displayed, the reader is left wondering. I think it would be very helpful to present a more detailed analysis for some of these "surprising" clustering where the PCA disagrees with the clustering so that suspicions that e.g. low-coverage samples might be separated out more often could be laid to rest.

      One way the presentation could be improved would be to be more consistent in what a suitable reference data set is. The PCAs (Fig2, S1 and S2, and Fig6) argue that it makes most sense to present ancient data relative to present-day genetic variation, but the qpWave and qpAdm analysis compare the historic data to that of older populations. Granted, this is a common issue with ancient DNA papers, but the advantage of using a consistent reference data set is that the analyses become directly comparable, and the reader wouldn't have to wonder whether any discrepancies in the two ways of presenting the data are just due to the reference set.

      ## PCA over time<br /> It is a very interesting observation that the Fst-vs distance curve does not appear to change after the bronze age. However, I wonder if the comparison of the PCA to the projection could be solidified. In particular, it is not obvious to me how to compare Fig 6 B and C, since the data in C is projected onto that in Fig B, and so we are viewing the historic samples in the context of the present-day ones. Thus, to me, this suggests that ancient samples are most closely related to the folks that contribute to present-day people that roughly live in the same geographic location, at least for the middle east, north Africa and the Baltics, the three regions where the projections are well resolved.<br /> Ideally, it would be nice to have independent PCAs (something F-stats based, or using probabilistic PCA or some other framework that allows for missingness). Alternatively, it could be helpful to quantify the similarity and projection error.

    1. Reviewer #1 (Public Review):

      The authors used MD simulations to investigate the role of N-terminal myristoylation and the presence of two SH domains on the allosteric regulation of c-Abl kinase. Standard established MD simulation methods and analyses were applied, including the force distribution analysis (FDA) method developed by Grater et al. some time ago.

      The system is large and the conformational changes are complicated. In light of this, and aggravated by the fact that direct comparison with - and critical testing against - experimental data is not possible in the present case, I consider the overall simulation times to be rather short (several repeats, but only 500 ns). So there might be statistical convergence issues. Especially also because at least some of the starting structures were generated from available experimental structures after some modifications/modelling, and they might thus be out of equilibrium and need some time to fully relax during the MD simulations.

      Unfortunately, I cannot find any convergence tests concerning the length of the simulations, which are usually considered to be standard analyses (Appendix Fig. 5 shows the effect of different thermostats and capping of the peptide chain, but no tests concerning simulation time). This could be critical in the present case, where the authors acknowledge themselves (e.g., on p. 4) that there are only subtle differences between the different simulation systems and the variations within a given system are larger than the relevant (putative) differences between systems (Fig. 1 C, D, E).

      Issues with statistical convergence are expected not only for the standard MD simulations but also for the umbrella sampling simulations, as 50 ns sampling per window is nowadays not considered state of the art and is likely insufficient for quantitative binding free energy calculation, especially for membranes (see, e.g., DOI 10.1021/ct200316w). However, worrying about this latter aspect might neither be useful nor needed, because in our view the statement that myristoyl groups can bind to the membrane and that they can compete with binding in the hydrophobic protein pocket can hardly be considered a surprise and would not have required any simulation at all in my view because the experimental K_D values are available (Table 1). The very unfavourable K_d values for unbinding of Myr from both the hydrophobic protein pocket as well as from the membrane in fact show that this is not how it is expected to work in reality. The fully solvated state will be avoided due to its high free energy. Instead, isn't the myristoyl expected to directly transition from the pocket into the membrane, after membrane binding of the kinase in a proper orientation?

      Concerning the metadynamics simulations, these are usually done to obtain a free energy landscape. Why was this not attempted here? In the present case, the authors seemed to have used metadynamics only for generating starting structures, with different degrees of helicity of the alpha_I part, for subsequent standard MD simulations. Not surprisingly, nothing much happened during the latter, and conformers with kinked/partially unfolded alpha_I as well as conformers with straight alpha_I were both found to be "stable", at least on the short simulation time scale. It could also not be expected that the SH domain would spontaneously detach in response to helix straightening - again, this would require much longer simulation times than 500 ns. Nevertheless, alpha_I straightening might very well reduce the binding affinity towards SH - this can only be explicitly studied with free energy simulations, however.

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

      In this manuscript, Bilgic et al aim to identify the progenitor types (and their specific progeny) that underlie the expanded nature of gyrencephalic brains. To do this, they take a comparative scRNAseq (single cell transcriptomics) approach between neurodevelopment of the gyrencephalic ferret, and previously published primary human brain and organoid data.

      They first improve gene annotations of the ferret genome and then collect a time series of scRNAseq data of 6 stages of the developing ferret brain spanning both embryonic and post-natal development. Among the various cell types they identify are a small proportion of truncated radial glial cells (tRGs), a population known to be enriched in humans and macaques that emerges late in neurogenesis as the RGC scaffold splits into an oRGC that contact the pial surface and a tRG that contacts the ventricular surface. They find that the tRGs consist of three distinct subpopulations two of which are committed to ependymal and astroglial fates.

      By integrating these data with publicly available data of developing human brains and human brain organoids they make some important observations. Human and ferret tRGs have very similar transcriptional states, suggesting that the human tRGs too give rise to ependymal and astroglial fates. They also find that the current culture conditions of human brain organoids seem to lack tRGs, something that will need to be addressed if they are to be used to study tRGs. While the primary human data set did contain tRGs, the stage or the region sampled were likely not appropriate, and therefore, the number of cells they could retrieve was low.

      The authors have spent considerable efforts in improving gene modeling of the ferret genome, which will be important for the field. They've generated valuable time series data for the developing ferret brain, and have proposed the lineal progeny for the tRGs in the human brain. Whether tRGs actually do give rise to the ependymal and astrogial fates needs to be validated in future studies.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The current study examines the necessity of estrogen receptor alpha (ESR1) in GABA neurons located in the anteroventral and preoptic periventricular nuclei and the medial preoptic nucleus of the hypothalamus. This brain area is implicated in regulating the pre-ovulatory LH surge in females, but the identity of the estrogen-sensitive neurons that are required remains unknown. The data indicate that approximately 70% knockdown of ESR1 in GABA neurons resulted in variable reproductive phenotypes. However, when the ESR1 knockdown also results in a decrease in kisspeptin expression by these cells, the females had disrupted LH surges, but no alterations in pulsatile LH release. These data support the hypothesis that kisspeptin cells in this region are critical for the pre-ovulatory LH surge in females.

      Strengths:<br /> The current study examined the efficacy of two guide RNAs to knockdown ESR1 in GABA neurons, resulting in an approximate 70% reduction in ESR1 in GABA neurons. The efficacy of this knockdown was confirmed in the brain via immunohistochemistry and the reproductive outcomes were analyzed several ways to account for differences in guide RNAs or the precise brain region with the ESR1 knockdown. The analysis was taken one step further by grouping mice based on kisspeptin expression following ESR1 knockdown and examining the reproductive phenotypes. Overall, the aims of the study were achieved, the methods were appropriate, and the data were analyzed extensively. This data supports the hypothesis that kisspeptin neurons in the anterior hypothalamus are critical for the preovulatory LH surge.

      Weaknesses: One minor weakness in this study is the conclusion that the guide RNAs didn't seem to have unique effects on GnRH cFos expression or the reproductive phenotypes. Though the data indicate a 60-70% knockdown for both gRNA2 and gRNA3, 3 of the 4 gRNA2 mice had no cFos expression in GnRH neurons during the time of the LH surge, whereas all mice receiving gRNA3 had at least some cFos/GnRH co-expression. In addition, when mice were re-categorized based on reduction (>75%) in kisspeptin expression, most of the mice in the unilateral or bilateral groups received gRNA2, whereas many of the mice that received gRNA3 were in the "normal" group with no disruption in kisspeptin expression. Thus, additional experiments with increased sample sizes are needed, even if the efficacy of the ESR1 knockdown was comparable before concluding these 2 gRNAs don't result in unique reproductive effects.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This work examined transcription factor Meis2 in the development of mouse and chick DRG neurons, using a combination of techniques, such as the generation of a new conditional mutant strain of Meis2, behavioral assays, in situ hybridization, transcriptomic study, immunohistochemistry, and electrophysiological (ex vivo skin-nerve preparation) recordings. The authors found that Meis2 was selectively expressed in A fiber LTMRs and that its disruption affects the A-LTMRs' end-organ innervation, transcriptome, electrophysiological properties, and light touch-sensation.

      Strengths:<br /> 1) The authors utilized a well-designed mouse genetics strategy to generate a mouse model where the Meis2 is selectively ablated from pre- and post-mitotic mouse DRG neurons. They used a combination of readouts, such as in situ hybridization, immunhistochemistry, transcriptomic analysis, skin-nerve preparation, electrophysiological recordings, and behavioral assays to determine the role of Meis2 in mouse DRG afferents.

      2) They observed a similar preferential expression of Meis2 in large-diameter DRG neurons during development in chicken, suggesting evolutionarily conserved functions of this transcription factor.

      3) Conducted severe behavioral assays to probe the reduction of light-touch sensitivity in mouse glabrous and hairy skin. Their behavioral findings support the idea that the function of Meis2 is essential for the development and/or maturation of LTMRs.

      4) RNAseq data provide potential molecular pathways through which Meis2 regulates embryonic target-field innervation.

      5) Well-performed electrophysiological study using skin-nerve preparation and recordings from saphenous and tibial nerves to investigate physiological deficits of Meis2 mutant sensory afferents.

      6) Nice whole-mount IHC of the hair skin, convincingly showing morphological deficits of Meis2 mutant SA- and RA- LTMRs.

      Overall, this manuscript is well-written. The experimental design and data quality are good, and the conclusion from the experimental results is logical.

      Weaknesses:<br /> 1) Although the authors justify this study for the involvement of Meis2 in Autism and Autism associated disorders, no experiments really investigated Autism-like specific behavior in the Meis2 ablated mice.

      2) For mechanical force sensing-related behavioral assays, the authors performed VFH and dynamic cotton swabs for the glabrous skin, and sticky tape on the back (hairy skin) for the hairy skin. A few additional experiments involving glabrous skin plantar surfaces, such as stick tape or flow texture discrimination, would make the conclusion stronger.

      3) The authors considered von Frey filaments (1 and 1.4 g) as noxious mechanical stimuli (Figure 1E and statement on lines 181-183), which is questionable. Alligator clips or pinpricks are more certain to activate mechanical nociceptors.

      4) There are disconnections and inconsistencies among findings from morphological characterization, physiological recordings, and behavior assays. For example, Meis2 mutant SA-LTMRs show a deficiency in Merkel cell innervation in the glabrous skin but not in hairy skin. With no clear justification, the authors pooled recordings of SA-LTMRs from both glabrous and hairy skin and found a significant increase in mean vibration threshold. Will the results be significantly different if the data are analyzed separately? In addition, whole-mount IHC of Meissner's corpuscles showed morphological changes, but electrophysiological recordings didn't find significant alternation of RAI LTMRs. What does the morphological change mean then? Since the authors found that Meis2 mice are less sensitive to a dynamic cotton swab, which is usually considered as an RA-LTMR mediated behavior, is the SAI-LTMR deficit here responsible for this behavior? Connections among results from different methods are not clear, and the inconsistency should be discussed.

    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:<br /> In 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) studied 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) used an inactive control site to reproduce the sound without effective neuromodulation, while manipulating the volume of the auditory confound at different TUS 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 alerting on the potential auditory confound of online US. Their 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:<br /> 1. Despite the main conclusion of the authors stating that there is no dose-response effects 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.<br /> 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. Joint Public Review:

      The manuscript presented by Pabba et al. studied chromatin dynamics throughout the cell cycle. The authors used fluorescence signals and patterns of GFP-PCNA (GFP tagged proliferating cell nuclear antigen) and CY3-dUTP (which labels newly synthesized DNA but not the DNA template) to determine cell cycle stages in asynchronized HeLa (Kyoto) cells and track movements of chromatin domains. PCNA binds to replication forks and form replication foci during the S phase. The major conclusions are: (1) Labeled chromatin domains were more mobile in G1/G2 relative to the S-phase. (2) Restricted chromatin motion occurred at sites in proximity to DNA replication sites. (3) Chromatin motion was restricted by the loading of replisomes, independent of DNA synthesis. This work is based on previous work published in 2015, entitled "4D Visualization of replication foci in mammalian cells corresponding to individual replicons," in which the labeling method was demonstrated to be sound.

      Comments on latest version: The revised manuscript has included data from a diploid cell line IMR90 (fibroblasts isolated from normal lung tissue) to strengthen the conclusions. Overall, quality of the work is substantially improved.

    1. Reviewer #1 (Public Review):

      Segas et al. present a novel solution to an upper-limb control problem which is often neglected by academia. The problem the authors are trying to solve is how to control the multiple degrees of freedom of the lower arm to enable grasp in people with transhumeral limb loss. The proposed solution is a neural network based approach which uses information from the position of the arm along with contextual information which defines the position and orientation of the target in space. Experimental work is presented, based on virtual simulations and a telerobotic proof of concept.

      The strength of this paper is that it proposes a method of control for people with transhumeral limb loss which does not rely upon additional surgical intervention to enable grasping objects in the local environment. A challenge the work faces is that it can be argued that a great many problems in upper limb prosthesis control can be solved given precise knowledge of the object to be grasped, its relative position in 3D space and its orientation. It is difficult to know how directly results obtained in a virtual environment will translate to real world impact. Some of the comparisons made in the paper are to physical systems which attempt to solve the same problem. It is important to note that real world prosthesis control introduces numerous challenges which do not exist in virtual spaces or in teleoperation robotics.

      The authors claim that the movement times obtained using their virtual system, and a teleoperation proof of concept demonstration, are comparable to natural movement times. The speed of movements obtained and presented are easier to understand by viewing the supplementary materials prior to reading the paper. The position of the upper arm and a given target are used as input to a classifier, which determines the positions of the lower arm, wrist and the end effector. The state of the virtual shoulder in the pick and place task is quite dynamic and includes humeral rotations which would be challenging to engineer in a real physical prosthesis above the elbow. Another question related to the pick and place task used is whether or not there are cases where both the pick position and the place position can be reached via the same, or very similar, shoulder positions? i.e. with the shoulder flexion-extension and abduction-adduction remaining fixed, can the ANN use the remaining five joint angles to solve the movement problem with little to no participant input, simply based on the new target position? If this was the case, movements times in the virtual space would present a very different distribution to natural movements, while the mean values could be similar. The arguments made in the paper could be supported by including individual participant data showing distributions of movement times and the distances travelled by the end effector where real movements are compared to those made by an ANN.

      In the proposed approach users control where the hand is in space via the shoulder. The position of the upper arm and a given target are used as input to a classifier, which determines the positions of the lower arm, wrist and the effector. The supplementary materials suggest the output of the classifier occurs instantaneously, in that from the start of the trial the user can explore the 3D space associated with the shoulder in order to reach the object. When the object is reached a visual indicator appears. In a virtual space this feedback will allow rapid exploration of different end effector positions which may contribute to the movement times presented. In a real world application, movement of a distal end-effector via the shoulder is not to be as graceful and a speed accuracy trade off would be necessary to ensure objects are grasped, rather than knocked or moved.

      Another aspect of the movement times presented which is of note, although it is not necessarily incorrect, is that the virtual prosthesis performance is close too perfect. In that, at the start of each trial period, either pick or place, the ANN appears to have already selected the position of the five joints it controls, leaving the user to position the upper arm such that the end effector reaches the target. This type of classification is achievable given a single object type to grasp and a limited number of orientations, however scaling this approach to work robustly in a real world environment will necessitate solving a number of challenges in machine learning and in particular computer vision which are not trivial in nature. On this topic, it is also important to note that, while very elegant, the teleoperation proof of concept of movement based control does not seem to feature a similar range of object distance from the user as the virtual environment. This would have been interesting to see and I look forward to seeing further real world demonstrations in the authors future work.

    1. Joint Public Review:

      The authors present a carefully controlled set of experiments that demonstrate an additional complexity for GPCR signaling in that endosomal signaling may be different when beta-arrestin is or isn't associated with a G protein-bound V2 vasopressin receptor. It uses state of the art biosensor-based approaches and beta-arrestin KO lines to assess this. It adds to a growing body of evidence that G proteins and beta-arresting can associate with GPCR complexes simultaneously. They also demonstrate the possibility that Gq might also be activated by the V2 receptor. My sense is one thing they may need to be considered is the possibility of such "megacomplexes" might actually involve receptor dimers or oligomers. They have added significant amounts of new data to address concerns I had about the sole use of mini-genes to assess G protein coupling and broadened discussions about the possible mechanisms underlying their observations. I would still argue that receptor oligomers are a more obvious way for such megacomplexes to be organized around.

    1. Reviewer #1 (Public Review):

      Summary:<br /> This manuscript by Estevam et al. reports new insights into the regulation of the receptor tyrosine kinase MET gained from two deep mutational scanning (DMS) datasets. In this paper, the authors use a classic selection system for oncogenic kinase signaling, the murine Ba/F3 cell line, to assess the functional effects of thousands of mutations in the kinase domains of MET in two contexts: (1) fusion of the whole MET intracellular region to the dimerization domain TPR, and (2) the same fusion protein, but with exon 14, which encodes part of the juxtamembrane region of MET, skipped. Critically, exon 14 skipping yields a version of MET that is found in many cancers and has higher signaling activity than the canonical MET isoform. The authors extensively analyze their DMS data to very convincingly show that their selection assay reports on kinase activity, by illustrating that many functionally important structural components of the kinase domain are not tolerant of mutation. Then, they turn their attention to a helical region of the juxtamembrane region (αJM), immediately after exon 14, which is posited to play a regulatory role in MET. Their DMS data illustrate that the strength and mutational tolerance of interactions between αJM and the key αC helix in the kinase domain depends on the presence or absence of exon 14. They also identify residues in the N-lobe of the kinase, such as P1153, which are not conserved across tyrosine kinases but appear to be essential for MET and MET-like kinases. Finally, the authors analyze their DMS data in the context of clinically-observed mutations and drug-resistance mutations.

      Overall, this manuscript is exciting because it provides new insights into MET regulation in general, as well as the role of exon 14. It also reveals ways in which the JM region of MET is different from that of many other receptor tyrosinekinases. The exon 14-skipped fusion protein DMS data is somewhat underexplored and could be discussed in greater detail, which would elevate excitement about the work. Furthermore, some of the cell biological validation experiments and the juxtaposition with clinical data are perhaps not assessed/interpreted as clearly they could be. Some constructive suggestions are given below to enhance the impact of the manuscript.

      Strengths:<br /> The main strengths of this paper, also summarized above in the summary, are as follows:

      1. The authors very convincingly show that Ba/F3 cells can be coupled with deep mutational scanning to examine MET mutational effects. This is most clearly shown by highlighting how all of the known kinase structure and regulatory elements are highly sensitive to mutations, in accordance with a few other DMS datasets on other kinases.

      2. A highlight of this paper is the juxtaposition of two DMS datasets for two different isoforms of the MET receptor. Very few comparisons like this exist in the literature, and they show how small changes to the overall architecture of a protein can impact its regulation and mutational sensitivity.

      3. Another exciting advance in this manuscript is the deep structural analysis of the MET juxtamembrane region with respect to that of other tyrosine kinases - guided by the striking effect of mutations in the juxtamembrane helical region. The authors illustrate how the JM region of MET differs from that of other tyrosine kinases.

      4. Overall, this manuscript will provide a resource for interpreting clinically relevant MET mutations.

      Weaknesses:<br /> 1. The manuscript is front-loaded with extensive analysis of the first DMS dataset, in which exon 14 is present, however, the discussion and analysis of the exon 14-skipped dataset is somewhat limited. In particular, a deeper discussion of the differences between the two datasets is warranted, to lay out the full landscape of mutations that have different functional consequences in the two isoforms. Rather, the authors only focus on differences in the JM region. What are the broader structural effects of exon 14 skipping across the whole kinase domain?

      2. It is unclear if gain-of-function mutations can actually be detected robustly in this specific system. This isn't a problem at face value, as different selection assays have different dynamic ranges. However, the authors don't discuss the statistical significance and reproducibility of gain- vs loss-of-function mutations, and none of the gain-of-function mutations are experimentally validated (some appear to show loss-of-function in their cellular validation assay with full-length MET). The manuscript would benefit from deeper statistical analysis (and discussion in the text) of gain-of-function mutations, as well as further validation of a broad range of activity scores in a functional assay. For the latter point, one option would be to express individual clones from their library in Ba/F3 cells and blot for MET activation loop phosphorylation (which is probably a reasonable proxy for activity/activation).

      3. In light of point 2, above, much of the discussion about clinically-relevant gain-of-function mutations feels a bit stretched - although this section is definitely very interesting in premise. A clearer delineation of gain-of-function, with further statistical support and ideally also some validation, would greatly strengthen the claims in this section.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The article by Siachisumo, Luzzi and Aldalaquan et al. describes studies of RBMX and its role in maintaining proper splicing of ultra-long exons. They combine CLIP, RNA-seq, and individual example validations with manipulation of RBMX and its family members RBMY and RBMXL2 to show that the RBMX family plays a key role in maintaining proper splicing of these exons.

      Strengths:<br /> I think one of the main strengths of the manuscript is its ability to explore a unique but interesting question (splicing of ultra-long exons), and derive a relatively simple model from the resulting genomics data. The results shown are quite clean, suggesting that RBMX plays an important role in the proper regulation of these exons. The ability of family members to rescue this phenotype (as well as only particular domains) is also quite intriguing and suggests that the mechanisms for keeping these exons properly spliced may be a quite important and highly conserved mechanism.

      Weaknesses:<br /> I think my main critique is that a lot of the conclusions in the text are written with very broad and general claims, but these are often based on either a small number of examples or a non-transcriptome-wide analysis that I think would be necessary for such broad conclusions. For example, pg 5 - "The above data indicated that RBMX has a major role in repressing cryptic splicing patterns in human somatic cells." is based on seeing ~120 exons with a 2:1 ratio of exclusion to inclusion (and doesn't include analysis confirming which of these are cryptic). Similarly, on page 15, line 31 "The above experiments showed that RBMXL2 is able to globally replace RBMX activity" and Fig 5 as well - I think the 'globally' term here is a bit too much with most of the analysis derived from n=3 events.

      Another weakness I think is the lack of context in the paper, where the basic description of how many 'long' and 'ultra-long' exons there are and what percent of those are bound by and/or regulated by RBMX is missing. The text (including the title, which to me is written to imply a general role for ultra-long exons') seems to imply that RBMX maintains proper splicing of ultra-long exons globally (which to me implies a significant percent of them), but I don't think the manuscript succeeds in strongly proving this global role.

      Along those same lines, I think the manuscript claims that the described principles are specific to the RBMX family, but I think the lack of negative examples weakens the strength of this claim. For example, for Figure 2 (D/E/F/G), what I think would make this more powerful is a negative example - if you take CLIP + knockdown/RNA-seq data for a different splicing regulator (RBFOX2, PTBP1, etc.), do you not see this size difference? I think the analysis described here is sufficient to show that there is an overlap for RBMX, but I think this would make it much stronger in confirming it's not just a 'longer genes get more CLIP tags' artifact.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Here, Boor et al focus on the regulation of daf-7 transcription in the ASJ chemosensory neurons, which has previously been shown to be sensitive to a variety of external and internal signals. Interestingly, they find that soluble (but not volatile) signals released by food activate daf-7 expression in ASJ, but that this is counteracted by signals from the ASIC channels del-3 and del-7, previously shown to detect the ingestion of food in the pharynx. Importantly, the authors find that ASJ-derived daf-7 can promote exploration, suggesting a feedback loop that influences locomotor states to promote feeding behavior. They also implicate signals known to regulate exploratory behavior (the neuropeptide receptor PDFR-1 and the neuromodulator serotonin) in the regulation of daf-7 expression in ASJ. Additionally, they identify a novel role for a pathway previously implicated in C. elegans sensory behavior, HEN-1/SCD-2, in the regulation of daf-7 in ASJ, suggesting that the SCD-2 homolog ALK may have a conserved role in feeding and metabolism.

      Strengths:<br /> The studies reported here, particularly the quantitation of gene expression and the careful behavioral analysis, are rigorously done and interpreted appropriately. The results suggest that, with respect to food, DAF-7 expression encodes a state of "unmet need" - the availability of nearby food to animals that are not currently eating. This is an interesting finding that reinforces and extends our understanding of the neurobiological significance of this important signaling pathway. The identification of a role for ASJ-derived daf-7 in motor behavior is a valuable advance, as is the finding that SCD-2 acts in the AIA interneurons to influence daf-7 expression in ASJ.

      Weaknesses:<br /> A limitation of the work is that some mechanistic relationships between the identified signaling pathways are not carefully examined, but this provides interesting opportunities for future work. A minor weakness concerns the experiment in which daf-7 is conditionally deleted from ASJ. This is an ideal approach for probing the function of daf-7, but these experiments seem to be carried out in the well-fed, on-food condition in which control animals should express little or no daf-7 in ASJ. Thus, the experimental design does not allow an assessment of the role of daf-7 under conditions in which its expression is activated (e.g., in animals exposed to un-ingestible food). An additional minor issue concerns the interpretation of the scd-2 experiments. The authors' findings do support a role for scd-2 signaling in the activation of daf-7 expression by un-ingestible food, but the data also suggest that scd-2 signaling is not essential for this effect, as there is still an effect in scd-2 mutants (Figure 4B).

    1. Joint Public Review:

      This study describes a group of CRH-releasing neurons, located in the paraventricular nucleus of the hypothalamus, which, in mice, affects both the state of sevoflurane anesthesia and a grooming behavior observed after it. PVH-CRH neurons showed elevated calcium activity during the post-anesthesia period. Optogenetic activation of these PVH-CRH neurons during sevoflurane anesthesia shifts the EEG from burst-suppression to a seemingly activated state (an apparent arousal effect), although without a behavioral correlate. Chemogenetic activation of the PVH-CRH neurons delays sevoflurane-induced loss of righting reflex (another apparent arousal effect). On the other hand, chemogenetic inhibition of PVH-CRH neurons delays recovery of the righting reflex and decreases sevoflurane-induced stress (an apparent decrease in the arousal effect). The authors conclude that PVH-CRH neurons are a common substrate for sevoflurane-induced anesthesia and stress. The PVH-CRH neurons are related to behavioral stress responses, and the authors claim that these findings provide direct evidence for a relationship between sevoflurane anesthesia and sevoflurane-mediated stress that might exist even when there is no surgical trauma, such as an incision. In its current form, the article does not achieve its intended goal.

      Strengths<br /> The manuscript uses targeted manipulation of the PVH-CRH neurons, and is technically sound. Also, the number of experiments is substantial.

      Weaknesses<br /> The most significant weaknesses are a) the lack of consideration and measurement of GABAergic mechanisms of sevoflurane anesthesia, b) the failure to use another anesthetic as a control, c) a failure to document a compelling post-anesthesia stress response to sevoflurane in humans, d) limitations in the novelty of the findings. These weaknesses are related to the primary concerns described below:

      Concerns about the primary conclusion, that PVH-CRH neurons mediate "the anesthetic effects and post-anesthesia stress response of sevoflurane GA".

      1) Just because the activity of a given neural cell type or neural circuit alters an anesthetic's response, this does not mean that those neurons play a role in how the anesthetic creates its anesthetic state. For example, sevoflurane is commonly used in children. Its primary mechanism of action is through enhancement of GABA-mediated inhibition. Children with ADHD on Ritalin (a dopamine reuptake inhibitor) who take it on the day of surgery can often require increased doses of sevoflurane to achieve the appropriate anesthetic state. The mesocortical pathway through which Ritalin acts is not part of the mechanism of action of sevoflurane. Through this pathway, Ritalin is simply increasing cortical excitability making it more challenging for the inhibitory effects of sevoflurane at GABAergic synapses to be effective. Similarly, here, altering the activity of the PVHCRH neurons and seeing a change in anesthetic response to sevoflurane does not mean that these neurons play a role in the fundamental mechanism of this anesthetic's action. With the current data set, the primary conclusions should be tempered.

      2) It is important to compare the effects of sevoflurane with at least one other inhaled ether anesthetic. Isoflurane, desflurane, and enflurane are ether anesthetics that are very similar to each other, as well as being similar to sevoflurane. It is important to distinguish whether the effects of sevoflurane pertain to other anesthetics, or, alternatively, relate to unique idiosyncratic properties of this gas that may not be a part of its anesthetic properties.

      For example, one study cited by the authors (Marana et al.. 2013) concludes that there is weak evidence for differences in stress-related hormones between sevoflurane and desflurane, with lower levels of cortisol and ACTH observed during the desflurane intraoperative period. It is not clear that this difference in some stress-related hormones is modeled by post-sevoflurane excess grooming in the mice, but using desflurane as a control could help determine this.

      Concerns about the clinical relevance of the experiments<br /> In anesthesiology practice, perioperative stress observed in patients is more commonly related to the trauma of the surgical intervention, with inadequate levels of antinociception or unconsciousness intraoperatively and/or poor post-operative pain control. The authors seem to be suggesting that the anesthetic itself is causing stress, but there is no evidence of this from human patients cited. We were not aware that this is a documented clinical phenomenon. It is important to know whether sevoflurane effectively produces behavioral stress in the recovery room in patients that could be related to the putative stress response (excess grooming) observed in mice. For example, in surgeries or procedures that required only a brief period of unconsciousness that could be achieved by administering sevoflurane alone (comparable to the 30 min administered to the mice), is there clinical evidence of post-operative stress?

      Patients who receive sevoflurane as the primary anesthetic do not wake up more stressed than if they had had one of the other GABAergic anesthetics. If there were signs of stress upon emergence (increased heart rate, blood pressure, thrashing movements) from general anesthesia, the anesthesiologist would treat this right away. The most likely cause of post-operative stress behaviors in humans is probably inadequate anti-nociception during the procedure, which translates into inadequate post-op analgesia and likely delirium. It is the case that children receiving sevoflurane do have a higher likelihood of post-operative delirium. Perhaps the authors' studies address a mechanism for delirium associated with sevoflurane, but this is not considered. Delirium seems likely to be the closest clinical phenomenon to what was studied.

      Concerns about the novelty of the findings<br /> CRH is associated with arousal in numerous studies. In fact, the authors' own work, published in eLife in 2021, showed that stimulating the hypothalamic CRH cells leads to arousal and their inhibition promotes hypersomnia. In both papers, the authors use fos expression in CRH cells during a specific event to implicate the cells, then manipulate them and measure EEG responses. In the previous work, the cells were active during wakefulness; here- they were active in the awake state that follows anesthesia (Figure 1). Thus, the findings in the current work are incremental.

      The activation of CRH cells in PVN has already been shown to result in grooming by Jaideep Bains (cited as reference 58). Thus, the involvement of these cells in this behavior is expected. The authors perform elaborate manipulations of CRH cells and numerous analyses of grooming and related behaviors. For example, they compare grooming and paw licking after anesthesia with those after other stressors such as forced swim, spraying mice with water, physical attack, and restraint. However, the relevance of these behaviors to humans and generalization to other types of anesthetics is not clear.

    1. Reviewer #1 (Public Review):

      Like the "preceding" co-submitted paper, this is again a very strong and interesting paper in which the authors address a question that is raised by the finding in their co-submitted paper - how does one factor induce two different fates. The authors provide an extremely satisfying answer - only one subset of the cells neighbors a source of signaling cells that trigger that subset to adopt a specific fate. The signal here is Delta and the read-out is Notch, whose intracellular domain, in conjunction with, presumably, SuH cooperates with Bsh to distinguish L4 from L5 fate (L5 is not neighbored by signal-providing cells). Like the back-to-back paper, the data is rigorous, well-presented and presents important conclusions. There's a wealth of data on the different functions of Notch (with and without Bsh). All very satisfying.

      I have again one suggestion that the authors may want to consider discussing. I'm wondering whether the open chromatin that the author convincingly measure is the CAUSE or the CONSEQUENCE of Bsh being able to activate L4 target genes. What I mean by this is that currently the authors seem to be focused on a somewhat sequential model where Notch signaling opens chromatin and this then enables Bsh to activate a specific set of target genes. But isn't it equally possible that the combined activity of Bsh/Notch(intra)/SuH opens chromatin? That's not a semantic/minor difference, it's a fundamentally different mechanism, I would think. This mechanism also solves the conundrum of specificity - how does Notch know which genes to "open" up? It would seem more intuitive to me to think that it's working together with Bsh to open up chromatin, with chromatin accessibility than being a "mere" secondary consequence. If I'm not overlooking something fundamental here, there is actually also a way to distinguish between these models - test chromatin accessibility in a Bsh mutant. If the author's model is true, chromatin accessibility should be unchanged.<br /> I again finish by commending the authors for this terrific piece of work.

    1. Reviewer #1 (Public Review):

      In this very strong and interesting paper the authors present a convincing series of experiments that reveal molecular mechanism of neuronal cell type diversification in the nervous system of Drosophila. The authors show that a homeodomain transcription factor, Bsh, fulfills several critical functions - repressing an alternative fate and inducing downstream homeodomain transcription factors with whom Bsh may collaborate to induce L4 and L5 fates (the author's accompanying paper reveals how Bsh can induce two distinct fates). The authors make elegant use of powerful genetic tools and an arsenal of satisfying cell identity markers.

      I believe that this is an important study because it provides some fundamental insights into the conservation of neuronal diversification programs. It is very satisfying to see that similar organizational principles apply in different organisms to generate cell type diversity. The authors should also be commended for contextualizing their work very well, giving a broad, scholarly background to the problem of neuronal cell type diversification.

      My one suggestion for the authors is to perhaps address in the Discussion (or experimentally address if they wish) how they reconcile that Bsh is on the one hand: (a) continuously expressed in L4/L4, (b) binding directly to a cohort of terminal effectors that are also continuously expressed but then, on the other hand, is not required for their maintaining L4 fate? A few questions: Is Bsh only NOT required for maintaining Ap expression or is it also NOT required for maintaining other terminal markers of L4? The former could be easily explained - Bsh simply kicks of Ap, Ap then autoregulates, but Bsh and Ap then continuously activate terminal effector genes. The second scenario would require a little more complex mechanism: Bsh binding of targets (with Notch) may open chromatin, but then once that's done, Bsh is no longer needed and Ap alone can continue to express genes. I feel that the authors should be at least discussing this. The postmitotic Bsh removal experiment in which they only checked Ap and depression of other markers is a little unsatisfying without further discussion (or experiments, such as testing terminal L4 markers). I hasten to add that this comment does not take away from my overall appreciation for the depth and quality of the data and the importance of their conclusions.

    1. Joint Public Review:

      Anthoney et al. provide an honorable attempt at furthering our understanding of the different sleep stages that may exist in Drosophila. The establishment of definable sleep stages/state in the fly model should be seen as a central goal in the field and this study represents an important step toward that goal. In particular, managing to draw parallels between sleep stages in flies and humans would make it relevant to use the power of fly genetics to better understand the molecular and cellular basis of these sleep stages. The authors use behavioral, physiological and transcriptomics approaches to describe the differences that exist between sleep triggered by optogenetic stimulation of dFB neurons and sleep induced by consumption of the sleep-promoting drug Gaboxadol (THIP). While there are still concerns regarding the interpretation of the major results, the authors have, in general, adequately responded to the reviewers' concerns.

      The strengths of this work are:<br /> 1- The article is easy to read, and the figures are mostly informative.<br /> 2- The authors employ state-of-the-art techniques to measure neuronal activity and locomotion in a single assay.<br /> 3- The analysis of transcriptomic data is appropriate.<br /> 4- The authors identify many new genes regulated in response to specific methods for sleep induction. These are all potentially interesting candidates for further studies investigating the molecular basis of sleep. It would be interesting to know which of these genes are already known to display circadian expression patterns.

      Concerns:<br /> 1- The fact that flies with dFB activation seem to keep a basal level of locomotor activity whereas THIP-treated ones don't is quite striking. Is it possible that there is an error with Figure 4C-D? Based on this, it is hard to believe that dFB stimulation and THIP consumption have similar behavioral effects on sleep.<br /> 2- The authors seem satisfied with the 'good correspondence' between their RNA-seq and qPCR results, this is true for only ~9/19 genes in Fig 6G and 2/6 genes in Fig 7G. The variability between the three biological replicates is not represented in the figure.

      Comments<br /> 1- The fact that THIP-induced sleep persists long after THIP removal (Fig 3D) is intriguing. This suggests that the drug might trigger a sleep-inducing pathway that, once activated, can continue on its own without the drug.<br /> 2- The claim that induction of the two forms (active/quiet) of sleep produce distinct transcriptomic and physiological effects while producing highly similar behavioral effects makes it difficult to understand the relationships between the former changes with sleep state. In their response the authors argue that sleep behavior, as currently measured using duration of inactivity, is not necessarily expected to allow for a differentiation between active and quiet sleep. This further argues for a need for better physiological/molecular correlates of sleep state in the fly (a laudable goal of this very study). However, until clear behavioral correlates can be strongly associated with physiological/molecular correlates, we will be limited to speculation about this important issue.<br /> 3- The authors suggest that the duration of the periods of inactivity may not be particularly useful for defining sleep states/stages in the fly. If this is the case, it is certainly an important issue as this measurement is the behavioral criteria for defining sleep in the field. In this regard, it raises the question of the relationship between the active and quiet sleep states examined in this study and the growing evidence for a deep sleep state (characterized by, among other things, a lowered metabolism).<br /> 4- Although the methodological concerns regarding the dose of Gaboxadol and the controls for the optogenetic/transcriptomic experiments remain, the authors have explained the rationale for the experimental design they used.<br /> 5- Overall, the authors have managed to clearly illustrate the differences between dFB stimulation and THIP consumption on behavior, neuronal activity, and gene expression. In this regard, they have achieved what they claim in the title of the article. Overall, the results support the conclusions, however the main point to consider is that the methods employed here are artificial, and there is no guarantee at this stage that 'spontaneous' sleep has the same effects on the transcriptome than what is presented here.<br /> 5- This article represents an interesting attempt at addressing very important questions, and some of the data presented here, especially the RNA-seq, can be very useful for others. However, because of the lack of definitive conclusion about whether the results presented are applicable to 'natural' sleep, the impact of this article may remain limited beyond a relatively small field.

      Comments to authors

      The authors have suitably addressed all comments from this section.

    1. Reviewer #1 (Public Review):

      The study describes a new computational method for unsupervised (i.e., non-artificial intelligence) segmentation of objects in grayscale images that contain substantial noise, to differentiate object, no object, and noise. Such a problem is essential in biology because they are commonly confronted in the analysis of microscope images of biological samples and recently have been resolved by artificial intelligence, especially by deep neural networks. However, training artificial intelligence for specific sample images is a difficult task and not every biological laboratory can handle it. Therefore, the proposed method is particularly appealing to laboratories with little computational background. The method was shown to achieve better performance than a threshold-based method for artificial and natural test images. To demonstrate the usability, the authors applied the method to high-power confocal images of the thalamus for the identification and quantification of immunostained potassium ion channel clusters formed in the proximity of large axons in the thalamic neuropil and verified the results in comparison to electron micrographs. 

      Strengths: <br /> The authors claim that the proposed method has higher pixel-wise accuracy than the threshold-based method when applied to gray-scale images with substantial noises.

      Since the method does not use artificial intelligence, training and testing are not necessary, which would be appealing to biologists who are not familiar with machine learning technology.

      The method does not require extensive tuning of adjustable parameters (trying different values of "Moran's order") given that the size of the object in question can be estimated in advance.

      Weaknesses:<br /> It is understood that the strength of the method is that it does not depend on artificial intelligence and therefore the authors wanted to compare the performance with another non-AI method (i.e. the threshold-based method; TBM). However, the TBM used in this work seems too naive to be fairly compared to the expensive computation of "Moran's I" used for the proposed method. To provide convincing evidence that the proposed method advances object segmentation technology and can be used practically in various fields, it should be compared to other advanced methods, including AI-based ones, as well.

      This method was claimed to be better than the TBM when the noise level was high. Related to the above, TBMs can be used in association with various denoising methods as a preprocess. It is questionable whether the claim is still valid when compared to the methods with adequate complexity used together with denoising. Consider for example, Weigert et al. (2018) https://doi.org/10.1038/s41592-018-0216-7; or Lehtinen et al (2018) https://doi.org/10.48550/arXiv.1803.04189.

      The computational complexity of the method, determined by the convolution matrix size (Moran's order), linearly increases as the object size increases (Fig. S2b). Given that the convolution must be run separately for each pixel, the computation seems quite demanding for scale-up, e.g. when the method is applied for 3D image volumes. It will be helpful if the requirement for computer resources and time is provided.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this well-designed study, the authors of the manuscript have analyzed the impact of individually silencing 90 lipid transfer proteins on the overall lipid composition of a specific cell type. They confirmed some of the evidence obtained by their own and other research groups in the past, and additionally, they identified an unreported role for ORP9-ORP11 in sphingomyelin production at the trans-Golgi. As they delved into the nature of this effect, the authors discovered that ORP9 and ORP11 form a dimer through a helical region positioned between their PH and ORD domains.

      Strengths:<br /> This well-designed study presents compelling new evidence regarding the role of lipid transfer proteins in controlling lipid metabolism. The discovery of ORP9 and ORP11's involvement in sphingolipid metabolism invites further investigation into the impact of the membrane environment on sphingomyelin synthase activity.

      Weaknesses:<br /> There are a couple of weaknesses evident in this manuscript. Firstly, there's a lack of mechanistic understanding regarding the regulatory role of ORP9-11 in sphingomyelin synthase activity. Secondly, the broader role of hetero-dimerization of LTPs at ER-Golgi membrane contact sites is not thoroughly addressed. The emerging theme of LTP dimerization through coiled domains has been reported for proteins such as CERT, OSBP, ORP9, and ORP10. However, the specific ways in which these LTPs hetero and/or homo-dimerize and how this impacts lipid fluxes at ER-Golgi membrane contact sites remain to be fully understood.

      Regardless of the unresolved points mentioned above, this manuscript presents a valuable conceptual advancement in the study of the impact of lipid transfer on overall lipid metabolism. Moreover, it encourages further exploration of the interplay among LTP actions across various cellular organelles.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this study, the authors present a new mouse model with a deletion of the Tmem263 gene, also known as C12orf23, which encodes the transmembrane protein 263.

      Strengths:<br /> The study indicates that Tmem263 interacts with growth hormone, and possibly participates in controlling body growth.

      Weaknesses (Major):<br /> The current study confirms previous findings using a mouse model of TMEM263 gene deletion. However, it remains descriptive and does not provide critical insights into the mechanism of action of TMEM263 protein. While the study demonstrates Tmem263-mediated reduction in GHR gene expression in the liver and subsequent growth retardation, it does not elucidate the pathway through which Tmem263 affects GHR expression.

      Weaknesses (minor):<br /> 1. Since GH-resistant dwarfism is typically associated with increased body adiposity and hepatic lipid accumulation, considering the high expression levels of Tmem263 in adipose tissue (Figure 1C), it would be valuable to measure body adiposity and hepatic lipid content.

      2. It would be helpful to specify the age at which the growth plate parameters were tested. Additionally, were there any differences observed between male and female mice (Figure 3 L-N)? Information on the local (growth plate) expression of IGF-1, IGF-1R, and GHR would also be beneficial.

      3. Given the low levels of blood glucose and serum insulin, it would be relevant to know if the mice were challenged with ITT or GTT.

      4. The liver transcriptomics data presented in the study is impressive. However, there seems to be a missed opportunity to delve into the data and identify potential factors involved in the regulation of GHR expression.

      5. The effects of whole-body Tmem263 nullification on osteoblast differentiation and function, as well as on osteoclastogenesis, were not investigated in the study. Considering the potential impact on bone health, these aspects could be explored in future research.

    1. Reviewer #1 (Public Review):

      In this manuscript, Huang and colleagues explored the role of iron in bacterial therapy for cancer. Using proteomics, they revealed the upregulation of bacterial genes that uptake iron, and reasoned that such regulation is an adaptation to the iron-deficient tumor microenvironment. Logically, they engineered E. Coli strains with enhanced iron-uptake efficiency, and showed that these strains, together with iron scavengers, suppress tumor growth in a mouse model. Lastly, they reported the tumor suppression by IroA-E. Coli provides immunological memory via CD8+ T cells. In general, I find the findings in the manuscript novel and the evidence convincing.

      1. Although the genetic and proteomic data are convincing, would it be possible to directly quantify the iron concentration in (1) E. Coli in different growth environments, and (2) tumor microenvironment? This will provide the functional consequences of upregulating genes that import iron into the bacteria.

      2. Related to 1, the experiment to study the synergistic effect of CDG and VLX600 (lines 139-175) is very nice and promising, but one flaw here is a lack of the measurement of iron concentration. Therefore, a possible explanation could be that CDG acts in another manner, unrelated to iron uptake, that synergizes with VLX600's function to deplete iron from cancer cells. Here, a direct measurement of iron concentration will show the effect of CDG on iron uptake, thus complementing the missing link.

      3. Lines 250-268: Although statistically significant, I would recommend the authors characterize the CD8+ T cells a little more, as the mechanism now seems quite elusive. What signals or memories do CD8+ T cells acquire after IroA-E. Coli treatment to confer their long-term immunogenicity?

      4. Perhaps this goes beyond the scope of the current manuscript, but how broadly applicable is the observed iron-transport phenomenon in other tumor models? I would recommend the authors to either experimentally test it in another model or at least discuss this question.

    1. Reviewer #1 (Public Review):

      This work reports the use of pulsed high-intensity (1-3 W/cm2) 1 MHz ultrasonic waves to stimulate the secretion of extracellular vesicles (EVs) from skeletal muscle cells (C2C12 myotubes) through the modulation of intracellular calcium, and that these, in turn, regulate the inflammatory response in macrophages.

      The authors first checked that the ultrasonic irradiation did not have any adverse effect on the structure (protein content) and function (proliferation and metabolic activity) of the myotubes, and showed an up to twofold increase in EV secretion, which they attributed to an increase in Ca2+ uptake into the cell. Finally, the authors show that the myotubes exposed to the ultrasonic irradiation wherein the EV concentration was found to be elevated led to a significant decrease in expression of IL-1b and IL-6 pro-inflammatory cytokines, therefore leading the authors to assert the potential of the use of ultrasonic irradiation for promoting anti-inflammatory effects on macrophages.

      While the manuscript was reasonably clearly written and the methodology and results sound, it is not clear what the real contribution of the work is. The authors' findings - that ultrasonic stimulation is capable of altering intracellular Ca2+ to effect an increase in EV secretion from cells as long as the irradiation does not affect cell viability-is well established (see, for example, Ambattu et al., Commun Biol 3, 553, 2020; Deng et al., Theranostics, 11, 9 2021; Li et al., Cell Mol Biol Lett 28, 9, 2023). Moreover, the authors' own work (Maeshige et al., Ultrasonics 110, 106243, 2021) using the exact same stimulation (including the same parameters, i.e., intensity and frequency) and cells (C2C12 skeletal myotubes) reported this. Similarly, the authors themselves reported that EV secretion from C2C12 myotubes has the ability to regulate macrophage inflammatory response (Yamaguchi et al., Front Immunol 14, 1099799, 2023). It would then stand to reason that a reasonable and logical deduction from both studies is that the ultrasonic stimulation would lead to the same attenuation of inflammatory response in macrophages through enhanced secretion of EVs from the myotubes.

      The authors' claim that 'the role of Ca2+ in ultrasound-induced EV release and its intensity-dependency are still unclear', and that the aim of the present work is to clarify the mechanism, is somewhat overstated. That ultrasonic stimulation alters intracellular Ca2+ to lead to EV release, therefore establishing their interdependency and hence demonstrating the mechanism by which EV secretion is enhanced by the ultrasonic stimulation, was detailed in Ambattu et al., Commun Biol 3, 553, 2020. While this was carried out at a slightly higher frequency (10 MHz) and slightly different form of ultrasonic stimulation, the same authors have appeared to since establish that a universal mechanism of transduction across an entire range of frequencies and stimuli (Ambattu, Biophysics Rev 4, 021301, 2023).

      Similarly, the anti-inflammatory effects of EVs on macrophages have also been extensively reported (Li et al., J Nanobiotechnol 20, 38, 2022; Lo Sicco et al., Stem Cells Transl Med 6, 3, 2017; Hu et al., Acta Pharma Sin B 11, 6, 2021), including that by the authors themselves in a recent study on the same C2C12 myotubes (Yamaguchi et al., Front Immunol 14, 1099799, 2023). Moreover, the authors' stated aim for the present work - clarifying the mechanism of the anti-inflammatory effects of ultrasound-induced skeletal muscle-derived EVs on macrophages - appears to be somewhat redundant given that they simply repeated the microRNA profiling study they carried out in Yamaguchi et al., Front Immunol 14, 1099799, 2023. The only difference was that a fraction of the EVs (from identical cells) that they tested were now a consequence of the ultrasound stimulation they imposed.

      That the authors have found that their specific type of ultrasonic stimulation maintains this EV content (i.e., microRNA profile) is novel, although this, in itself, appears to be of little consequence to the overall objective of the work which was to show the suppression of macrophage pro-inflammatory response due to enhanced EV secretion under the ultrasonic irradiation since it was the anti-inflammatory effects were attributed to the increase in EV concentration and not their content.

    1. Reviewer #1 (Public Review):

      I have a major conceptual problem with this manuscript: How can the full deletion of a gene (PARG) sensitize a cell to further inhibition by its chemical inhibitor (PARGi) since the target protein is fully absent?

      The authors state in the discussion section: "The residual PARG dePARylation activity observed in PARG KO cells likely supports cell growth, which can be further inhibited by PARGi". What does this statement mean? Is the authors' conclusion that their PARG KOs are not true KOs but partial hypomorphic knockdowns? Were the authors working with KO clones or CRISPR deletion in populations of cells?

      Are there splice variants of PARG that were not knocked down? Are there PARP paralogues that can complement the biochemical activity of PARG in the PARG KOs? The authors do not discuss these critical issues nor engage with this problem.

      These issues have to be dealt with upfront in the manuscript for the reader to make sense of their work.

    1. Reviewer #1 (Public Review):

      The authors consider data by the Heisenberg group on rheological properties of non-confluent tissue in zebrafish embryos. These data had shown a steep increase and subsequent saturation in viscosity with cell density. The authors introduce a physical agent-based model of such tissues that accounts for the dispersion in cell size and the softness of the cells. The model is inspired by previous models to study glassy dynamics and reveals essential physical features that can explain the observed behavior. It goes beyond previous studies that had analysed the observations in terms of a percolation problem. The numerics is thoroughly done and could have a deep impact on how we describe non-confluent tissues.

    1. Reviewer #1 (Public Review):

      This paper provides new evidence on the relationship between genetic/chromosome divergence and capacity for asexual reproduction (via unreduced, clonal gametes) in hybrid males or females. Whereas previous studies have focussed just on the hybrid combinations that have yielded asexual lineages in nature, the authors take an experimental approach, analysing meiotic processes in F1 hybrids for combinations of species spanning different levels of divergence, whether or not they form asexual lineages in nature. As such, the findings here are a substantial advance towards understanding how new asexual lineages form.

      The quality of the work is high, the analyses are sound, and the authors sensibly link their observations to the speciation continuum. I should also add that the cytogenetic work here is just beautiful!

      A key finding is that the precondition for asexual reproduction - the formation of unreduced gametes - is not unusual among hybrid females, so that we have to consider other factors to explain the rarity of asexual species - a major unresolved issue in evolutionary biology. This work also highlights a previously overlooked effect of chromosome organisation on speciation.

    1. Reviewer #1 (Public Review):

      The manuscript "Diffusive lensing as a mechanism of intracellular transport and compartmentalization", explores the implications of heterogeneous viscosity on the diffusive dynamics of particles. The authors analyze three different scenarios:

      (i) diffusion under a gradient of viscosity,

      (ii) clustering of interacting particles in a viscosity gradient, and

      (iii) diffusive dynamics of non-interacting particles with circular patches of heterogeneous viscous medium.

      The implications of a heterogeneous environment on phase separation and reaction kinetics in cells are under-explored. This makes the general theme of this manuscript very relevant and interesting. However, the analysis in the manuscript is not rigorous, and the claims in the abstract are not supported by the analysis in the main text.

      Following are my main comments on the work presented in this manuscript:

      (a) The central theme of this work is that spatially varying viscosity leads to position-dependent diffusion constant. This, for an overdamped Langevin dynamics with Gaussian white noise, leads to the well-known issue of the interpretation of the noise term. The authors use the Ito interpretation of the noise term because their system is non-equilibrium.

      One of the main criticisms I have is on this central point. The issue of interpretation arises only when there are ill-posed stochastic dynamics that do not have the relevant timescales required to analyze the noise term properly. Hence, if the authors want to start with an ill-posed equation it should be mentioned at the start. At least the Langevin dynamics considered should be explicitly mentioned in the main text. Since this work claims to be relevant to biological systems, it is also of significance to highlight the motivation for using the ill-posed equation rather than a well-posed equation. The authors refer to the non-equilibrium nature of the dynamics but it is not mentioned what non-equilibrium dynamics to authors have in mind. To properly analyze an overdamped Langevin dynamics a clear source of integrated timescales must be provided. As an example, one can write the dynamics as<br /> Eq. (1) \dot x = f(x) + g(x) \eta , which is ill-defined if the noise \eta is delta correlated in time but well-defined when \eta is exponentially correlated in time. One can of course look at the limit in which the exponential correlation goes to a delta correlation which leads to Eq. (1) interpreted in Stratonovich convention. The choice to use the Ito convention for Eq. (1) in this case is not justified.

      (b) Generally, the manuscript talks of viscosity gradient but the equations deal with diffusion which is a combination of viscosity, temperature, particle size, and particle-medium interaction. There is no clear motivation provided for focus on viscosity (cytoplasm as such is a complex fluid) instead of just saying position-dependent diffusion constant. Maybe authors should use viscosity only when talking of a context where the existence of a viscosity gradient is established either in a real experiment or in a thought experiment.

      (c) The section "Viscophoresis drives particle accumulation" seems to not have new results. Fig. 1 verifies the numerical code used to obtain the results in the later sections. If that is the case maybe this section can be moved to supplementary or at least it should be clearly stated that this is to establish the correctness of the simulation method. It would also be nice to comment a bit more on the choice of simulation methods with changing hopping sizes instead of, for example, numerically solving stochastic ODE.

      A minor comment, the statement "the physically appropriate convention to use depends upon microscopic parameters and timescale hierarchies not captured in a coarse-grained model of diffusion." is not true as is noted in the references that authors mention, a correct coarse-grained model provides a suitable convention (see also Phys. Rev. E, 70(3), 036120., Phys. Rev. E, 100(6), 062602.).

      (d) The section "Interaction-mediated clustering is affected by viscophoresis" makes an interesting statement about the positioning of clusters by a viscous gradient. As a theoretical calculation, the interplay between position-dependent diffusivity and phase separation is indeed interesting, but the problem needs more analysis than that offered in this manuscript. Just a plot showing clustering with and without a gradient of diffusion does not give enough insight into the interplay between density-dependent diffusion and position-dependent diffusion. A phase plot that somehow shows the relative contribution of the two effects would have been nice. Also, it should be emphasized in the main text that the inter-particle interaction is through a density-dependent diffusion constant and not a conservative coupling by an interaction potential.

      (e) The section "In silico microrheology shows that viscophoresis manifests as anomalous diffusion" the authors show that the MSD with and without spatial heterogeneity is different. This is not a surprise - as the underlying equations are different the MSD should be different. There are various analogies drawn in this section without any justification:<br /> (i) "the saturation MSD was higher than what was seen in the homogeneous diffusion scenario possibly due to particles robustly populating the bulk milieu followed by directed motion into the viscous zone (similar to that of a Brownian ratchet, (Peskin et al., 1993))."<br /> (ii) "Note that lensing may cause particle displacements to deviate from a Gaussian distribution, which could explain anomalous behaviors observed both in our simulations and in experiments in cells (Parry et al., 2014)."<br /> Since the full trajectory of the particles is available, it can be analyzed to check if this is indeed the case.

      (f) The final section "In silico FRAP in a heterogeneously viscous environment ... " studies the MSD of the particles in a medium with heterogeneous viscous patches which I find the most novel section of the work. As with the section on inter-particle interaction, this needs further analysis.

      To summarise, as this is a theory paper, just showing MSD or in silico FRAP data is not sufficient. Unlike experiments where one is trying to understand the systems, here one has full access to the dynamics either analytically or in simulation. So just stating that the MSD in heterogeneous and homogeneous environments are not the same is not sufficient. With further analysis, this work can be of theoretical interest. Finally, just as a matter of personal taste, I am not in favor of the analogy with optical lensing. I don't see the connection.

    1. Reviewer #1 (Public Review):

      Summary: Yin Luo and colleagues describe a new regulatory mode of TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand) -induced apoptosis of tumor cells. The work is timely because high expectations existed on the possibility to induce tumor-specific apoptosis by activation of TRAIL-receptors DR4 and DR5. So far, however, attempted TRAIL-based anti-tumor therapies failed, and several tumor types were found to resist TRAIL-induced apoptosis. The work is also important because it aims at a better mechanistic understanding of TRAIL-induced apoptosis and TRAIL resistance of some tumor types.

      Strengths: The major novel finding of this study is that extracellular heparan sulfate (HS) acts as a positive regulator of TRAIL-induced tumor cell apoptosis, and that HS expression of different tumor cell lines correlates with their capacity to induce cell death. The authors first show by affinity chromatography and SPR that murine and human TRAIL bind strongly to heparin (heparin is a highly sulfated, and thus strongly negatively charged form of HS that is derived from connective tissue type mast cells), and identify three basic amino acids on the TRAIL N-terminus that are required for the interaction. Size exclusion chromatography (SEC) and multiangle light scattering (MALS) revealed that TRAIL exists as a trimer that requires a minimum heparin length of 8 sugar residues for binding, and small angle X-ray scattering (SAXS) showed that TRAIL interaction with longer oligosaccharides induced higher order multimerization of TRAIL. Consistent with these biochemical and biophysical analyses, HS on tumor cells contributes to TRAIL-binding to their cell surface and subsequent apoptosis. Minor novel findings include domain swapping observed by TRAIL trimer crystallization and different degrees of HS core protein and DR receptor expression in different tumor cell types. These findings are well supported and together with the advanced and established methodology used by the authors are the strengths of this paper. The paper will be of great interest to medical biologists studying TRAIL-resistance of tumors, to biologists interested in DR4 and DR5 receptor function and the effects of receptor internalization, and to glycobiologists aiming to understand the multiple important roles that HS plays in development and disease. The authors also raise the important point (and support it well) that routine heparin treatment of cancer patients potentially interferes with TRAIL-based therapies, providing one possible reason for their failure.

      Weaknesses: Despite the importance and the clear strengths of the paper, some of its aspects could have been developed further to better support the authors claims and their hypothesis that HS downregulation may represent one strategy to explain tumor resistance to TRAIL-induced apoptosis.

      1) The authors demonstrate that HS at the tumor surface promotes TRAIL binding, and that HS promotes TRAIL-induced breast cancer and myeloma cell apoptosis. These findings are based on pre-treatment with heparinase to remove cell-surface HS prior to TRAIL-treatment, or presence of soluble heparin to compete with cell-surface HS for TRAIL binding. A more direct way to establish such new HS function could have been genetic manipulation of cancer cells to overexpress HS (e.g. by Syndecan 1 core-protein transgene expression in resistant cell types, e.g. IM-9 cells) or to express less or undersulfated HS (by interfering with the biosynthetic pathway in apoptosis-prone cells, for example by targeted inactivation/RNAi of the HS co-polymerase or sulfotransferases in RPMI-8226 cells). Changed susceptibility of these cells to TRAIL-induced apoptosis would greatly support the authors claim and suggest one promising new strategy to decrease tumor resistance against TRAIL-induced apoptosis.

      2) The mechanistics of TRAIL-induced, HS-modulated tumor cell apoptosis could be more clearly defined. For example, the authors demonstrate convincingly that cell surface HS is essential for TRAIL-induced apoptosis in MDA-MB-453 breast cancer cells, and later show that a tumor cell line (IM-9 cells) that expresses HS and the core protein to which HS is attached to only limited degrees is the most resistant to TRAIL-induced apoptosis. Yet, these findings suggest, but do not prove a dose-dependent role of HS in TRAIL-induced apoptosis. Indeed, the authors later report that cell surface HS promotes TRAIL-induced myeloma cell apoptosis regardless of the sensitivity levels, and that other factors - the degree of TRAIL multimerization or DR4/DR5 receptor internalization - are also important. A cartoon could be added to the final figure to sort through these possible mechanisms and their relative importance.

      3) The authors show that RPMI-8226 cell-surface HS promotes DR5 internalization despite the absence of direct DR5/heparin interactions. This is important because, as the authors are certainly aware of, HS manipulation at the cell surface (for example by heparinase treatment) changes a plethora of signaling pathways that may also indirectly affect apoptosis. HS-dependent internalization of TRAIL-receptor complexes, however, provides an important direct link from HS expression to TRAIL-induced apoptosis. To further support this possible link, it would therefore be worthwhile to include the binding characteristics and HS-dependent internalization of DR4 into this study.

    1. Reviewer #1 (Public Review):

      Aquaporin-0 forms 2D crystals in the lens of the eye. This propensity to form 2D crystals was originally exploited to solve the structure of aquaporin-0 reconstituted in membranes. Existing structures do not explain why the proteins spontaneously form these arrays, however. In this work the authors investigate the hypothesis that the main lipids in the native membranes, sphingomyelin and cholesterol, contribute to lattice formation. By titrating the cholesterol: sphingomyelin ratio, the authors identify cholesterol binding sites of increasing stability. The authors identify a cholesterol that interacts with adjacent tetramers and is bound at an unusual membrane depth. Computational simulations suggest that this cholesterol is only stable in the context of adjacent tetramers (ie lattice formation) and that the presence of the cholesterol increases the stability of that interface. The exact mechanism is not clear, but the authors propose that the so-called "deep cholesterol" improves shape complementarity between adjacent tetramers and modulates the kinetics of protein-protein interactions. Finally, the authors provide a reasonable model for the role of cholesterol in

      Strengths of this manuscript include the analysis of multiple structures determined with different lipid compositions and lipid:cholesterol ratios. For each these, multiple lipids can be modelled, giving a good sense of the lipid specificity at various favorable lipid binding positions. In addition, multiple hypotheses are tested in a very thorough computational analysis that provides the framework for interpreting the structural observations. The authors also provide a thorough scholarly discussion that connects their work with other studies of membrane protein-cholesterol interactions.

      The model presented by the authors is consistent with the data described. Further testing of this model, for example by mutating the deep cholesterol binding site, would strengthen the model. However, such experiments might be challenging due to the relatively non-specific/hydrophobic nature of the deep cholesterol binding site.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Pulfer et al., describe the development and testing of a transformer-based deep learning architecture called ADeS, which the authors use to identify apoptotic events in cultured cells and live animals. The classifier is trained on large datasets and provides robust classification accuracies in test sets that are comparable to and even outperform existing deep learning architectures for apoptosis detection. Following this validation, the authors also design use cases for their technique both in vitro and in vivo, demonstrating the value of ADeS to the apoptosis research space.

      Strengths:<br /> ADeS is a powerful tool in the arsenal of cell biologists interested in the spatio-temporal co-ordinates of apoptotic events in vitro, since live cell imaging typically generates densely packed fields of view that are challenging to parse by manual inspection. The authors also integrate ADeS into the analysis of data generated using different types of fluorescent markers in a variety of cell types and imaging modalities, which increases its adaptability by a larger number of researchers. ADeS is an example of the successful deployment of activity recognition (AR) in the automated bioimage analysis space, highlighting the potential benefits of AR to quantifying other intra- and intercellular processes observable using live cell imaging.

      Weaknesses:<br /> A major drawback was the lack of access to the ADeS platform for the reviewers; the authors state that the code is available in the code availability section, which is missing from the current version of the manuscript. This prevented an evaluation of the usability of ADeS as a resource for other researchers. The authors also emphasize the need for label-free apoptotic cell detection in both their abstract and their introduction but have not demonstrated the performance of ADeS in a true label-free environment where the cells do not express any fluorescent markers. While Pulfer et al., provide a wealth of information about the generation and validation of their DL classifier for in vitro movies, and the utility of ADeS is obvious in identifying apoptotic events among FOVs containing ~1700 cells, the evidence is not as strong for in vivo use cases. They mention the technical challenges involved in identifying apoptotic events in vivo, and use 3D rotation to generate a larger dataset from their original acquisitions. However, it is not clear how this strategy would provide a suitable training dataset for understanding the duration of apoptotic events in vivo since the temporal information remains the same. The authors also provide examples of in vivo acquisitions in their paper, where the cell density appears to be quite low, questioning the need for automated apoptotic detection in those situations. In the use cases for in vivo apoptotic detection using ADeS (Fig 8), it appears that the location of the apoptotic event itself was obvious and did not need ADeS, as in the case of laser ablation in the spleen and the sparse distribution of GFP labeled neutrophils in the lymph nodes. Finally, the authors also mention that video quality altered the sensitivity of ADeS in vivo (Fig 6L) but fail to provide an example of ADeS implementation on a video of poor quality, which would be useful for end users to assess whether to adopt ADeS for their own live cell movies.

    1. Reviewer #1 (Public Review):

      The prevalence of primary angle closure glaucoma (PACG) is high in Asia compared to all over the world. This study focused on characterizing the metabolite profile associated with PACG, identifying potential blood diagnostic markers, assessing their specificity for PACG, and verifying their applicability to predict the progression of visual field loss. To this end, Li et al. implemented a 5-phases multicenter prospective study to identify novel candidate biomarkers of PACG. A total of 616 individuals were recruited, identifying 1464 distinct metabolites in the serum by metabolomics and chemiluminescence immunoassays. By applying different machine learning algorithms the metabolite androstenedione showed good discrimination between PACG and control subjects, in both the discovery and validation phases. This metabolite also showed alterations in the aqueous humor and higher levels of androstenedione seemed to be associated with faster loss of visual field. Overall, the authors claimed that serum androstenedione levels may provide a new biomarker for early detection and monitoring/predicting PACG severity/progression.

      Strengths:<br /> • Omics research on glaucoma is constrained by inadequate sample sizes, a dearth of validation sets to corroborate findings, and an absence of specificity analyses. The 5-phases study was designed to try to overcome these limitations. The study design is very robust, with well well-described discovery set (1 and 2), validation phase (1 and 2), supplemental phase, and cohort phase. Large and well-characterized patients with adequate control subjects contributed to the robustness of the results.<br /> • Combining untargeted and targeted metabolomics using mass spectrometry instruments (high resolution and low resolution) with an additional chemiluminiscence immunoassay determining androstenedione levels<br /> • Androstenedione achieved better diagnostic accuracy across the discovery and validation sets, with AUC varying between 0.85 and 1.0. Interestingly, baseline androstenedione levels can predict glaucoma progression via visual field loss results.<br /> • A positive correlation was observed between levels of androstenedione in serum and aqueous humor of PACG patients.<br /> • A level higher of 1.66 ng/mL of the metabolite androstenedione seems to imply a high risk of visual field loss. Androstenedione may serve as a predictor of glaucomatous visual field progression.

      Weakness:<br /> • A single biomarker seems very unlikely to be of much help in the detection of glaucoma due to the etiological heterogeneity of the disease, the existence of different subtypes, and the genetic variability among patients. Rather, a panel of biomarkers may provide more useful information for clinical prediction, including better sensitivity and specificity. The inclusion of additional metabolites already identifying in the study, in combination, may provide more reliable and correct assignment results.<br /> • The number of samples in the supplementary phase is low, larger sample sizes are mandatory to confirm the diagnostic accuracy.<br /> • Cohorts from different populations are needed to verify the applicability of this candidate biomarker.<br /> • Sex hormones seem to be associated also with other types of glaucoma, such as primary open-angle glaucoma (POAG), although the molecular mechanisms are unclear (see doi:10.1167/iovs.17-22708). The inclusion of patients diagnosed with other subtypes of glaucoma, like POAG, may contribute to determining the sensitivity and specificity of the proposed biomarker. Androstenedione levels should be determined in POAG, NTG, or PEXG patients.<br /> • In addition, the levels of androstenedione were found significantly altered during other diseases as described by the authors or by conditions like polycystic ovary syndrome, limiting the utility of the proposed biomarker.<br /> • Uncertainty of the androstenedione levels compromises its usefulness in clinical practice.

    1. Reviewer #1 (Public Review):

      The authors found that nifuroxazide has the potential to augment the efficacy of radiotherapy in HCC by reducing PD-L1 expression. This effect may be attributed to increased degradation of PD-L1 through the ubiquitination-proteasome pathway. The paper provides new ideas and insights to improve treatment effectiveness, however, there are additional points that could be addressed.

      -The paper highlights that the combination of nifuroxazide increases tumor cell apoptosis. A discussion regarding the potential crosstalk or regulatory mechanisms between apoptotic pathways and PD-L1 expression would be valuable.

      -The benefits and advantages of nifuroxazide combination could be compared to the current clinical treatment options.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The data clearly demonstrate that arpin is important for vessel barrier function, yet its genetic loss via a CRISPR strategy was not lethality, but led to viable animals in C57Blk strain at 12 weeks of age, albeit with leaky blood vessels. Pharmacological approaches were employed to demonstrate that loss of arpin led to ROCK1-dependent stress fiber formation that promoted increased permeability.

      Strengths:<br /> The results clearly demonstrate that arpin is expressed in the endothelium of blood vessels and its deficiency leads to leaky blood vessels in in vivo and in vitro models.

      Weaknesses:<br /> They conclude vessel leak was not related to enhanced Arp2/3 function through arpin deficiency, but no direct evidence of Arp2/3 activity is provided to support this conclusion. Instead, the authors concluded that ROCK1 activity was elevated in arpin knockdown cells and caused robust stress fiber formation. This idea could be strengthened by testing if ROCK1 inhibition by pharmacological block in arpin KO mice leads to less vascular leakage while pharmacological inhibition of Arp2/3 does not attenuate increased vessel permeability.

    1. Reviewer #1 (Public Review):

      Summary: The authors of this study sought to define a role for IgM in responses to house dust mites in the lung.

      Strengths:

      Unexpected observation about IgM biology<br /> Combination of experiments to elucidate function

      Weaknesses:

      Would love more connection to human disease

    1. Reviewer #1 (Public Review):

      Summary: The authors identified that genetically and pharmacological inhibition of CERS1, an enzyme implicated in ceramides biosynthesis worsen muscle fibrosis and inflammation during aging.

      Strengths: the study points out an interesting issue on excluding CERS1 inhibition as a therapeutic strategy for sarcopenia. Overall, the article it's well written and clear.

      Weaknesses: Many of the experiments confirmed previous published data, which also show a decline of CERS1 in ageing and the generation and characterization of a muscle specific knockout mouse line. The mechanistic insights of how the increased amount of long ceramides (cer c24) and the decreased of shorter ones (cer c18) might influence muscle mass, force production, fibrosis and inflammation in aged mice have not been addressed.

    1. Reviewer #1 (Public Review):

      The mechanisms underlying the generation and maintenance of LLPCs have been one of the unresolved issues. Recently, four groups have independently generated new genetic tools that allow fate tracing of murine plasma cells and have addressed how LLPCs are generated or maintained in homeostatic conditions or upon antigen immunization or viral infection. Here, Jing et al. have established another, but essentially the same, PC time stamping system, and tried to address the issues above. The question is whether the findings reported here provide significant conceptual advances from what has already been published.

      1) Some of the observations in this manuscript have already been made by other studies (Xu et al. 2020, Robinson et al. 2022, Liu et al. 2022, Koike et al. 2023, Robinson et al. 2023). In my opinion, however, genetic analysis of the role of CXCR4 on PC localization or survival in BM (Figure 5) was well performed and provided some new aspects which have not been addressed in previous reports. The motility of CXCR4 cKO plasma cells in BM is not shown, but it could further support the idea that reduced mobility or increased clustering is required for longevity.

      2) The combination of the several surface markers shown in Figure 3&4 doesn't seem to be practically applicable to identify or gate on LLPCs, because differential expression of CD81, CXCR4, CD326, CD44, or CD48 on LLPCs vs bulk PCs was very modest. EpCAMhi/CXCR3-, Ly6Ahi/Tigit- (Liu et al. 2022), B220lo/MHC-IIlo (Koike et al. 2023), or SLAMF6lo/MHC-IIlo (Robinson et al. 2023) has been reported as markers for LLPC population. It is unclear that the combination of surface markers presented here is superior to published markers. In addition, it is unclear why the authors did not use their own gene expression data (Fig.6), instead of using public datasets, for picking up candidate markers.

    1. Reviewer #1 (Public Review):

      The authors investigate the alpha chain TCR landscape in conventional vs regulatory CD4 T cells. Overall I think it is a very well thought out and executed study with interesting conclusions. The authors have investigated CDR3 alpha repertoires coupled with a transgenic fixed CDR3beta in a mouse system.

      Strengths:<br /> - One of a kind evidence and dataset.

      - State-of-the-art analyses using tools that are well-accepted in the literature.

      - Interesting conclusions on the breadth of immune response to challenges across different types of challenges (tumor, viral and parasitic).

      Weaknesses:<br /> - Some conclusions regarding the eCD4->eTreg transition are not so strong using only the data.

      - Some formatting issues.

    1. Joint Public Review:

      In this study, Cacho-Navas et al. describe the role of ICAM-1 expressed on the apical membrane of bile canaliculi and its function to control the bile canaliculi (BCs) homeostasis. This is a previously unrecognized function of this protein in hepatocytes. The same authors have previously shown that basolateral ICAM-1 plays a role in controlling lymphocyte adhesion to hepatocytes during inflammation and that this interaction is responsible for the loss of polarity of hepatocytes during disease states.

      This new study shows that ICAM-1 is mainly localized in the apical domain of the BC and in association with EBP-50, communicates with the subapical acto-myosin ring to regulate the size and morphology of the BC. They used the well-known immortal cell line of liver cells (HepG2) in which they deleted ICAM-1 gene by CRISPR-Cas9 editing and hepatic organoids derived from WT and ICAM-1-KO mice. alternating KO as well as rescue experiments. They show that in the absence of apical ICAM-1, the BC become dilated.

      The data sufficiently support the conclusions of the study.

    1. Reviewer #1 (Public Review):

      In this report, Hariharan and colleagues describe a protocol based on machine learning to differentiate wild type and DMD myofibers differentiated on a micropattern. They use images generated by immunofluorescence against the proteins of the DAPC complex, (which is disrupted in absence of dystrophin) to train a classifier to recognize healthy and diseased myofibers. They show that combining images generated with utrophin and alpha-sarcoglycan provide the most effective discrimination. They then validate the approach by applying their pipeline to wild type cells treated with DMD siRNA to mimic the DMD mutation or with antisense oligonucleotides to restore the DMD coding frame by exon skipping. They show that their strategy groups the siRNA treated myofibers with the DMD ones efficiently. Grouping of the oligonucleotide treated DMD myofibers with the healthy also works but is less efficient.

      Overall the work is well done and I don't have any significant technical critique. I found this highly technical study interesting for a specialized audience.

    1. Joint Public Review:

      The study by Ding et al demonstrated that microbial metabolite I3A reduced western diet induced steatosis and inflammation in mice. They showed that I3A mediates its anti-inflammatory activities through AMP-activated protein kinase (AMPK)-dependent manner in macrophages. Translationally, they proposed that I3A could be a potential therapeutic molecule in preventing the progression of steatosis to NASH.

      Major strengths<br /> • Authors clearly demonstrated that the Western Diet (WD)-induced steatosis and I3A treatment reduced steatosis and inflammation in pre-clinical models. Data clearly supports these statements.<br /> • I3A treatment rescued WD-altered bile acids as well as partially rescued the metabolome, proteome in the liver.<br /> • I3A treatment reduced the levels of enzymes in fatty acid transport, de novo lipogenesis and β-oxidation<br /> • I3A mediates its anti-inflammatory activities through AMP-activated protein kinase (AMPK)-dependent manner in macrophages.

      Minor<br /> I agree with the authors that investigating known other AhR ligands in comparison may be beyond the scope of this study.

    1. Reviewer #1 (Public Review):

      Summary:

      This study by Lee et al. is a direct follow-up on their previous study that described an evolutionary conservancy among placental mammals of two motifs (a transmembrane motif and a juxtamembrane palmitoylation site) in CD4, an antigen co-receptor, and showed their relevance for T-cell antigen signaling. In this study, they describe the contribution of these two motifs to the CD4-mediated antigen signaling in the absence of CD4-LCK binding. Their approach was the comparison of antigen-induced proximal TCR signaling and distal IL-2 production in 58-/- T-cell hybridoma expressing exogenous truncated version of CD4 (without the interaction with LCK), called T1 with T1 version with the mutations in either or both of the conserved motifs. They show that the T1 CD4 can support signaling to the extend similar to WT CD4, but the mutation of the conserved motifs substantially reduced the signaling. The authors conclude that the role of these motifs is independent of the LCK-binding.

      Strengths:<br /> The authors convincingly show that T1 CD4, lacking the interaction with LCK supports the TCR signaling and also that the two studied motifs have a significant contribution to it.

      Weaknesses:<br /> The study has several weaknesses.

      1. The whole study is based on a single experimental system, genetically modified 58-/- hybridoma. It is unclear at this moment, how the molecular motifs studied here contribute to the signaling in a real T cell. The evolutionary conservancy suggests that these motifs are important for T cell biology. However, the LCK-binding motif is conserved as well (perhaps even more) and it plays a very minor role in their model. Without verifying their results in primary cells, the quantitative, but even qualitative, importance of these motifs for T-cell signaling and biology is unclear. Although the authors discuss this issue in the Discussion, it should be noted in all important parts of the manuscript, where conclusions are made (abstract, end of introduction, perhaps also in the title) that the results are coming from the hybridoma cells.

      2. Many of the experiments lack the negative control. I believe that two types of negative controls should be included in all experiments. First, hybridoma cells without CD4 (or with CD4 mutant unable to bind MHCII). Second, no peptide control, i.e., activation of the hybridoma cells with the APC not loaded with the cognate peptide. These controls are required to distinguish the basal levels of phoshorylation and CD4-independent antigen-induced phosphorylation to quantify, what is the contribution of the particular motifs to the CD4-mediated support. Although these controls are included in some of the experiments, they are missing in other ones. The binding mutant appears in some FC results as a horizontal bar (without any error bar/variability), showing that CD4 does not give a huge advantage in these readouts. Why don't the authors show no peptide controls here as well? Why the primary FC data (histograms) are not shown? Why neither of these two controls is shown for the % of responders plots? Although the IL-2 production is a very robust and convincing readout, the phosphoflow is much less sensitive. It seems that the signaling is elevated only marginally. Without the mentioned controls and showing the raw data, the precise interpretation is not possible.

      3. The processing of the data is not clear. Some of the figures seem to be overprocessed. For instance, I am not sure what "Normalized % responders of pCD3zeta" means (e.g., Fig. 1C and elsewhere)? Why do not the authors show the actual % of pCD3zeta+ cells including the gating strategy? Why do the authors subtract the two histograms in Fig. 2- Fig.S3? It is very unusual.

      4. The manuscript lacks Materials and Methods. It only refers to the previous paper, which is very unusual. Although most of the methods are the same, they still should be mentioned here. Moreover, some of the mutants presented here were not generated in the previous study, as far as I understand. Perhaps the authors plan to include Materials and Methods during the revision...

      5. Membrane rafts are a very controversial topic. I recommend the authors stick to the more consensual term "detergent resistant microdomains" in all cases/occurances.

      6. Last, but not least, the mechanistic explanation (beyond the independence of LCK binding) of the role of these motifs is very unclear at the moment.

    1. Reviewer #1 (Public Review):

      Summary: Zai et al test if songbirds can recover the capacity to sing auditory targets without singing experience or sensory feedback. Past work showed that after the pitch of targeted song syllables is driven outside of birds' preferred target range with external reinforcement, birds revert to baseline (i.e. restore their song to their target). Here the authors tested the extent to which this restoration occurs in muted or deafened birds. If these birds can restore, this would suggest an internal model that allows for sensory-to-motor mapping. If they cannot, this would suggest that learning relies entirely on feedback-dependent mechanisms, e.g. reinforcement learning (RL). The authors find that deafened birds exhibit moderate but significant restoration, consistent with the existence of a previously under-appreciated internal model in songbirds.

      Strengths:<br /> The experimental approach of studying vocal plasticity in deafened or muted birds is innovative, technically difficult, and perfectly suited for the question of feedback-independent learning. The finding in Figure 4 that deafened birds exhibit subtle but significant plasticity toward restoration of their pre-deafening target is surprising and important for the songbird and vocal learning fields, in general.

      Weaknesses:<br /> The evidence and analyses related to the directed plasticity in deafened birds are confusing, and the magnitude of the plasticity is far less than the plasticity observed in control birds with intact feedback. The authors acknowledge this difference in a two-system model of vocal plasticity, but one wonders why the feedback-independent model, which could powerfully enhance learning speed, is weak in this songbird system.

      There remains some confusion about the precise pitch-change methods used to study the deafened birds, including the possibility that a critical cohort of birds was not suitably balanced in a way where deafened birds were tested on their ability to implement both pitch increases and decreases toward target restoration.

    1. Reviewer #1 (Public Review):

      This is an elegant didactic exposition showing how dendritic plateau potentials can enable neurons to perform reliable 'binary' computations in the face of realistic spike time jitter in cortical networks. The authors make many good arguments, and the general concept underlying the paper is sound. A strength is their systematic progression from biophiysical to simplified models of single neurons, and their parallel investigation of spiking and binary neural networks, with training happening in the binary neural network.

    1. Reviewer #1 (Public Review):

      Summary: In the manuscript by Chen et al. entitled, "The retina uncouples glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle", the authors look to provide insight on retinal metabolism and substrate utilization by using a murine explant model with various pharmacological treatments in conjunction with metabolomics. The authors conclude that photoreceptors, a specific cell within the explant, which also includes retinal pigment epithelium (RPE) and many other types of cells, are able to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: 1) the mini-Krebs-cycle, fueled by glutamine and branched-chain amino acids; 2) the alanine-generating Cahill-cycle; and 3) the lactate-releasing Cori-cycle. While intriguing if determined to be true, these cell-specific conclusions are called into question due to the ex vivo experimental setup with the inclusion of RPE, the fact that the treatments were not cell-specific nor targeted at an enzyme specific to a certain cell within the retina, and no stable isotope tracing nor mitochondrial function assays were performed. Hence, without significant cell-specific methods and future experimentation, the primary claims are not supported.

      Strengths: This study attempts to improve on the issues that have limited the results obtained from previous ex vivo retinal explant studies by culturing in the presence of the RPE, which is a major player in the outer retinal metabolic microenvironment. Additionally, the study utilizes multiple pharmacologic methods to define retinal metabolism and substrate utilization.

      Weaknesses: A major weakness of this study is the lack of in vivo supporting data. Explant cultures remove the retina from its dual blood supply. Typically, retinal explant cultures are done without RPE. However, the authors included RPE in the majority of experimental conditions herein. However, it is unclear if the metabolomics samples included the RPE or not. The inclusion of the RPE, which is metabolically active and can be altered by the treatments investigated herein, further confounds the claims made regarding the neuroretina. Considering the pharmacologic treatments utilized with the explant cultures are not cell-specific and/or have significant off-target effects, it is difficult to ascertain that the metabolic changes are secondary to the effects on photoreceptors alone, which the authors claim. Additionally, the explants are taken at a very early age when photoreceptors are known to still be maturing. No mention or data is presented on how these metabolic changes are altered in retinal explants after photoreceptors have fully matured. Likewise, significant assumptions are made based on a single metabolomics experiment with no stable isotope tracing to support the pathways suggested. While the authors use immunofluorescence to support their claims at multiple points, demonstrating the presence of certain enzymes in the photoreceptors, many of these enzymes are present throughout the retina and likely the RPE. Finally, the claims presented here are in direction contradiction to recent in vivo studies that used cell-specific methods when examining retinal metabolism. No discussion of this difference in results is attempted.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this manuscript, Abe and colleagues employ in vivo 2-photon calcium imaging of dopaminergic axons in the mPFC. The study reveals that these axons primarily respond to unconditioned aversive stimuli (US) and enhance their responses to initially-neutral stimuli after classical association learning. The manuscript is well-structured and presents results clearly. The utilization of a refined prism-based imaging technique, though not entirely novel, is well-implemented. The study's significance lies in its contribution to the existing literature by offering single-axon resolution functional insights, supplementing prior bulk measurements of calcium or dopamine release. Given the current focus on neuromodulator neuron heterogeneity, the work aligns well with current research trends and will greatly interest researchers in the field.

      However, I would like to highlight that the authors could further enhance their manuscript by addressing study limitations more comprehensively and by providing essential details to ensure the reproducibility of their research. In light of this, I have a number of comments and suggestions that, if incorporated, would significantly contribute to the manuscript's value to the field.

      Strengths:<br /> -Descriptive.<br /> -Utilization of a well-optimized prism-based imaging method.<br /> -Provides valuable single-axon resolution functional observations, filling a gap in existing literature.<br /> -Timely contribution to the study of neuromodulator neuron heterogeneity.

      Weaknesses:<br /> 1. It's important to fully discuss the fact that the measurements were carried out only on superficial layers (30-100um), while major dopamine projections target deep layers of the mPFC as discussed in the cited literature (Vander Weele et al., 2018) and as illustrated in FigS1B,C. This limitation should be explicitly acknowledged and discussed in the manuscript, especially given the potential functional heterogeneity among dopamine neurons in different layers. This potential across-layer heterogeneity could also be the cause of discrepancy among past recording studies with different measurement modalities. Also, mentioning technical limitations would be informative. For example: how deep the authors can perform 2p-imaging through the prism? was the "30-100um" maximum depth the authors could get?

      2. In the introduction, it seems that the authors intended to refer to Poulin et al. 2018 regarding molecular/anatomical heterogeneity of dopamine neurons, but they inadvertently cited Poulin et al. 2016 (a general review on scRNAseq). Additionally, the statement that "dopamine neurons that project to the PFC show unique genetic profiles (line 85)" requires clarification, as Poulin et al. 2018 did not specifically establish this point. Instead, they found at least the Vglut2/Cck+ population projects into mPFC, and they did not reject the possibility of other subclasses projecting to mPFC. Rather, they observed denser innervation with DAT-cre, suggesting that non-Vglut2/Cck populations would also project to mPFC. Discuss the potential molecular heterogeneity among mPFC dopamine axons in light of the sampling limitation mentioned earlier.

      3. I find the data presented in Figure 2 to be odd. Firstly, the latency of shock responses in the representative axons (right panels of G, H) is consistently very long - nearly 500ms. It raises a query whether this is a biological phenomenon or if it stems from a potential technical artifact, possibly arising from an issue in synchronization between the 2-photon imaging and stimulus presentation. My reservations are compounded by the notable absence of comprehensive information concerning the synchronization of the experimental system in the method section. Secondly, there appear to be irregularities in Panel J. While the authors indicate that "Significant axons were classified as either reward-preferring (cyan) or aversive-preferring (magenta), based on whether the axons are above or below the unity line of the reward/aversive scatter plot (Line 566)," a cyan dot slightly but clearly deviates above the unity line (around coordinates (x, y) = (20, 21)). This needs clarification. Lastly, when categorizing axons for analysis of conditioning data in Fig3 (not Fig2), the authors stated "The color-coded classification (cyan/magenta) was based on k-means clustering, using the responses before classical conditioning (Figure 2J)". I do not understand why the authors used different classification methods for two almost identical datasets.

      4. In connection with Point 3, conducting separate statistical analyses for aversive and rewarding stimuli would offer a fairer approach. This could potentially reveal a subset of axons that display responses to both aversive and appetitive stimuli, aligning more accurately with the true underlying dynamics. Moreover, the characterization of Figure 2J as a bimodal distribution while disregarding the presence of axons responsive to both aversive and appetitive cues seems somewhat arbitrary and circular logic. A more inclusive consideration of this dual-responsive population could contribute to a more comprehensive interpretation.

      5. The contrast in initialization to novel cues between aversive and appetitive axons mirrors findings in other areas, such as the tail-of-striatum (TS) and ventral striatum (VS) projecting dopamine neurons (Menegas et al., 2017, not 2018). You might consider citing this very relevant study and discussing potential collateral projections between mPFC and TS or VS.

      6. The use of correlation values (here >0.65) to group ROIs into axons is common but should be justified based on axon density in the FOV and imaging quality. It's important to present the distribution of correlation values and demonstrate the consistency of results with varying cut-off values. Also, provide insights into the reliability of aversive/appetitive classifications for individual ROIs with high correlations. Importantly, if you do the statistical testing and aversive/appetitive classifications for individual ROIs with above-threshold high correlation (to be grouped into the same axon), do they always fall into the same category? How many false positives/false negatives are observed?
<br /> "Our results remained similar for different correlation threshold values (Line 556)" (data not shown) is obsolete.

    1. Joint Public Review:

      Neuropeptide signaling is an important component of nervous systems, where neuropeptides typically act via G-protein coupled receptors (GPCRs) to regulate many physiological and behavioral processes. Neuropeptides and their cognate GPCRs have been extensively characterized in bilaterian animals, revealing that a core set of neuropeptide signaling systems originated in common ancestors of extant Bilateria. Neuropeptides have also been identified in cnidarians, which are a sister group to the Bilateria. However, the GPCRs that mediate the effects of neuropeptides in cnidarians have not been identified.

      In this paper the authors perform a phylogenetic analysis of GPCRs in metazoans and report that the orthologs of bilaterian neuropeptide receptors are not found in cnidarians. This indicates that neuropeptide signaling systems have largely evolved independently in cnidarians and bilaterians. To accomplish this, they generated a library of putative and known neuropeptides computationally identified in the genome of the cnidarian sea anemone Nematostella vectensis. These peptides were systematically screened for their ability to activate any of the 161 putative Nematostella GPCRs.

      This work identified 31 validated GPCRs. These, together with GPCRs that cluster with them, were then used to demonstrate the independent expansion of GPCRs in cnidarian and bilaterian lineages. The authors then mapped validated receptors and ligands to the Nematostella single cell data to provide an overview of the cell types expressing these signaling genes. In addition, the authors have begun to analyze neuropeptide signaling networks in N. vectensis by showing potential signaling connections between cell types expressing neuropeptides and cell types expressing cognate receptors.

      This work is the most extensive pharmacological characterization of neuropeptide GPCRs in a cnidarian to date and thus represents an important accomplishment, and is one that will improve our understanding of how peptidergic signaling evolved in animals and its impact on evolution of nervous systems."

      The reviewers did not detect any weaknesses in the work but ask that the authors comment on the following points:<br /> 1- It was not clear why the phylogenetic analysis included non-validated GPCRs that clustered with the validated peptidergic receptors. Would restricting the phylogenetic analyses only to confirmed peptidergic GPCRs alter the topology of the tree and subsequent conclusions of independent expansion?<br /> 2- Clearly, other neuropeptide signaling systems in cnidarians remain to be discovered but this paper represents a huge step forward.<br /> 3- There are limitations in what can be interpreted from single cell transcriptomic data but the data nevertheless provide the foundations for future studies involving i). detailed anatomical analysis of neuropeptide and neuropeptide receptor expression in N. vectensis using mRNA in situ hybridization and/or immunohistochemical methods and ii). functional analysis of the physiological/behavioral roles of neuropeptide signaling systems in N. vectensis

    1. Reviewer #1 (Public Review):

      Summary:

      This research study utilizes a realistic motoneuron model to explore the potential to trace back the appropriate levels of excitation, inhibition, and neuromodulation in the firing patterns of motoneurons observed in in-vitro and in-vivo experiments in mammals. The research employs high-performance computing power to achieve its objectives. The work introduces a new framework that enhances understanding of the neural inputs to motoneuron pools, thereby opening up new avenues for hypothesis testing research.

      Strengths:<br /> The significance of the study holds relevance for all neuroscientists. Motoneurons are a unique class of neurons with known distribution of outputs for a wide range of voluntary and involuntary motor commands, and their physiological function is precisely understood. More importantly, they can be recorded in-vivo using minimally invasive methods, and they are directly impacted by many neurodegenerative diseases at the spinal cord level.<br /> The computational framework developed in this research offers the potential to reverse engineer the synaptic input distribution when assessing motor unit activity in humans, which holds particular importance.<br /> Overall, the strength of the findings focuses on providing a novel framework for studying and understanding the inputs that govern motoneuron behavior, with broad applications in neuroscience and potential implications for understanding neurodegenerative diseases. It highlights the significance of the study for various research domains, making it valuable to the scientific community.

      Weaknesses: The exact levels of inhibition, excitation, and neuromodulatory inputs to neural networks are unknown. Therefore the work is based on fine-tuned measures that are indirectly based on experimental results. However, obtaining such physiological information is challenging and currently impossible. From a computational perspective it is a challenge that in theory can be solved. Thus, although we have no ground-truth evidence, this framework can provide compelling evidence for all hypothesis testing research and potentially solve this physiological problem with the use of computers.

    1. Reviewer #1 (Public Review):

      Summary:<br /> I read the paper by Parrotta et al with great interest. The authors are asking an interesting and important question regarding pain perception, which is derived from predictive processing accounts of brain function. They ask: If the brain indeed integrates information coming from within the body (interoceptive information) to comprise predictions about the expected incoming input and how to respond to it, could we provide false interoceptive information to modulate its predictions, and subsequently alter the perception of such input? To test this question, they use pain as the input and the sounds of heartbeats (falsified or accurate) as the interoceptive signal.

      Strengths:<br /> I found the question well-established, interesting, and important, with important implications and contributions for several fields, including neuroscience of prediction-perception, pain research, placebo research, and health psychology. The paper is well-written, the methods are adequate, and the findings largely support the hypothesis of the authors. The authors carried out a control experiment to rule out an alternative explanation of their finding, which was important.

      Weaknesses:<br /> I will list here one theoretical weakness or concern I had, and several methodological weaknesses.

      The theoretical concern regards what I see as a misalignment between a hypothesis and a result, which could influence our understanding of the manipulation of heartbeats, and its meaning: The authors indicate from prior literature and find in their own findings, that when preparing for an aversive incoming stimulus, heartbeats *decrease*. However, in their findings, manipulating the heartbeats that participants hear to be slower than their own prior to receiving a painful stimulus had *no effect* on participants' actual heartbeats, nor on their pain perceptions. What authors did find is that when listening to heartbeats that are *increased* in frequency - that was when their own heartbeats decreased (meaning they expected an aversive stimulus) and their pain perceptions increased.

      This is quite complex - but here is my concern: If the assumption is that the brain is collecting evidence from both outside and inside the body to prepare for an upcoming stimulus, and we know that *slowing down* of heartbeats predicts an aversive stimulus, why is it that participants responded in a change in pain perception and physiological response when listened to *increased heartbeats* and not decreased? My interpretation is that the manipulation did not fool the interoceptive signals that the brain collects, but rather the more conscious experience of participants, which may then have been translated to fear/preparation for the incoming stimulus. As the authors indicate in the discussion (lines 704-705), participants do not *know* that decreased heartbeats indicate upcoming aversive stimulus, and I would even argue the opposite - the common knowledge or intuitive response is to increase alertness when we hear increased heartbeats, like in horror films or similar scenarios. Therefore, the unfortunate conclusion is that what the authors assume is a manipulation of interoception - to me seems like a manipulation of participants' alertness or conscious experience of possible danger. I hope the (important) distinction between the two is clear enough because I find this issue of utmost importance for the point the paper is trying to make. If to summarize in one sentence - if it is decreased heartbeats that lead the brain to predict an approaching aversive input, and we assume the manipulation is altering the brain's interoceptive data collection, why isn't it responding to the decreased signal? --> My conclusion is, that this is not in fact a manipulation of interoception, unfortunately.

      I will add that the control experiment - with an exteroceptive signal (knocking of wood) manipulated in a similar manner - could be seen as evidence of the fact that heartbeats are regarded as an interoceptive signal, and it is an important control experiment, however, to me it seems that what it is showing is the importance of human-relevant signals to pain prediction/perception, and not directly proves that it is considered interoceptive. For example, it could be experienced as a social cue of human anxiety/fear etc, and induce alertness.

      Several additional, more methodological weaknesses include the very small number of trials per condition - the methods mention 18 test trials per participant for the 3 conditions, with varying pain intensities, which are later averaged (and whether this is appropriate is a different issue). This means 6 trials per condition, and only 2 trials per condition and pain intensity. I thought that this number could be increased, though it is not a huge concern of the paper. It is, however, needed to show some statistics about the distribution of responses, given the very small trial number (see recommendations for authors). The sample size is also rather small, on the verge of "just right" to meet the required sample size according to the authors' calculations. Finally, and just as important, the data exists to analyze participants' physiological responses (ECG) after receiving the painful stimulus - this could support the authors' claims about the change in both subjective and objective responses to pain. It could also strengthen the physiological evidence, which is rather weak in terms of its effect. Nevertheless, this is missing from the paper.

      I have several additional recommendations regarding data analysis (using an ANOVA rather than multiple t-tests, using raw normalized data rather than change scores, questioning the averaging across 3 pain intensities) - which I will detail in the "recommendations for authors" section.

      Conclusion:<br /> To conclude, the authors have shown in their findings that predictions about an upcoming aversive (pain) stimulus - and its subsequent subjective perception - can be altered not only by external expectations, or manipulating the pain cue, as was done in studies so far, but also by manipulating a cue that has fundamental importance to human physiological status, namely heartbeats. Whether this is a manipulation of actual interoception as sensed by the brain is - in my view - left to be proven.<br /> Still, the paper has important implications in several fields of science ranging from neuroscience prediction-perception research, to pain and placebo research, and may have implications for clinical disorders, as the authors propose. Furthermore, it may lead - either the authors or someone else - to further test this interesting question of manipulation of interoception in a different or more controlled manner.

      I salute the authors for coming up with this interesting question and encourage them to continue and explore ways to study it and related follow-up questions.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The question at hand is whether astrocytes contribute to the mechanism of long-term synaptic potentiation (LTP) at synaptic contacts between excitatory glutamatergic neurons and inhibitory neurons (E-I synapses). This is a legitimate query considering the immense body of work that has now established synaptic plasticity (LTP, LTD and spike-timing dependent plasticity) as an astrocyte-dependent process at excitatory synapses and, by contrast, the lack of knowledge on whether and how astrocytes control IN activity. Taking direct inspiration from that same body of work, authors recapitulate a number of experiments and approaches from prior seminal studies and provide evidence that E-I synapses in the stratum radiatum of the hippocampus display NMDAR-dependent plasticity, which can be suppressed by pharmacologically hindering astrocytes physiology, preventing astrocyte Ca2+ transients or blocking endocannabinoid CB1 receptors. Under any of these conditions, LTP can still be rescued by exogenously applying D-serine, a naturally occurring co-agonist of NMDARs primarily released by astrocytes. Coincidently, authors show that the conditions used to elicit LTP also cause a transient increase in NMDAR co-agonist site occupancy. Lastly, based on some evidence that gamma-CaMKII is predominantly expressed in INs rather than excitatory neurons, authors conducted AAV-mediated IN-specific gamma-CaMKII shRNA experiments and found that this is sufficient to suppress LTP at E-I synapses. They found that this approach also impairs contextual fear learning in behaving mice. Authors conclude that astrocytes gate LTP at E-I synapses via a mechanism wherein neuronal depolarization during LTP induction elicits endocannabinoid release which drives CB1-dependent astrocyte Ca2+ activity, causing the release of the NMDAR co-agonist D-serine (required for NMDAR activation).

      Strengths:<br /> This is an important question and the experimental work seems to have been conducted at high standards. The electrophysiology traces are impeccable, the experiments are well powered, including the behavioral testing, and multiple controls and validations are provided throughout. The figures are clear and easy to understand. Overall, the conclusions from the study are consistent, or partially consistent, by the findings.

      Main Weaknesses:<br /> 1- A major point of concern is the lack of proper acknowledgment of the seminal studies that were mimicked in this manuscript, notably Henneberger et al, Nature 2010, Adamsky et al, Cell 2018; and Robin et al., Neuron 2017. The entire study design is a replica of these landmark studies: it isn't built upon or inspired from them, it exactly repeats the experiments and methods performed in them, coming dangerously close to being simply a hidden attempt to plagiarize published work. The resemblance goes as far as using an identical figure display (see Fig4.D vs Fig 2D of Ref#4). The issue is that authors frame the problem, scientist logic, reasoning, technical tricks, approaches, and interpretations as their own whereas, in reality, they were taken verbatim out of previous work and applied to a (shockingly) similar problem. The probity of the present study is thus in question. Authors need to clearly acknowledge, in all relevant instances, that the work presented here recapitulates the approach, reasoning and methodology used in past seminal studies that tackled the mechanisms of astrocyte regulation of LTP.

      2-Relatedly, in past work, field recordings were used to monitor LTP in hippocampal slices (refs 4, 26 and others). This method captures indiscriminately all excitatory synapses where glutamate is released to cause AMPAR-dependent (and NMDAR) transmembrane flux of cations in the postsynaptic element, including E-I synapses and not just E-E synapse like the authors claim. Therefore, a strong argument can be made that there is no actual ground to differentiate the present results from past ones.

      3-There is a general lack of excitement about this study. One reason is that it replicates almost identically past work, as mentioned above. Another is that the scientific question and importance of the findings are not framed appropriately. The work is presented as an astrocyte-focused investigation, but it has very limited value to the astrocyte field. The findings are, in all accounts, identical to those unveiled by previous work especially because E-I synapses are, in fact, excitatory synapses. Where this study does bring value, however, is to the field of interneurons, but it would need to be reframed to shift the emphasis from astrocytes to E-I connections. Authors would need to elevate the text by framing their work around relevant considerations, such as IN diversity, mechanisms of LTP in IN subtypes, role of E-I connections in hippocampal circuit function, information processing, behavior, spatial learning, navigation, or grid cells activity etc...

      4-A clear weakness of the study is that it fails to consider the molecular and functional diversity of interneurons in the stratum radiatum and provides no insights or considerations related to it. Authors provide no information on what type of IN were patched, or the location of their cell body in the s.r., effectively treating all patched IN as a homogeneous ensemble of cells - which they are not. Relatedly, the study is extremely evasive on the importance of the results in the context of inhibitory interneurons. This renders the significance of the insights highly uncertain and dampens both the impact of the study and the excitement it generates. Hippocampal interneurons are very diverse in molecular identity, sub-anatomical location, morphology, projections, connectivity and functional importance. Some experts go as far as recognizing 29 subtypes in the CA1, including 9 in the stratum radiatum alone (based on the location of their soma). However, this is neither addressed nor acknowledged by the authors, with the exception of a statement (line 659) where they claim to have "focused on a subpopulation of interneurons in the stratum radiatum" without providing any precision or evidence to corroborate this assertion. This diversity, alone, could explain why not all cells showed LTP, or why the mechanisms authors describe in the radiatum do not seem to be at play in the oriens. Hence, carefully considering the diversity of INs in the present work is necessary. It would refine and augment the conclusions of the paper. Instead of a sub-region specificity, the study might fuel the notion of an IN subtype specificity of LTP mechanisms, which is more useful to the field.

      5-Authors take several shortcuts. Some of the conclusions are a leap from the experiments and are only acceptable due to the close analogy with very similar investigations conducted in the past that provided identical results. For instance, the present study provides no evidence of any sort that D-serine is involved - rather, it provides evidence that the pathway at hand contributes to increasing the occupancy of the co-agonist binding site of NMDARs. Considering the absence of work demonstrating that D-serine is the endogenous co-agonist of NMDARs at E-I synapses, most of the authors claims on D-serine are unfounded. This would necessitate using tools such as the canonical D-serine scavengers DAAS or DsDA, serine racemase KO mice etc. Similarly, authors provide no compelling evidence that endocannabinoid CB1 receptors involved in this pathway are located on astrocytes

      6-An important caveat in this study is the protocol employed to induce LTP, which includes steps of sustained depolarization of the patched IN to -10mV. Neuronal depolarization is known to induce endocannabinoids production. In several instances, this was shown to 'activate' astrocytes and elicit the release of astrocyte-derived transmitters at nearby synapses. This implies that the endocannabinoid-dependent pathway described in the study is, most likely, artificially engaged by the protocol itself. Hence, the present work only provides evidence that an astrocyte-dependent, CB1-D-serine-pathway can be artificially called upon with this specific LTP protocol, but does not convincingly demonstrate that it is naturally occurring or necessary for plasticity at E-I synapses. Authors would need to thoroughly address this caveat by replicating some of their key findings (AM251, calcium-clamp, D-serine and CaMKII shRNA) using a protocol that does not entail the artificial depolarization of the patched interneuron.

      7-Reading and understanding are hindered by a rather vast array of issues with the text itself. It needs thorough editing for typos, misnomers, meaning-altering errors in syntax, and a number of issues with English.

    1. Reviewer #1 (Public Review):

      This paper presents a set of experiments designed to test whether gravity in people's intuitive physics engine is implemented as a simple deterministic representation of gravity or as a Gaussian distribution. The work shows experimentally that the probabilistic representation of gravity does a better job at capturing both human judgments, including biases in stability inferences. The work further shows that Gaussian representations of gravity can evolve in a simple agent-environment reinforcement learning problem setup.

      Strengths:<br /> The paper approaches the problem from three different angles in an impressive way. The first is through a direct comparison of human judgments against model predictions. The second is through an analysis of whether the model correctly predicts cognitive illusions. The third is through a computational exploration of how these representations emerge in a reinforcement-learning setup. The idea of approaching the same problem from multiple independent angles, and seeking confirming evidence is laudable.

      Weaknesses:<br /> There are two differences between the "natural gravity" account and the "mental gravity" account. The first difference lies in the implementation of gravity. The second, however, is simply that the mental gravity model is integrating more uncertainty into the simulator. In my understanding, adding small amounts of noise to computational models will often increase their fit to human judgments (with softmaxing perhaps being the most common example of this). While counter-intuitive, this is because 'noiseless' models have perfect representations of the stimuli, which is an unrealistic assumption. In the case of intuitive physics, people might have noisy perceptual representations of exactly how flat the table is, the exact location of each block, what small disturbances might be happening in the environment, and so on. The absence of these sources of uncertainty in deterministic models can make them perform in a non-human-like manner.

      While all the data presented in the paper is consistent with the possibility that people have a stochastic representation of gravity. It is possible that people have uncertainty over what unobservable forces a block tower might be under (e.g., wind, bumps to the table, etc). Therefore, even if you have a firm belief that gravity goes down, you may want to add noise in your simulations to account for the fact that, in the real world, gravity is almost never the only force acting on an object that has started to move. While the paper acknowledges that such an account would be mathematically equivalent, it does not acknowledge that this raises the question of whether people actually have stochastic representations of gravity.

      This alternative account could be particularly important because I believe it might be a more accurate representation of what people believe. I may be wrong, but I believe that it is common to emphasize the probabilistic nature of the models and the importance of implementing forces as distributions (e.g., the concept of 'noisy newtons').

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors made significant updates to Hippacampome.org including 50 new cell types.

      Strengths:<br /> The authors have been thorough in basing their views on peer-reviewed literature. They have made the data highly accessible and the user has the ability to control what is included.

      Weaknesses:<br /> There are many inconsistencies in the literature regarding cell types and how these are incorporated into hippocampome.org is not clear.

      Properties are often a result of modeling and not biological data, and caveats to this approach, and other assumptions are unclear.

      Several interneuron subtypes in the dentate gyrus do not appear to be listed, such as neurogliaform cells.

      The nomenclature HIPROM should be distinguished or made synonymous with HIPP. Same for MOCAP and MOPP/HICAP.

      Dorsal ventral and sex differences are not mentioned.

    1. Joint Public Review:

      Summary: This study follows up on previous work showing a female-specific enhancer region of PAX1 is associated with adolescent idiopathic scoliosis (AIS). This new analysis combines human GWAS analysis from multiple countries to identify a new AIS-associated coding variant in the COL11A1 gene. Two nonsynonymous variants were found to be significantly associated with AIS: MMP14 p.Asp273Asn and COL11A1 p.Pro1335Leu, the latter of which had the more robust association and remained significant when females were tested independent of males. Using a Pax1 knockout mouse they go on to find that PAX1 and Collagen XI protein are expressed in the intervertebral discs (IVDs) and robustly in the growth plate, showing that COL11A1 expression is reduced in Pax1 mutant growth plate. Moreover, other AIS-associated genes, Gpr126 and Sox6, were also reduced in Pax1 mutant mice, suggesting a common pathway is involved in AIS. The proposed implication of a Pax1-Col11a1-Mmp3 signaling axis modulated by estrogen signaling suggests a potential mechanism by which young women have more severe scoliosis than young men, as is observed in humans.

      Strengths: This work integrates a large cohort of human genetic data from AIS patients and controls from diverse ethnic backgrounds, across the globe. This work attempts to functionally test their findings in vivo and by use of cell culture. The authors propose an interesting model which warrants in depth investigation.

      Weaknesses: There are concerns regarding the candidacy of COL11A1 p.Pro1335Leu that need to be addressed and clarified. Many of the main functional work was done in cell culture and not in vivo. Moreover, the evidence linking COL11A1 p.Pro1153Leu to AIS is indirect, making unclear whether impaired COL11A1 function can cause scoliosis in the mouse model, thus diminishing the strength of the conclusions regarding the proposed pathogenicity of COL11A1 p.Pro1335Leu.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this manuscript, Seba et al., investigate the mechanism of chromosome organization by the MukBEF complex in E. coli. They use a combination of Hi-C and ChIP analysis to understand the steps of MukBEF regulation: its unloading from DNA (how MukBEF activity is prevented in the terminus regions of the chromosome by MatP), and its loading onto DNA (how DNA replication influences MukBEF association with the chromosome). Seba et al., induce chromosomal rearrangements to flip the sections of the ter region, thus perturbing matS site numbers and position. They find that MukBEF activity is prevented around matS sites, and that higher matS density has greater effect on MukBEF. Separately, using replication mutants and inducible MukBEF expression, they find that MukBEF can associate with the chromosome even in the absence of replication (as seen by the emergence of long-range contacts). However, ChIP data suggests that MukBEF binding to DNA is enriched on newly replicated DNA.

      Strengths:<br /> Altogether, this work provides a valuable and comprehensive view of MukBEF-mediated chromosome organization, with insights on the mechanism of the exclusion of MukBEF from the terminus region of the chromosome. The use of the programmed genetic rearrangements is powerful, and allows the authors to provide clear and convincing evidence for MukBEF exclusion from ter by matS sites. It is particularly striking to see that MukBEF can promote long-range contacts even in chromosomal regions between two matS, but the complex is excluded from the matS 'zones'. Experiments using cells blocked for replication show that MukBEF can influence chromosome organization in the absence of replication as well. While previous studies have reported some evidence in support of both of the above conclusions, the experiments described here offer clear and direct demonstration of the same.

      Limitations:<br /> A few control experiments are required to strengthen conclusions. Additionally, the discussion section is lacking many references and key papers have not been cited (paragraph 1 of discussion for example has no references). The possibility that SMC-ScpAB and MukBEF can act independent of replication has been suggested previously, but are not cited or discussed. Similarly, there is some evidence for SMC-ScpAB association with newly replicated DNA (PMID 21923769).

    1. Reviewer #1 (Public Review):

      Summary:<br /> The authors set out to determine how chemical variation on kinase inhibitors determines the selection of Erk2 conformations and how inhibitor binding affects ERk2 structure and dynamics.

      Strengths:<br /> The study is beautifully presented both verbally and visually. The NMR experiments and the HDX experiments complement each other for the study of Erk2 solution dynamics. X-ray crystallography of Erk2 complexes with inhibitors shows small but distinct structural changes that support the proposed model for the impact of inhibitor binding.

      Weaknesses:<br /> A discussion of compound residence time for the different compounds and kinase constructs and how it could affect the very slow HDX rates might be helpful. For example, could any of the observed effects in Figure 4 be due to slow compound dissociation rather than slowed down kinase dynamics? What would be the implications?

    1. Reviewer #1 (Public Review):

      The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

      The study provides a wealth of interesting observation of behavior and much of this data constitute a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further. However, the data they provide does not support strong conclusion statements arguing that these chirps are used for localization purposes and is even less convincing at rejecting previously established hypotheses on the communication purpose of the chirps. I would suggest thoroughly revising the manuscript to provide a neutral description of the results and leaving any speculations and interpretations for the discussion where the authors should be careful to separate strongly supported hypotheses from more preliminary speculations. I detail below several instances where the argumentation and/or the analysis are flawed.

      - They analyze chirp patterning and show that, most likely, a chirp by an individual is followed by a chirp in the same individual. They argue that it is rare that a chirp elicits a "response" in the other fish. Even if there are clearly stronger correlations between chirps in the same individual, they provide no statistical analysis that discards the existence of occasional "response" patterns. The fact that these are rare, and that the authors don't do an appropriate analysis of probabilities, leads to this unsupported conclusion.<br /> - One of the main pieces of evidence that chirps can be used to enhance conspecific localization is based on their "interference" measure. The measure is based on an analysis of "inter-peak-intervales". This in itself is a questionable choice. The nervous system encodes all parts of the stimulus, not just the peak, and disruption occurring at other phases of the beat might be as relevant. The interference will be mostly affected by the summed duration of intervals between peaks in the chirp AM. They do not explain why this varies with beat frequency. It is likely that the changes they see are simply an artifact of the simplistic measure. A clear demonstration that this measure is not adequate comes from the observation in Fig7E-H. They show that the interference value changes as the signal is weaker. This measure should be independent of the strength of the signal. The method is based on detecting peaks and quantifying the time between peaks. The only reason this measure could be affected by signal strength is if noisy recordings affect how the peak detection occurs. There is no way to argue that this phenomenon would happen the same way in the nervous system. Furthermore, they qualitatively argue that patterns of chirp production follow patterns of interference strength. No statistical demonstration is done. Even the qualitative appraisal is questionable. For example, they argue that there are relatively few chirps being produced for DFs of 60 or -60 Hz. But these are DF where they have only a very small sample size. The single pair of fish that they recorded at some of these frequencies might not have chirped by chance and a rigorous statistical analysis is necessary. Similarly, in Fig 5C they argue that the position of the chirps fall on areas of the graph where the interferences are strongest (darker blue) but this is far from obvious and, again, not proven.<br /> - They relate the angle at which one fish produces chirps relative to the orientation of the mesh enclosing. They argue that this is related to the orientation of electric field lines by doing a qualitative comparison with a simplified estimate of field lines. To be convincing this analysis should include a quantitative comparison using the exact same body position of the two fish when the chirps are emitted.<br /> -They show that the very vast majority of chirps in Fig 6 occur when the fish are within a few centimeters (e.g. very large first bin in Fig6E-Type2). This is a situation when the other fish signal will be strongest and localization will be the easiest. It is hard to understand why the fish would need a mechanism to enhance localization in these conditions (this is the opposite of difficult conditions e.g. the "cluttered" environment).<br /> - The argumentation aimed at rejecting the well-established role of chirp in communication is weak at best. First, they ignored some existing data when they argue that there is no correlation between chirping and behavioral interactions. Particularly, Hupe and Lewis (2008) showed a clear temporal correlation between chirps and a decrease in bites during aggressive encounters. It could be argued that this is "causal evidence" (to reuse their wording) that chirps cause a decrease in attacks by the receiver fish (see Fig 8B of the Hupe paper and associated significant statistics). Also, Oboti et al. argue that social interactions involve "higher levels of locomotion" which would explain the use of chirps since they are used to localize. But chirps are frequent in "chirp chamber" paradigms where no movement is involved. They also point out that social context covaries with beat frequency and thus that it is hard to distinguish which one is linked to chirping propensity and then say that it is hard to disentangle this from "biophysical features of EOD fields affecting detection and localization of conspecific fish". But they don't provide any proof that beat frequency affects detection and localization so their argument is not clear. Last, they argue that tests in one species shouldn't be extrapolated to other species. But many of the studies arguing for the role of chirps in communication was done on brown ghost. In conclusion of this point, they do not provide any strong argument that rejects the role of chirps as a communication signal. A perspective that would be better supported by their data and consistent with past research would be to argue that, in addition to a role in communication, chirps could sometimes be used to help localize conspecifics.<br /> -The discussion they provide on the possible mechanism by which chirps could help with localization of the conspecific is problematic. They imply that chirps cause a stronger response in the receptors. For most chirps considered here, this is not true. For a large portion of the beat frequencies shown in this paper, chirps will cause a de-synchronization of the receptors with no increase in firing rate. They cannot argue that this represents an enhanced response. They also discuss a role for having a broader frequency spectrum -during the chirp- in localization by making a parallel with pulse fish. There is no evidence that a similar mechanism could even work in wave-type fish.<br /> -They write the whole paper as if males and females had been identified in their experiments. Although EOD frequency can provide some guess of the sex the method is unreliable. We can expect a non-negligible percentage of error in assigning sex.

    1. Reviewer #1 (Public Review):

      Numerous neurodegenerative diseases are thought to be driven by the aggregation of proteins into insoluble filaments known as "amyloids". Despite decades of research, the mechanism by which proteins convert from the soluble to insoluble state is poorly understood. In particular, the initial nucleation step is has proven especially elusive to both experiments and simulation. This is because the critical nucleus is thermodynamically unstable, and therefore, occurs too infrequently to directly observe. Furthermore, after nucleation much faster processes like growth and secondary nucleation dominate the kinetics, which makes it difficult to isolate the effects of the initial nucleation event. In this work Kandola et al. attempt to surmount these obstacles using individual yeast cells as microscopic reaction vessels. The large number of cells, and their small size, provides the statistics to separate the cells into pre- and post-nucleation populations, allowing them to obtain nucleation rates under physiological conditions. By systematically introducing mutations into the amyloid-forming polyglutamine core of huntingtin protein, they deduce the probable structure of the amyloid nucleus. This work shows that, despite the complexity of the cellular environment, the seemingly random effects of mutations can be understood with a relatively simple physical model. Furthermore, their model shows how amyloid nucleation and growth differ in significant ways, which provides testable hypotheses for probing how different steps in the aggregation pathway may lead to neurotoxicity.

      In this study Kandola et al. probe the nucleation barrier by observing a bimodal distribution of cells that contain aggregates; the cells containing aggregates have had a stochastic fluctuation allowing the proteins to surmount the barrier, while those without aggregates have yet to have a fluctuation of suitable size. The authors confirm this interpretation with the selective manipulation of the PIN gene, which provides an amyloid template that allows the system to skip the nucleation event.

      In simple systems lacking internal degrees of freedom (i.e., colloids or rigid molecules) the nucleation barrier comes from a significant entropic cost that comes from bringing molecules together. In large aggregates this entropic cost is balanced by attractive interactions between the particles, but small clusters are unable to form the extensive network of stabilizing contacts present in the larger aggregates. Therefore, the initial steps in nucleation incur an entropic cost without compensating attractive interactions (this imbalance can be described as a surface tension). When internal degrees of freedom are present, such as the conformational states of a polypeptide chain, there is an additional contribution to the barrier coming from the loss of conformational entropy required to the adopt aggregation-prone state(s). In such systems the clustering and conformational processes do not necessarily coincide, and a major challenge studying nucleation is to separate out these two contributions to the free energy barrier. Surprisingly, Kandola et al. find that the critical nucleus occurs within a single molecule. This means that the largest contribution to the barrier comes from the conformational entropy cost of adopting the beta-sheet state. Once this state is attained, additional molecules can be recruited with a much lower free energy barrier.

      There are several considerations in interpreting this result. First, the height of the nucleation barrier(s) comes from the relative strength of the entropic costs compared to the binding affinities. This balance determines how large a nascent nucleus must grow before it can form interactions comparable to a mature aggregate. In amyloid nuclei the first three beta strands form immature contacts consisting of either side chain or backbone contacts, whereas the fourth strand is the first that is able to form both kinds of contacts (as in a mature fibril). This study used relatively long polypeptides of 60 amino acids. This is greater than the 20-40 amino acids found in amyloid-forming molecules like ABeta or IAPP. As a result, Kandola et al.'s molecules are able to fold enough times to create four beta strands and generate mature contacts intramolecularly. This authors make the plausible claim that these intramolecular folds explain the well-known length threshold (L~35) observed in polyQ diseases. The intramolecular folds reduce the importance of clustering multiple molecules together and increase the importance of the conformational states. Similarly, manipulating the sequence or molecular concentrations will be expected to manipulate the relative magnitude of the binding affinities and the clustering entropy, which will shift the relative heights of the entropic barriers.

      The authors make an important point that the structure of the nucleus does not necessarily resemble that of the mature fibril. They find that the critical nucleus has a serpentine structure that is required by the need to form four beta strands to get the first mature contacts. However, this structure comes at a cost because residues in the hairpins cannot form strong backbone or zipper interactions. Mature fibrils offer a beta sheet template that allows incoming molecules to form mature contacts immediately. Thus, it is expected that the role of the serpentine nucleus is to template a more extended beta sheet structure that is found in mature fibrils.

      A second point of consideration is the striking homogeneity of the nucleus structure they describe. This homogeneity is likely to be somewhat illusory. Homopolymers, like polyglutamine, have a discrete translational symmetry, which implies that the hairpins needed to form multiple beta sheets can occur at many places along the sequence. The asparagine residues introduced by the authors place limitations on where the hairpins can occur, and should be expected to increase structural homogeneity. Furthermore, the authors demonstrate that polyglutamine chains close to the minimum length of ~35 will have strict limitations on where the folds must occur in order to attain the required four beta strands.

      A novel result of this work is the observation of multiple concentration regimes in the nucleation rate. Specifically, they report a plateau-like regime at intermediate regimes in which the nucleation rate is insensitive to protein concentration. The authors attribute this effect to the "self-poisoning" phenomenon observed in growth of some crystals. This is a valid comparison because the homogeneity observed in NMR and crystallography structures of mature fibrils resemble a one-dimensional crystal. Furthermore, the typical elongation rate of amyloid fibrils (on the order of one molecule per second) is many orders of magnitude slower than the molecular collision rate (by factors of 10^6 or more), implying that the search for the beta-sheet state is very slow. This slow conformational search implies the presence of deep kinetic traps that would be prone to poisoning phenomena. However, the observation of poisoning in nucleation during nucleation is striking, particularly in consideration of the expected disorder and concentration sensitivity of the nucleus. Kandola et al.'s structural model of an ordered, intramolecular nucleus explains why the internal states responsible for poisoning are relevant in nucleation.

      To achieve these results the authors used a novel approach involving a systematic series of simple sequences. This is significant because, while individual experiments showed seemingly random behavior, the randomness resolved into clear trends with the systematic approach. These trends provided clues to build a model and guide further experiments.

      There has been discussion in the review process about whether a monomeric nucleus is consistent with established properties of huntingtin aggregation. I do not see a problem with an energetically unfavorable conformational state preceding a concentration-dependent growth step. The authors make the case for this sequence using a schematic free energy landscape (Fig 6) that has many similarities to a free energy landscape derived from models of polyQ nucleation (Phan et al. 2022, see Fig. 6). The theory does not consider molecules large enough to form the conformational state described by Kandola et al., but the transition state is otherwise very similar.

    1. Reviewer #1 (Public Review):

      The current manuscript provides a timely contribution to the ongoing discussion about the mechanism of the apical sodium/bile acid transporter (ASBT) transporters. Recent structures of the mammalian ASBT transporters exhibited a substrate binding mode with few interactions with the core domain (classically associated with substrate binding), prompting an unusual proposal for the transport mechanism. Early structures of ASBT homologues from bacteria also exhibit unusual substrate binding in which the core substrate binding domain is less engaged than expected. Due to the ongoing questions of how substrate binding and mechanism are linked in these transporters, the authors set out to deepen our understanding of a model ABST homolog from bacteria N. meningitidis (ABST-NM).

      The premise of the current paper is that the bacterial ASBT homologs are probably not physiological bile acid transporters, and that structural elucidation of a natively transported substrate might provide better mechanistic information. In the current manuscript, the authors revisit the first BASS homologue to be structurally characterized, ABST-NM. Based on bacteriological assays in the literature, the authors identify the coenzyme A precursor pantoate as a more likely substrate for ABST-NM than taurocholate, the substrate in the original structure. A structure of ASBT-NM with pantoate exhibits interesting differences in structure. The structures are complemented with MD simulations, and the authors propose that the structures are consistent with a classical elevator transport mechanism.

      The structural experiments are convincing. The binding and molecular dynamics experiments provide intriguing insights into the transporter's conformational changes. However, it is nonetheless a soft spot in the story that a transport assay is not readily available for this substrate. Mechanistic proposals, like the proposed role of T112 in unlocking the transporter, would be better supported by transport data.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The manuscript proposes an alternative method by SDS-PAGE calibration of Halo-Myo10 signals to quantify myosin molecules at specific subcellular locations, in this specific case filopodia, in epifluorescence datasets compared to the more laborious and troublesome single molecule approaches. Based on these preliminary estimates, the authors developed further their analysis and discussed different scenarios regarding myosin 10 working models to explain intracellular diffusion and targeting to filopodia.

      Strengths:<br /> Overall, the paper is elegantly written and the data analysis is appropriately presented.

      Weaknesses:<br /> While the methodology is intriguing in its descriptive potential and could be the beginning of an interesting story, a good portion of the paper is dedicated to the discussion of hypothetical working mechanisms to explain myosin diffusion, localization, and decoration of filopodial actin that is not accompanied by the mandatory gain/loss of function studies required to sustain these claims.

    1. Reviewer #1 (Public Review):

      Summary: Bloodstream stages of the parasitic protist, Trypanosoma brucei, exhibit very high rates of constitutive endocytosis, which is needed to recycle the surface coat of Variant Surface Glycoproteins (VSGs) and remove surface immune complexes. While many studies have shown that the endo-lysosomal systems of T. brucei BF stages contain canonical domains, as defined by classical Rab markers, it has remained unclear whether these protists have evolved additional adaptations/mechanisms for sustaining these very high rates of membrane transport and protein sorting. The authors have addressed this question by reconstructing the 3D ultrastructure and functional domains of the T. brucei BF endosome membrane system using advanced electron tomography and super-resolution microscopy approaches. Their studies reveal that, unusually, the BF endosome network comprises a continuous system of cisternae and tubules that contain overlapping functional subdomains. It is proposed that a continuous membrane system allows higher rates of protein cargo segregation, sorting and recycling than can otherwise occur when transport between compartments is mediated by membrane vesicles or other fusion events.

      Strengths: The study is a technical tour-de-force using a combination of electron tomography, super-resolution/expansion microscopy, immune-EM of cryo-sections to define the 3D structures and connectivity of different endocytic compartments. The images are very clear and generally support the central conclusion that functionally distinct endocytic domains occur within a dynamic and continuous endosome network in BF stages.

      Weaknesses: The authors suggest that this dynamic endocytic network may also fulfil many of the functions of the Golgi TGN and that the latter may be absent in these stages. Although plausible, this comment needs further experimental support. For example, have the authors attempted to localize canonical makers of the TGN (e.g. GRIP proteins) in T. brucei BF and/or shown that exocytic carriers bud directly from the endosomes?

    1. Reviewer #1 (Public Review):

      Summary: Planar cell polarity core proteins Frizzled (Fz)/Dishevelled (Dvl) and Van Gogh-like (Vangl)/Prickle (Pk) are localized on opposite sides of the cell and engage in reciprocal repression to modulate cellular polarity within the plane of static epithelium. In this interesting manuscript, the authors explore how the anterior core proteins (Vangl/Pk) inhibit the posterior core protein (Dvl). The authors propose that Pk assists Vangl2 in sequestering both Dvl2 and Ror2, while Ror2 is essential for Dvl to transition from Vangl to Fz in response to non-canonical Wnt signaling. There are several points that affect the strength of the author's conclusions.

      Strengths: The strengths of the manuscript are in the very interesting and new concept for a model of how non-canonical Wnt induces Dvl to transition from Vangl to Fz. Prickle and Vangl2 are proposed to play an opposing role to suppress Dvl activity during convergent extension movements, whereas Ror antagonizes Vangl and may be required for the transition.

      Weaknesses: The weaknesses are in the clarity and resolution of the data that forms the basis of the model. In addition to whole embryo morphology that is used as evidence for convergent extension (CE) defects, two forms of data are presented, co-expression and IP, as well as a strong reliance on IF of exogenously expressed proteins. Thus, it is critical that both forms of evidence be very strong and clear, and this is where there are deficiencies; 1) For vast majority of experiments general morphology and LWR was used as evidence of effects on convergent extension movements rather than Keller explants or actual cell movements in the embryo. 2) The study would benefit from high or super resolution microscopy, since in many cases the differences in protein localization are not very pronounced. 3) The IP and Western analysis data often show subtle differences, and not apparent in some cases. 4) It is not clear how many biological repeats were performed or how and whether statistical analyses were performed.

    1. Joint Public Review:

      This paper's strengths are the interesting analysis of TLR signaling in hair follicle stem cell activation and the striking phenotype of the TLR2 cKO mice (but note below). The functional interrogation parts using HFSC-specific TLR2 genetic deletion are solid, and an endogenous regulator, CEP, is identified. The experiments reported in this manuscript are well-designed and presented. The authors provided extensive evidence supporting the roles of TLR2 signaling in regulating hair follicle stem cell functions. Importantly, the findings from this paper may have sustained impacts on our understanding of the roles of innate immunity in regulating tissue regeneration in the absence of inflammation.

      The main evidence for the mechanistic analysis is based on fluorescence using immunohistochemistry, and here the expression analysis is not convincing. In addition, additional assays beyond immunolandscaping are needed to confirm the findings. The reviewers felt that your data substantiating the mechanism of interaction between TLR2 and BMP pathway needs bolstering.

    1. Reviewer #1 (Public Review):

      Authors performed a meta-analysis of GC concentrations and metabolic rates in birds and mammals. They found close associations for all studies showing a positive association between these two traits. As GCs have been viewed with close links to "stress," authors suggest that this overlooks the importance of metabolism and perhaps GC variation does not relate to "stress" per se but an increase in metabolism instead.

      This is an important meta-analysis, as most researchers acknowledge the link between GCs and metabolism, metabolism is often overlooked in studies. The field of conservation physiology is especially focused on GCs being a "stress" hormone, which overlooks the importance of GCs in mediating energy balance, i.e., an animal that has high GC concentrations may not be doing that poorly compared to an animal with low GC concentrations, it might just be expending more energy, e.g., caring for young. The results, with overwhelming directionality and strong effect sizes, support the link for a positive association with these two variables.

      My main concern lies in that most of the studies come from a few labs, therefore there may be limited data to test this relationship. I would include lab as a random effect to see how strong this effect might be. Furthermore, I would like to see a test of the directionality of the two variables. Authors suggest that changes in metabolism affect GC levels but likely changes in GC levels would affect metabolism. Why not look into studies that have altered GC levels experimentally and see the effect on metabolism? Based on the close link, authors suggest that GCs may not play a role outside of "stress" beyond the stressor's effect on metabolic rate. However, if they were to investigate manipulations of GCs on metabolic rate, the link may or may not be there, which would be interesting to look at. I firmly believe that GCs are tightly linked to metabolism; however, I also think that GCs have a range of effects outside of metabolism as well, depending on the course and strength of the stressor.

      This work helps in the thinking that GCs are not the same as a "stress" hormone or labelling hormones with only one function. As hormones are naturally pleiotropic, the view of any one hormone being X is overly simplistic.

    1. Reviewer #1 (Public Review):

      Summary: This work by Zhang et al. provides new strategies to improve the efficiency of precise Prime Editing (PE) in zebrafish embryos. The authors test how two simple changes impact PE efficiency: first, by refolding the pegRNA before complexing with Cas9 nickase-reverse transcriptase PE2, and second, by introducing mutations to the pegRNA intended to reduce its autoinhibitory activity by disrupting complementarity between the 5' spacer sequence and the 3' PBS-RTT (Primer Binding Site-Reverse Transcriptase Template).

      Strengths: The authors tested multiple loci in the zebrafish genome to determine how pegRNA refolding and point mutations in the RTT would impact overall mutagenesis efficiency and precise PE at the target sites. The impact on efficiency was tested with three types of pegRNAs designed to introduce base substitutions, insertions or deletions. Next-generation sequencing of amplicons from pooled, injected embryos provided robust measurement of mutagenesis and editing. Insertion and deletion pegRNAs were overall more efficient than substitution pegRNAs, which may be useful information in considering experimental design strategy for introducing a specific variant. There is potential for further improvement by combining the authors' methods with previously published strategies to improve pegRNAs through design and chemical modification.

      Weaknesses: The observed increases in the frequency of precise PE were relatively minor and inconsistent across the multiple pegRNAs tested. The substitution pegRNAs showed very low precise PE, at levels less than 1 percent, therefore the fold changes reported were still representative of 10 percent or less of overall edits. Overall mutagenesis frequency, as measured by indel formation, increased along with increased precise PE. The approach produces highly genetically mosaic embryos, therefore the utility for transient studies in injected zebrafish embryos is unclear. Data on improved germline transmission frequency of precise PE alleles would strengthen the study and be of wide interest in the zebrafish community.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The preprint by Laganowsky and co-workers describes the use of mutant cycles to dissect the thermodynamic profile of specific lipid recognition by the ABC transporter MsbA. The authors use native mass spectrometry with a variable temperature source to monitor lipid binding to the native protein dimer solubilized in detergent. Analysis of the peak intensities (that is, relative abundance) of 1-3 bound lipids as a function of solution temperature and lipid concentration yields temperature-dependent Kds. The authors use these to then generate van't Hoff plots, from which they calculate the enthalpy and entropy contributions to binding of one, two, and in some cases, three lipids to MsbA.<br /> The authors then employ mutant cycles, in which basic residues involved in headgroup binding are mutated to alanine. By comparing the thermodynamic signatures of single and double (and in one instance triple) mutants, they aim to identify cooperativity between the different positions. They furthermore use inward and outward locking conditions which should control access to the different binding sites determined previously.<br /> The main conclusion is that lipid binding to MsbA is driven mainly by energetically favorable entropy increase upon binding, which stems from the release of ordered water molecules that normally coordinate the basic residues, which helps to overcome the enthalpic barrier of lipid binding. The authors also report an increase in lipid binding at higher temperatures which they attribute to a non-uniform heat capacity of the protein. Although they find that most residue pairs display some degree of cooperativity, particularly between the inner and outer lipid binding sites, they do not provide a structural interpretation of these results.

      Strengths:<br /> The use of double mutant cycles and mass spectrometry to dissect lipid binding is novel and interesting. For example, the observation that mutating a basic residue in the inner and one in the outer binding site abolishes lipid binding to a greater extent than the individual mutations is highly informative even without having to break it down into thermodynamic terms (see "weaknesses" section). In this sense, the method and data reported here opens new avenues for the structure/activity relationship of MsbA. The "mutant cycle" approach is in principle widely applicable to other membrane proteins with complex lipid interactions.

      Weaknesses:<br /> The use of double mutant cycles to dissect binding energies is well-established, and has, as the authors point out, been employed in combination with mass spectrometry to study protein-protein interactions. Its application to extract thermodynamic parameters is robust in cases where a single binding event is monitored, e.g. the formation of a complex with well-defined stoichiometry, where dissociation constants can be determined with high confidence. It is, however, complicated significantly by the fact that for MsbA-lipid interactions, we are not looking at a single binding event, but a stochastic distribution of lipids across different sites. Even if the protein is locked in a specific conformation, the observation of a single lipid adduct does not guarantee that the one lipid is always bound to a specific site. In some of the complexes detected by MS, the lipid is likely bound somewhere else. Lipid binding Kds from mass spectrometry, although helpful in some instances as a proxy for global binding affinities, should therefore be taken with a grain of salt.

      The authors analyze the difference in binding upon mutating binding sites (ddG etc). Here, another complicating factor comes into play, the fact that mutation of a binding site (which the authors show reduces lipid binding) may instead allow the lipid to bind to a lower-affinity site elsewhere. Unfortunately, the authors do not specify the protein concentration, but assuming it is in the single-digit micromolar range, as common for native MS experiments, lipid and protein concentrations are almost equal for most of the data points, resulting in competition between binding sites for free lipids. As a rule of thumb, for Kd measurements, the concentration of the constant component, the protein, should be far below the Kd, to avoid working in the "titration" regime rather than the "binding" regime (see Jarmoskaite et al, eLife 2020). I cannot determine whether this is the case here. The way I understand the double mutant cycle approach, reliable Kd measurements are required to accurately determine dH and TdS, so I would encourage the authors to confirm their Kd values using complementary methods before in-depth interpretations of the thermodynamic components.

      It is somewhat counterintuitive that for many double mutants, and the triple mutant, the entropic component becomes more favorable compared to the WT protein. If the increase in entropy upon lipid binding comes from the release of ordered water molecules around the basic residues (a reasonable assumption) why does this apply even more in proteins where several basic residues have been changed to alanine, which coordinate far fewer water molecules?

      The authors could devote more attention to the fact that they use detergent micelles as a vehicle for lipid binding studies. To a limited extent, detergents compete with lipids for binding, and are present in extreme excess over the lipid. The micelle likely changes its behavior in response to temperature changes. For example, the packing around the protein loosens up upon heating, which may increase the chance for lipids to bind. In this case, the increase in binding at higher temperatures may not be related to a change in heat capacity. This question could be addressed by MD simulations, if it's not already in the literature.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria is still not fully understood, particularly substrate recognition and insertion by beta-assembly machinery (BAM). In the studies, the authors present their studies that in addition to recognition by the last strand of an OMP, sometimes referred to as the beta-signal, an additional signal upstream of the last strand is also important for OMP biogenesis.

      Strengths:<br /> 1. Overall the manuscript is well organized and written, and addresses an important question in the field. The idea that BAM recognizes multiple signals on OMPs has been presented previously, however, it was not fully tested.

      2. The authors here re-address this idea and propose that it is a more general mechanism used by BAM for OMP biogenesis.

      3. The notion that additional signals assist in biogenesis is an important concept that indeed needs fully tested in OMP biogenesis.

      4. A significant study was performed with extensive experiments reported in an attempt to address this important question in the field.

      5. The identification of important crosslinks and regions of substrates and Bam proteins that interact during biogenesis is an important contribution that gives clues to the path substrates take en route to the membrane.

      Weaknesses:

      Major critiques (in no particular order):

      1. The title indicates 'simultaneous recognition', however no experiments were presented that test the order of interactions during OMP biogenesis.

      2. Aspects of the study focus on the peptides that appear to inhibit OmpC assembly, but should also include an analysis of the peptides that do not to determine this the motif(s) present still or not.

      3. The b-signal is known to form a b-strand, therefore it is unclear why the authors did not choose to chop OmpC up according to its strands, rather than by a fixed peptide size. What was the rationale for how the peptide lengths were chosen since many of them partially overlap known strands, and only partially (2 residues) overlap each other? It may not be too surprising that most of the inhibitory peptides consist of full strands (#4, 10, 21, 23).

      4. It would be good to have an idea of the propensity of the chosen peptides to form b-stands and participate in b-augmentation. We know from previous studies with darobactin and other peptides that they can inhibit OMP assembly by competing with substrates.

      5. The recognition motifs that the authors present span up to 9 residues which would suggest a relatively large binding surface, however, the structures of these regions are not large enough to accommodate these large peptides.

      6. The authors highlight that the sequence motifs are common among the inhibiting peptides, but do not test if this is a necessary motif to mediate the interactions. It would have been good to see if a library of non-OMP related peptides that match this motif could also inhibit or not.

      7. In the studies that disrupt the motifs by mutagenesis, an effect was observed and attributed to disruption of the interaction of the 'internal signal'. However, the literature is filled with point mutations in OMPs that disrupt biogenesis, particular those within the membrane region. F280, Y286, V359, and Y365 are all residues that are in the membrane region that point into the membrane. Therefore, more work is needed to confirm that these mutations are in parts of a recognition motif rather than on the residues that are disrupting stability/assembly into the membrane.

      8. The title of Figure 3 indicates that disrupting the internal signal motif disrupts OMP assembly, however, the point mutations did not seem to have any effect. Only when both 280 and 286 were mutated was an effect observed. And even then, the trimer appeared to form just fine, albeit at reduced levels, indicating assembly is just fine, rather the rate of biogenesis is being affected.

      9. In Figure 4, the authors attempt to quantify their blots. However, this seems to be a difficult task given the lack of quality of the blots and the spread of the intended signals, particularly of the 'int' bands. However, the more disturbing trend is the obvious reduction in signal from the post-urea treatment, even for the WT samples. The authors are using urea washes to indicate removal of only stalled substrates. However a reduction of signal is also observed for the WT. The authors should quantify this blot as well, but it is clear visually that both WT and the mutant have obvious reductions in the observable signals. Further, this data seems to conflict with Fig 3D where no noticeable difference in OmpC assembly was observed between WT and Y286A, why is this the case?

      10. The pull down assays with BamA and BamD should include a no protein control at the least to confirm there is no non-specific binding to the resin. Also, no detergent was mentioned as part of the pull downs that contained BamA or OmpC, nor was it detailed if OmpC was urea solubilized.

      11. The neutron reflectometry experiments are not convincing primarily due to the lack controls to confirm a consistent uniform bilayer is being formed and even if so, uniform orientations of the BamA molecules across the surface. Further, no controls were performed with BamD alone, or with OmpC alone, and it is hard to understand how the method can discriminate between an actual BamA/BamD complex versus BamA and BamD individually being located at the membrane surface without forming an actual complex. Previous studies have reported difficulty in preparing a complex with BamA and BamD from purified components. Additionally, little signal differences were observed for the addition of OmpC. However, an elongated unfolded polypeptide that is nearly 400 residues long would be expected to produce a large distinct signal given that only the C-terminal portion is supposedly anchored to BAM, while the rest would be extended out above the surface. The depiction in Figure 5D is quite misleading when viewing the full structures on the same scales with one another.

      12. In the crosslinking studies, the authors show 17 crosslinking sites (43% of all tested) on BamD crosslinked with OmpC. Given that the authors are presenting specific interactions between the two proteins, this is worrisome as the crosslinks were found across the entire surface of BamD. How do the authors explain this? Are all these specific or non-specific?

      13. The study in Figure 6 focuses on defined regions within the OmpC sequence, but a more broad range is necessary to demonstrate specificity to these regions vs binding to other regions of the sequence as well. If the authors wish to demonstrate a specific interaction to this motif, they need to show no binding to other regions.

      14. The levels of the crosslinks are barely detectable via western blot analysis. If the interactions between the two surfaces are required, why are the levels for most of the blots so low?

      15. Figure 7 indicates that two regions of BamD promote OMP orientation and assembly, however, none of the experiments appears to measure OMP orientation? Also, one common observation from panel F was that not only was the trimer reduced, but also the monomer. But even then, still a percentage of the trimer is formed, not a complete loss.

      16. The experiment in Fig 7B would be more conclusive if it was repeated with both the Y62A and R197A mutants and a double mutant. These controls would also help resolve any effect from crowding that may also promote the crosslinks. Further, the mutation of R197 is an odd choice given that this residue has been studied previously and was found to mediate a salt bridge with BamA. How was this resolved by the authors in choosing this site since it was not one of the original crosslinking sites?

      17. As demonstrated by the authors in Fig 8, the mutations in BamD lead to reduction in OMP levels for more than just OmpC and issues with the membrane are clearly observable with Y62A, although not with R197A in the presence of VCN. The authors should also test with rifampicin which is smaller and would monitor even more subtle issues with the membrane. Oddly, no growth was observed for the Vec control in the lower concentration of VCN, but was near WT levels for 3 times VCN, how is this explained?

      18. While Fig 8I indeed shows diminished levels for FY as stated, little difference was observed for the trimer for the other mutants compared to WT, although differences were observed for the dimer. Interestingly, the VY mutant has nearly WT levels of dimer. What do the authors postulate is going on here with the dimer to trimer transition? How do the levels of monomer compare, which is not shown?

      19. In the discussion, the authors indicate they have '...defined an internal signal for OMP assembly', however, their study is limited and only investigates a specific region of OmpC. More is needed to definitively say this for even OmpC, and even more so to indicate this is a general feature for all OMPs.

      20. In the proposed model in Fig 9, it is hard to conceive how 5 strands will form along BamD given the limited surface area and tight space beneath BAM. More concerning is that the two proposal interaction sites on BamD, Y62 and R197, are on opposite sides of the BamD structure, not along the same interface, which makes this model even more unlikely. As evidence against this model, in Figure 9E, the two indicates sites of BamD are not even in close proximity of the modeled substrate strands.

    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.

      Weaknesses:

      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.

    1. Reviewer #1 (Public Review):

      Precision guided sterile insect technology (pgSIT) is a means of mosquito vector control that aims to simultaneously kill females while generating sterile males for field release. These sterile males are expected to mate with 'wild' females resulting in very few eggs being laid or low hatching rates. Repeated releases are expected to result in the suppression of the mosquito population. This method avoids cumbersome sex-sorting while generating the sterile males. Importantly, until release, the two genetic elements that bring about female lethality and male sterility - the Cas9 and the gRNA carrying mosquitoes - are maintained as separate lines. They are crossed only prior to release, and therefore, this approach is considered to be more safe than gene drives.

      The authors had made a version of this pgSIT in their 2021 paper where they targeted *β-Tubulin 85D*, which is only expressed in the male testes and its loss-of-function results in male sterility. In that pgSIT, they did not have female lethality, but generated flightless females by simultaneously targeted *myosin heavy chain,* which is expressed only in the female wings. Here the authors argue, that the survival of females is not ideal, and so modify their 2021 approach to achieve female lethality/sterility.

      To do this, they target two genes - the female specific isoform of Dsx and intersex. They use multiple gRNAs against these genes and validate their ability to cause female lethality/sterility. Having verified that these do indeed affect female fertility, they combine gRNAs against Dsx and ix to generate female lethality/sterility and use *β-Tubulin 85D* to generate male sterility (previously validated). When these gRNA mosquitoes are crossed to Cas9 and the progeny crossed to WT (the set-up for pgSIT), they find that very few eggs are laid, larval death is high, and what emerges are males or intersex progeny that are sterile.

      As this is the requirement for pgSIT, the authors then test if it is able to induce population suppression. To do this, they conduct cage trials and find that only when they use 20:1 or 40:1 ratio of pgSIT:WT cages, does the population crash in 4-5 generations. They model this pgSIT's ability to suppress a population in the wild. Unfortunately, I was not able to assess what parameters from their pgSIT were used in the model and therefore the predicted efficacy of their pgSIT, (though the range of 0-.1 is not great, given that the assessment is between 0-0.15).

      Finally, they also develop a SENSR with a rapid fluorescence read-out for detecting the transgene in the field. They show that this sensor is specific and sensitive, detecting low copy numbers of the transgene. This would be important for monitoring any release.

      Overall, the data are clear and well presented. The manuscript is well written (albeit likely dense for the uninitiated!). I had concerns about the efficacy of generating the pgSIT animals - the overall number of eggs hatched from the gRNA (X) Cas9 cross appears to be low, therefore, very large numbers of parental animals would have to be reared and crossed to obtain enough sterile males for the SIT. In addition to this, I was concerned about the intersex progeny that can blood-feed. These could potentially contribute to the population and it would be useful to see the data that suggest that these numbers are low and the animals will not be competent in the field.

    1. Reviewer #1 (Public Review):

      Despite durable viral suppression by antiretroviral therapy (ART), HIV-1 persists in cellular reservoirs in vivo. The viral reservoir in circulating memory T cells has been well characterized, in part due to the ability to safely obtain blood via peripheral phlebotomy from people living with HIV-1 infection (PWH). Tissue reservoirs in PWH are more difficult to sample and are less well understood. In this small (n=3) autopsy study, Sun and colleagues use an advanced genetic sequencing technique to characterize HIV-1 that persists in human tissues despite antiretroviral therapy. The authors describe isolation and genetic characterization of HIV-1 reservoirs from a variety of tissues including the central nervous system (CNS) obtained from three recently deceased individuals at autopsy. They identified clonally expanded proviruses in the CNS in all three individuals.

      Strengths of the work include the study of human tissues that are under-studied and difficult to access, and the sophisticated near-full length sequencing technique that allows for inferences about genetic intactness and clonality of proviruses. The small sample size (n=3) is a drawback. Furthermore, two individuals were on ART for just one year at the time of autopsy and had T cells compatible with AIDS, and one of these individuals had a low-level detectable viral load (Figure S1). This makes generalizability of these results to PWH who have been on ART for years or decades and have achieved durable viral suppression and immune reconstitution difficult.

      While anatomic tissue compartment and CNS region accompany these PCR results, it is unclear which cell types these viruses persist in. As the authors point out, it is possible that these reservoir cells might have been infiltrating T cells from blood present at the time of autopsy tissue sampling. Cell type identification would greatly enhance the impact of this work. Overall, this small, thoughtful study contributes to our understanding of the tissue distribution of persistent HIV-1, and informs the ongoing search for viral eradication.

    1. Joint Public Review:

      In this manuscript, Xue and colleagues investigate the fundamental aspects of cellular fate decisions and differentiation, focusing on the dynamic behaviour of gene regulatory networks. It explores the debate between static (noise-driven) and dynamic (signal-driven) perspectives within Waddington's epigenetic landscape, highlighting the essential role of gene regulatory networks in this process. The authors propose an integrated analysis of fate-decision modes and gene regulatory networks, using the Cross-Inhibition with Self-activation (CIS) network as a model. Through mathematical modelling, they differentiate two logic modes and their effect on cell fate decisions: requires both the presence of an activator and absence of a repressor (AA configuration) with one where transcription occurs as long the repressor is not the only species on the promoter (OO configuration).

      The authors establish a relationship between noise profiles, logic-motifs, and fate-decision modes, showing that defining any two of these properties allows the inference of the third. They also identify, under the signal-driven mode, two fundamental patterns of cell fate decisions: either prioritising progression or accuracy in the differentiation process. The authors apply this analysis to available high-throughput datasets of cell fate decisions in hematopoiesis and embryogenesis, proposing the underlying driving force in each case and utilising the observed noise patterns to nominate key regulators.

      The paper makes a substantial contribution by rigorously evaluating assumptions in gene regulatory network modelling. Notably, it extensively compares two model configurations based on different integration logic, illuminating the consequences of these assumptions in a clear, understandable manner. The practical simulation results effectively bridge theoretical models with real biological systems, adding relevance to the study's insights. With its potential to enhance our understanding of gene regulatory networks across biological processes, the paper holds promise. Its implications extend practically to synthetic circuit design, impacting biotechnology. The conclusions stand out, addressing cell fate decisions and noise's role in gene networks, contributing significantly to our understanding. Moreover, the adaptable approach proposed offers versatility for broader applications in diverse scenarios, solidifying its relevance beyond its current scope.

      However, the manuscript in its current form also has some important weaknesses, including the lack of clarity in the text and the questionable generality of specific observations. For instance, even when focusing on the CIS network, the effect of alternative model implementations is not discussed. Notably, the input signals are only considered as an additive effect over the differential equations, while signals can potentially affect each of the individual processes. The proposed model allows for a continuum of interactions/competition between transcription factors, yet only very restrictive scenarios are explored (strict AND/OR logic operations). Moreover, how the model parameters are chosen throughout the paper is not clear. Similarly, the concentration and time units are not clearly specified, making their comparison to experimental data troublesome.

      Regarding clarity, how the general model (equations 1-2) transforms into the specific cases evaluated in the paper is not clearly stated in the main text, nor are the positive and negative effects of individual transcription factors adequately explained. Similarly, in the main text and Figure 2, the authors refer to a Boolean model. However, they do not clearly explain how this relates to the differential equation model, nor its relevance to understanding the paper. Additionally, the term "noise levels" is generally used to refer to noise introduced in the "noise-driven" analysis (i.e., as an input or parameter in the models). Nonetheless, it is later claimed to be evaluated as an intrinsic property of the network (likely referring to expression level variability measured by the coefficient of variation). Finally, some jargon is introduced without sufficient context about its meaning (e.g., "temporal fully-connected stage").

      Additionally, proper discussion of previous work is also missing. For instance, the dynamics of the CIS network investigated by the authors have been extensively characterised (see e.g., Huang et al., Dev Biol, 2007), and how the author's results compare to this previous work should be discussed. In particular, the central assumptions behind the derivation of the model proposed in the manuscript must be assessed in the context of previous work.

    1. Reviewer #1 (Public Review):

      Gating of Kv10 channels is unique because it involves coupling between non-domain swapped voltage-sensing domains, a domain-swapped cytoplasmic ring assembly formed by the N- and C-termini, and the pore domain. Recent structural data suggests that activation of the voltage sensing domain relieves a steric hindrance to pore opening, but the contribution of the cytoplasmic domain to gating is still not well understood. This aspect is of particular importance because proteins like Calmodulin interact with the cytoplasmic domain to regulate channel activity. The effects of Calmodulin (CaM) in WT and mutant channels with disrupted cytoplasmic gating ring assemblies are contradictory, resulting in inhibition or activation, respectively. The underlying mechanism for these discrepancies is not understood. In the present manuscript, Reham Abdelaziz and collaborators use electrophysiology, biochemistry, and mathematical modeling to explore the mechanistic effects on gating of various mutations and deletions that disrupt inter-subunit interactions at the cytoplasmic gating ring assembly and the consequences for channel modulation by CaM. From the beginning, it becomes challenging for non-experts to grasp the structural basis of the perturbations that are introduced (ΔPASCap and E600R), because no structural data or schematic cartoons are provided to illustrate the rationale for those deletions or their potential mechanistic effects. In addition, the lack of additional structural information or illustrations, and a somewhat confusing discussion of the structural data, make it challenging for a reader to reconcile the experimental data and mathematical model with a particular structural mechanism for gating, limiting the impact of the work.

      By expressing mutants in oocytes and recording currents using Two Electrode Voltage-Clamp (TEV), it is found that both ΔPASCap and E600R mutants have biphasic voltage-activation curves, with two clear components contributing to activation and deactivation kinetics. Notably, the first component involving activation occurs at voltages where WT channels are mostly closed. Larger deletions at the N-terminus that further disrupt the cytoplasmic gating ring assembly accentuate the first component by heavily disfavoring the second one. The data can be well described by three components involving a closed state and two open states O1 and O2, in which the second component O2 is the one affected by the mutations and deletions. Based on the structural data, the first component is hypothesized to be associated with voltage sensor activation, whereas the second component is associated with conformational changes at the cytoplasmic ring. Consistent with this interpretation, a deletion construct where the covalent link between the voltage sensor and pore has been severed is shown to primarily affect that first component. Also consistent with the first component involving voltage-sensor activation, it is found that divalent cations that are known to stabilize the voltage sensor in its most deactivated conformations, shift the occupancy of the first component to more depolarizing potentials. Activation towards and closure from the first component is slow, whereas channels close rapidly from O2. A rapid alternating pulse protocol is used to take advantage of the difference in activation and deactivation kinetics between the two open components in the mutants and thus drive an increasing number of channels toward state O1. Currents activated by the alternating protocol reached larger amplitudes than those elicited by a long depolarization to the same voltage. This finding is interpreted as an indication that the first component (O1) has a larger conductance than the second (O2). It is shown that conditioning pulses to very negative voltages results in currents that are larger and activate more slowly than those elicited at the same voltage but starting from less negative conditioning pulses. In voltage-activated curves, the component corresponding to state O1 is shown to be favored by increasingly negative conditioning voltages as compared to less negative ones. This is interpreted as indicating that the first open component O1 is primarily accessed from so-called 'deeply closed' states in which voltage sensors are in their most deactivated position(s). Consistently, a mutation that destabilizes these deactivated states is shown to largely suppress the first component in voltage-activation curves for both ΔPASCap and E600R channels. It is also shown that stimulating calcium entry into the oocytes with ionomycin and thapsigargin, which is assumed to enhance CaM-dependent modulation, results in preferential potentiation of the first component in ΔPASCap and E600R, and this potentiation is attenuated by including an additional mutation that disfavors deeply closed states where voltage sensors are (mostly) deactivated. Together, these results are interpreted as an indication that calcium-CaM preferentially stabilizes O1 in mutant channels, thus favoring activation, whereas in WT channels lacking occupancy of O1, CaM stabilizes closed states and is therefore inhibitory. Moreover, it is found that the potentiation of ΔPASCap and E600R by CaM is more strongly attenuated by mutations in the channel that disrupt interaction with the C-terminal lobe of CaM than mutations affecting interaction with the N-terminal lobe. Finally, a mathematical model is proposed consisting of two layers involving two activation steps for the voltage sensor, and one conformational change in the cytoplasmic gating ring - completion of both sets of conformational changes is required to access state O2, but accessing state O1 only requires completion of the first voltage-sensor activation step in the four subunits. The model qualitatively reproduces most major findings on the mutants.

      There are several concerns associated with the analysis and interpretations that are provided. First, the conductance-voltage (G-V) relations for the mutants do not seem to saturate, and the absolute open probability is not quantified for any mutant under any condition. This makes it impossible to quantitatively compare the relative amplitudes of the two components because the amplitude of the second component remains undetermined. This makes it challenging to interpret results involving perturbations that affect the relative occupancy of O1 and O2, such as those in Figures 2, 6, and 7, and also raises concerns about the extent to which model parameters can be constrained. This issue is made even more serious by the observation that the currents in both key mutants (ΔPASCap and E600R) are extremely slow and do not appear to reach steady-state over the intervals that are studied. This reduces confidence in the parameters associated with G-V relations, as the shape and position of both components might change significantly if longer pulses were used. This is not addressed or acknowledged in the manuscript. Further, because the mutant channel currents do not saturate at the most positive potentials and time intervals examined, the kinetic characterization based on reaching 80% of the maximum seems inappropriate, because the 100% mark is arbitrary. Further, the kinetics for some of the other examined mutants (e.g. those in Fig. 2A) are not shown, making it difficult to assess the extent to which the data could be affected by having been measured before full equilibration. There are additional aspects associated with gating kinetics that are not appropriately explored. For example, I would expect that the enhanced current amplitudes from Figure 5 are only transient, ultimately reaching a smaller steady-state current magnitude that depends only on the stimulation voltage and is independent of the pre-pulse. The entire time course including the rise-time and decay is not examined experimentally. This raises concern on whether occupancy of state O1 might be overestimated under some experimental conditions if a fraction of the occupancy is only transient. The mathematical model is not utilized to examine some of these slower relaxations - this may be because the model does not reproduce these slow processes, which would represent a serious shortcoming given that the slow kinetics appear to be intrinsic to transitions around state O1. The significance of the results with the Δ2-10.L341Split is unclear. First, structural as well as functional data has established that the coupling of the voltage sensor and pore does not entirely rely on the S4-S5 linker, and thus the Split construct could still retain coupling through other mechanisms, which is consistent with the prominent voltage dependence that is observed. If both state O1 and O2 require voltage sensor activation, it is unclear why the Split construct would affect state O1 primarily, as suggested in the manuscript, as opposed to decreasing occupancy of both open states.

      The figure legends and text do not describe which solutions exactly were utilized for each experiment, and the rationale for choosing some solutions over others is not properly explained. The reversal potential for solutions used to measure voltage-activation curves falls right at the spot where occupancy of the first component peaks (e.g. see Figure 1B). Because no zero-current levels are shown on the current traces, it becomes very hard to determine which voltages correspond to each of the currents (see Fig. 1A). It is unclear whether any artifacts could have been introduced to the mutant activation curves at voltages close to the reversal potential. One key assumption that is not well-supported by the data pertains to the difference in single-channel conductance between states O1 and O2 - no analysis or discussion is provided on whether the data could also be well described by an alternative model in which O1 and O2 have the same conductance. No additional experimental evidence is provided related to the difference in conductance, which represents a key aspect of the mathematical model utilized to interpret the data. The CaM experiments are potentially very interesting and could have wide physiological relevance. However, the approach utilized to activate CaM is indirect and could result in additional non-specific effects on the oocytes that could affect the results.

      The description of the mathematical model that is provided is difficult to follow, and some key aspects are left unclear, such as the precise states from which state O1 can be accessed, and whether there is any direct connectivity between states O1 and O2 - different portions of the text appear to give contradictory information regarding these points. Several rate constants other than those explicitly mentioned to represent voltage sensor activation are also assigned a voltage dependence - the mechanistic basis of that voltage dependence is unclear. Finally, a clear mechanistic explanation for the full range of effects that the ΔPASCap and E600R mutants have on channel function is lacking, as well as a detailed description of how those newly uncovered transitions would influence the activity of the WT channel; this latter point is important when considering whether the findings in the manuscript advance our understanding of the gating mechanism of Kv10 channels in general, or are specific to the particular mutants that are studied. It is unclear, for example, how both the mutation or the deletion at the cytoplasmic gating ring enable conduction by state O1, especially when considering the hypothesis put forward in this study that transition to O1 exclusively involves transitions by the voltage sensor and not the cytoplasmic gating ring. It is also not clearly described whether a non-conducting state with the equivalent state-connectivity as O1 can be accessed in WT channels, or if a state like O1 can only be accessed in the mutant channels. Importantly, if a non-conducting state with the same connectivity to O1 were to be accessed in WT channels, it would be expected that an alternating pulse protocol as in Fig. 4 would result in progressively decreasing currents as the occupancy of the non-conducting state equivalent to O1 is increased. Because this is not the case, it means that mutation and deletion cause additional perturbations on the gating energetics relative to WT, which are not clearly fleshed out.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Human Abeta42 inhibits gamma-secretase activity in biochemical assays.

      Strengths:<br /> Determination of inhibitory concentration human Abeta42 on gamma-secretase activity in biochemical assays.

      Weaknesses:<br /> Human Abeta42 may concentrate up to microM order in endosomes. If so, production of Abeta42 would be attenuated then lead to less Abeta deposition in the brain. The authors finding is interesting but does not fit the physiological condition in the brain.<br /> It is not clear whether the FRET-based assay in living cells really reflect gamma-secretase activity.<br /> Processing of APP-CTF in living cells is not only the cleavage by gamma-secretase.

    1. Reviewer #1 (Public Review):

      In this manuscript, Tian et al. describe a novel modified version of the pro-drug triptolide, CK21, and provide evidence for its improved pharmacokinetics and its safety and efficacy in multiple xenograft models of pancreatic cancer. The authors performed transcriptomic analysis upon CK21 treatment which revealed that downregulation of NF-kB and mitochondrial dysfunction induce apoptosis and therefore lead to tumor regression. Downregulation of NF-kB and induction of apoptosis was then validated in vitro and in vivo. These findings have potential clinical significance as the efficacy of CK21 in preclinical PDAC models is compelling. However, there are also some limitations to their experiments and more validation studies are necessary to strengthen their findings regarding the mechanism of action of the drug. Specifically, the authors suggest that mitochondrial dysfunction is responsible for the observed apoptosis; however, this is not demonstrated. Additionally, side-by-side comparisons to other clinical triptolide analogs to show CK21 is at least as efficacious as other analogs in vivo would be valuable, especially since other analogs have been shown to synergize with conventional chemotherapy in PDAC mouse models, whereas CK21 does not appear to. Moreover, assessing whether CK21 is efficacious in syngeneic orthotopic PDAC models is critical, especially since CK21 was shown to have an impact on NF-KB which plays a major role in the immune compartment and triptolide has been shown to be immunosuppressive.

    1. Reviewer #1 (Public Review):

      Ruesseler and colleagues combine careful paradigm design, psychophysical and EEG analyses to determine whether information leakage during decision formation is strategically adjusted to meet changing task demands. Participants made motion direction judgments that required monitoring a continuous stream of dot motion for 'response periods' characterised by a sustained period of coherent motion in a leftward or rightward direction. Coherence was modulated on a frame-to-frame basis throughout the task furnishing a parametric regressor that could be used to interrogate the longevity of sensory samples in the decision process and their influence on corresponding EEG signals. Participants completed the task under varying conditions of response period length and frequency. Psychophysical kernel analyses suggest that sensory samples had a more short-lived impact on the participants' choices when response periods were rare, suggestive of greater information leakage. When the stimulus perturbations were regressed against the EEG data, it highlighted a centro-parietal component that showed increased responsiveness to large shifts in evidence when those shifts were more rare, suggestive of a role in representing surprise. An additional triphasic component was found to correlate with the time constant of integration as estimated from the kernel analyses.

      This is a very timely paper that addresses an important and difficult-to-address question in the decision-making field - the degree to which information leakage can be strategically adapted to optimise decisions in a task-dependent fashion. The authors apply a sophisticated suite of analyses that are appropriate and yield a range of very interesting observations. The paper centres on analyses of one possible model that hinges on certain assumptions about the nature of the decision process for this task which raises questions about whether leak adjustments are the only possible explanation for the current data. I think the conclusions would be greatly strengthened if they were supported by the application and/or simulation of alternative model structures.

      The behavioural trends when comparing blocks with frequent versus rare response periods seem difficult to tally with a change in the leak. The greater leak should result in a reduction in the rate of false alarms yet no significant differences were observed between these two conditions. Meanwhile, false alarms did vary as a function of short/long target durations which did not show any leak effect in the psychophysical kernel analyses. Are there other models that could reproduce such effects? For example, could a model in which the drift rate varies between Rare and Frequent trials do a similar or better job of explaining the data? This ties in to a related query about the nature of the task employed by the authors. Due to the very significant volatility of the stimulus, it seems likely that the participants are not solely making judgments about the presence/absence of coherent motion but also making judgments about its duration (because strong coherent motion frequently occurs in the inter-target intervals). If that is so, then could the Rare condition equate to less evidence because there is an increased probability that an extended period of coherent motion could be an outlier generated from the noise distribution? Note that a drift rate reduction would also be expected to result in fewer hits and slower reaction times, as observed.

      Some adjustment of the language used when discussing FAs seems merited. If I have understood correctly, the sensory samples encountered by the participants during the inter-response intervals can at times favour a particular alternative just as strongly (or more strongly) than that encountered during the response interval itself. In that sense, the responses are not necessarily real false alarms because the physical evidence itself does not distinguish the target from the non-target. I don't think this invalidates the authors' approach but I think it should be acknowledged and considered in light of the comment above regarding the nature of the decision process employed on this task.

      The authors report that preparatory motor activity over central electrodes reached a larger decision threshold for RARE vs. FREQUENT response periods. It is not clear what identifies this signal as reflecting motor preparation. Did the authors consider using other effector-selective EEG signatures of motor preparation such as beta-band activity which has been used elsewhere to make inferences about decision bounds? Assuming that this central ERP signal does reflect the decision bounds, the observation that it has a larger amplitude at the response on Rare trials appears to directly contradict the kernel analyses which suggest no difference in the cumulative evidence required to trigger commitment.

      P11, the "absolute sensory evidence" regressor elicited a triphasic potential over centroparietal electrodes. The first two phases of this component look to have an occipital focus. The third phase has a more centroparietal focus but appears markedly more posterior than the change in evidence component. This raises the question of whether it is safe to assume that they reflect the same process.

    1. Reviewer #1 (Public Review):

      The paper first demonstrates that heat-killed bacteria show little DAF-16 activation compared to live food. Of note, daf-16 survival is longer than WT when fed HK bacteria, giving important insights into the lethality of these mutants. Leakiness of the gut is assessed, which is induced by age and exacerbated by daf-16 mutation. The authors then go on to identify indole as the causal bacterial compound to drive daf-16 nuclear localization. The indole effect is fully daf-16 dependent. In searching for the indole sensor in the worm, TRPA-1 is identified and the authors argue that indole is sensed in neurons to modulate gut DAF-16. Closing the circle, lys genes are identified whose expression is upregulated by daf-16 and indole, and which are required to control bacterial growth in the gut with aging.

    1. Reviewer #1 (Public Review):

      The strongest aspect of the study is the identification of the probable Ric-8A/NCS-1 interface through the crystal structures of NCS-1 complexed with candidate peptide mimetics from Ric-8A. However, since the structures involve peptides, it is critical to validate this interface with mutational analysis of the full-length or truncated Ric-8A. Furthermore, the evidence for the complex structure based on cryo-EM reconstruction is weak. The low resolution does not allow for reliable modeling of the complex. Two analyses may support the authors' main conclusions: a) validation of the interface with mutational analysis of Ric-8A, and b) new optimized sample/grid preparation for cryo-EM data collection.

    1. Reviewer #1 (Public Review):

      In this study, the authors characterize regulatory control of embryonic genome activation in the allotetraploid, Xenopus laevis. By characterizing transcription from its L and S subgenomes, they determine that homeologous genes are differentially activated in the early embryo. It has recently been appreciated that homeologs may be differentially expressed in later embryonic development (Session and Rokhsar, Nature 2016). However, an unanswered question is whether vertebrate tetraploid genomes undergo differential induction at the onset of the major wave of zygotic genome activation (ZGA). This is a fertile area for research, that enables the study of gene regulatory network adaptations to changes in ploidy, limited by the constraints of gene dosage and an essential early developmental transition. Xenopus laevis, which recently underwent a tetraploidization event, approximately 18 million years ago, provides a very useful model embryonic system for the study of homeologous gene activation during vertebrate ZGA.

      To characterize differential subgenome activation the authors focused on the ~ 2600 maternally-regulated genes expressed in the first wave of widespread ZGA. They treated embryos with cycloheximide at Stage 8 to prevent the translation of zygotic factors that would further alter the transcriptome. They found a majority of these maternally-regulated genes have asymmetric expression between the two homeologs, with transcription often occurring from the L or S copy alone. This is a fascinating result from which to dig deeper into gene regulatory mechanisms. To understand whether cis-regulatory networks dictate the biased L/S homeolog expression in the late blastula, the authors performed CUT&RUN to map active chromatin marks, H3K4me3 and H3K27ac. However, they found no differences in promoter sequences of homeologs that would implicate differential recruitment of specific transcription factors. Instead, they focused on distal enhancers and additionally performed ATAC-seq on Stage 8 and 9 animal cap explants. Approximately 70% of enhancers for homeolog pairs exhibited differential H3K27ac enrichment and chromatin accessibility. The authors then searched for transcription factor binding motifs that distinguished active enhancers from their inactive homeolog. They found binding sequences for OCT4 and SOX2/3 were enriched in active L enhancers and active S enhancers. To assess the role of these pluripotency factors, they used antisense morpholinos to block their translation in the early embryo. MOs were complementary to both the L and S homeologs of pou5f3.3 and sox3, but not to their paralogs that are primarily expressed zygotically; pou5f3.1 and pou5f3.2. MO knockdown of both Pou5f3.3 and Sox3 was inhibited leading to significant downregulation of 62% of activated genes compared to embryos injected with a control morpholino. They also analyzed binding to the genome of V5-tagged, injected versions of these 2 transcription factors and found some evidence for differential binding around TSS of homeolog pairs and a correlation between binding and the overall level of transcription at ZGA. Finally, they compare enhancer marks and accessibility in tetraploid X.laevis subgenomes to homologous enhancers in the diploid X.tropicalis. They conclude conservation of active enhancers with X.tropicalis and even zebrafish when considering the combined data from X.laevis L and S subgenome.

      There are many strengths of this manuscript. In this interesting study, the authors identify what appears to be an evolutionary divergence of enhancers in a vertebrate tetraploid, that may underlie the differential expression of homeologs during the first major wave of ZGA. They generate CUT&RUN datasets of active chromatin marks during the early and late blastula. Additionally, they provide binding data for pluripotency factors OCT4 and SOX2/3 and demonstrate that their MO knockdown leads to reduced expression at ZGA. Their analyses identify correlations between differential homeolog expression and active or accessible chromatin. Further, they identify that active enhancers are enriched in OCT4 and SOX2/3. Enthusiasm is somewhat dampened by a lack of direct perturbation to differential subgenome activation or an understanding of the functional impacts of differential homeolog expression on subsequent development.

    1. Reviewer #1 (Public Review):

      The study by Akter et al demonstrates that astrocyte-derived L-lactate plays a key role in schema memory formation and promotes mitochondrial biogenesis in the Anterior Cingulate Cortex (ACC).

      The main tool used by the authors is the DREADD technology that allows to pharmacologically activate receptors in a cell-specific manner. In the study, the authors used the DREADD technique to activate appropriately transfected astrocytes, a subtype of muscarinic receptor that is not normally present in cells. This receptor being coupled to a Gi-mediated signal transduction pathway inhibiting cAMP formation, the authors could demonstrate cell-(astrocyte) specific decreases in cAMP levels that result in decreased L-lactate production by astrocytes.

      Behaviorally this pharmacological manipulation results in impairments of schema memory formation and retrieval in the ACC in flavor-place paired associate paradigms. Such impairments are prevented by co-administration of L-lactate.

      The authors also show that activation of Gi signaling resulting in L-lactate decreased release by astrocytes impairs mitochondrial biogenesis in neurons in an L-lactate reversible manner.

      By using MCT 2 inhibitors and an NMDAR antagonist the authors conclude that the molecular mechanisms underlying the observed effects are mediated by L-lactate entering neurons through MCT2 transporters and involve NMDAR.

      Overall, the article's conclusions are warranted by the experimental evidence, but some weak points could be addressed which would make the conclusions even stronger.

      The number of animals in some of the experiments is on the low side (4 to 6).<br /> The use of CIN to inhibit MCT2 is not optimal. Authors may want to decrease MCT2 expression by using antisense oligonucleotides.<br /> The experiment using AVP to block NMDAR only partially supports the conclusions. Indeed, blocking NMDAR will knock down any response that involves these receptors, whether L-lactate is necessary or not.<br /> Is inhibition of glycogenolysis involved in the observed effects mediated by Gi signaling? Indeed, L-lactate is formed both by glycolysis and glycogenolysis. The authors could test whether the glycogen metabolism-inhibiting drug DAB would mimic the effects of Gi activation.

    1. Reviewer #1 (Public Review):

      Zou et al. employ single-cell RNA sequencing of healthy skin, actinic keratosis (AK), squamous cell carcinoma in situ (SCCIS), and cutaneous squamous cell carcinoma (cSCC) to unravel the molecular events driving the progression of AK into cSCC (n=13 samples from 6 patients), thereby filling a gap of knowledge in skin cancer research. The authors identified several previously unreported candidate genes (including ALDH3A1, IGFBP2, MAGEA4, ITGA6, and LGALS1) involved in different stages of malignant progression, the expression of which was validated in situ in a large cohort. Functional in vitro experiments confirm a possible role for these genes in the transformation from benign to malignant skin lesions.

      Moreover, the authors identified epidermal cell subpopulations that may play an important role in the development from AK to cSCC, including an "early malignant cell" subpopulation within SCCIS basal cells with higher mutational load according to CNV analysis, which they characterized in more detail. For example, they found MAGEA4 strongly expressed in basal cells of (most) SCCIS and cSCC, as well as ITGA6. Functional assays in HaCaT and cSCC cell lines revealed that the knock-down of MAGEA4 and ITGA6 reduced proliferation, migration, and invasion but increased apoptosis in the cSCC cell lines.

      Finally, they describe the tumor microenvironment of a poorly differentiated cSCC sample, and scATAC sequencing of this poorly differentiated cSCC revealed that the majority of differentially accessible chromatin regions (DARs) were located in basal epidermal cells.

      Altogether, the authors provide a comprehensive transcriptional analysis of premalignant (AK, SCCIS) and malignant stages of cSCC. They suggest some key driver genes for each stage, the role of which are addressed in vitro and in situ in a large cohort. Thus, this study may provide novel biomarkers for tumor staging and diagnosis as well as potential targets for the prevention and treatment of cSCC.

    1. Reviewer #1 (Public Review):

      Zhao et al. investigated the molecular nature of the binding site for carbohydrates within the UDP-sugars known to activate the P2Y14 receptor. In order to do so, they built a molecular model of the hP2Y14, docked the corresponding agonists, and performed MD simulation on the resulting complexes. The modeling was used to identify the key molecular interactions with a cluster of charged residues in the extracellular side of the TM region of the receptor, which they show are conserved within the P2Y receptors. The binding site of the UDP region was, not surprisingly, overlapping with the analogous ADP binding site experimentally observed for the P2Y12 receptor, and consequently, the region that recognizes the sugars could be anticipated. Nevertheless, the detailed modeling and simulation work shows the consistency of this hypothesis and provides a quantification of the particular interactions involved, pinpointing specifically the residues candidate to be involved in the recognition of sugars.

      It follows the characterization, by functional assays, of the effect of single-point mutations of these residues in the efficacy of the different UDP-sugars. Here the results show a tendency to correlate with the molecular models, however some of the data has very low statistical significance and consequently the interpretation and conclusions extracted from this data should be taken with caution. This pertains to the particular role of the identified residues in the binding of the different sugars, which in some cases should be taken as a suggestion rather than a proof, though the general conclusion of the identification of the binding region for the sugar, its conservation among P2Y receptors and the role of some specific residues in sugar recognition seems convincing and the data are conveniently presented.

      Finally, the design of ADP-sugars that activate the P2Y12 receptor, based on the transferability of the observations with the UDP-sugars for the P2Y14 receptor, is a first indication that such a recognition is possible and should happen in an analogous binding region. However, the low potencies exhibited by the ATP-sugars, in the micromolar range, are too far from the ATP agonist and the relevance of this mechanism remains to be proved. The difference between P2Y12 and P2T14, with the last one showing much higher potencies for UDP-sugar derivatives than P2Y12 for the corresponding ADP-sugars, remains an interesting question not explored in this manuscript.

    1. Reviewer #1 (Public Review):

      Identifying compounds that can selectively inhibit protein kinases is of significant importance. Here, the authors describe a computational method to use existing kinome-wide profiling data to identify sets of compounds that, when combined, are more selective than any of the compounds on their own.

      The authors explain the methodology well and the methodology is well-supported. The outcome of the methodology is assessed using an assay orthogonal to the original profiling assays. It is hard to assess whether the methodology works when a different assay is used.

      The discussion of using this method for polypharmacology is naively discussed and under-supported.

    1. Joint Public Review

      In this manuscript, the authors develop a multi-scale agent-based model (ABM) capable of reproducing the self-organizing behavior observed in the intestinal crypt. By considering just the signaling pathways -previously reported as regulatory in the intestinal crypt- and local physical cell-to-cell interactions, the proposed model not only explains the emergence of the spatial organization, but also recapitulates cell composition dynamics in the crypt (proliferation, migration, and differentiation of cells), as previously characterized in the complex tissue of the small intestine epithelium in mice. The authors show that the self-organized system resulting from the model displays a stable composition over time. Additionally, the authors show how this model can be effectively used to test different conditions, such as biomedically relevant perturbations (e.g. stem cell ablation, cell cycle inhibition, and toxicity of particular drug treatments) and the posterior recovery, allowing to predict the safety of potential oncotherapies.

      In summary, the authors provide a powerful and versatile model, which can be applied to better understand the formation and response of the intestinal crypt, as well as the functional heterogeneity of the intestinal epithelium at multiple scales. The proposed mathematical model simulates features across scales in the intestinal crypt such as multiple signaling pathways, the mechanical environment and its forces, and cell cycle regulation. The model demonstrates the stability of the homeostatic crypt and recovery following stem cell ablation. The model also simulates the cell cycle protein network and demonstrates that CDK1 inhibition creates oversized cells. In sum, the model generated by the authors increases the understanding of how these biological processes take place in vivo, exploring not only healthy cell behavior but also cell response to injury by oncotherapies or other external factors. Additionally, the authors provide a series of fascinating movies that show the spatial organization of the crypt during these processes, and the manuscript has clear applications for the clinics.

      Nevertheless, in its current form, the manuscript has some weaknesses that are worth mentioning:

      (1) The developed model considers the interaction of multiple signaling networks that are essential for morphogenesis and homeostasis in the intestinal tissue, as well as other elements that had been proposed as relevant in the literature. Nevertheless, the details of how these interactions are modeled couldn't be evaluated in the current revision as the model was not shared with the reviewers and it is not available yet online, nor specified in any detail in the current manuscript. Additionally, how quantitative information from Wnt and BMP signaling pathways is incorporated in a quantitative way in the model is not clear.

      (2) Some conclusions by the authors are not properly justified in the text, as "Paneth cells are the main driver behind the differential mechanical environment in the niche", "Wnt-mediated feedback loop prevents the uncontrolled expansion of the niche", the specific effect of p27 in contrast with Wee1 phosphorylation over the cell cycle length, and "their recovery [absorptive progenitors] started before the end of the treatment, driven by a negative feedback loop from mature enterocytes to their progenitors".

      (3) Only the results of the "main" model are shown, with no information about its sensitivity to parameter values, and how their conclusions depend on specific decisions on the model. For example, the authors said that "an optimal crypt cell composition is achieved when BMP and Wnt differentiation thresholds result in progenitors dividing approximately four times before differentiating into enterocytes", but the results of alternative scenarios are not shown.

      (4) Regarding the construction of the model, the authors used "counts of Ki-67 positive cells recorded by position" while the original data reported "overall cell counts per crypt and villus". Some explanation about how this conversion was made, why it is valid, as well as any potential problems, is needed. Additionally, the model is based on experiments done by others in mouse models; the similarity to the response in human intestinal crypts is not discussed.

      (5) The authors imply that their mathematical model of the intestinal crypt is an improvement over those already published but there is no direct comparison or review of the literature to substantiate this claim.

      (6) The authors claim that the simulated data and the available mouse data match up. Nevertheless, the data vs the model still appear both quantitatively and qualitatively different (as presented in Figures 2E, F, and 5C, D). This puts in doubt how much the model can actually reproduce the experimental data. In conclusion, the model would benefit from further refinement, particularly if the goal is to use the model for predicting the dynamics of oncogenic drug candidates.

    1. Reviewer #1 (Public Review):

      In their manuscript, Brischigliaro et al. show that the disruption of respiratory complex assembly results in Drosophila melanogaster results in the accumulation of respiratory supercomplexes. Further, they show that the change in the supercomplex abundance does not impact respiratory function suggesting that the main role of supercomplex formation is structural. Overall, the manuscript is well written and the results and conclusion are supported. The D. melanogaster system, in which the abundance of supercomplexes can be altered through the genetic disruption of the assembly of the individual complexes, will be important for the field to discover the role of the supercomplexes. This manuscript will be of broad interest to the field of mitochondrial bioenergetics. The findings are valuable and the evidence is convincing.

      Strengths<br /> The system developed in which the relative levels of SCs can be varied will be extremely useful for studying SC physiology.

      The experiments are clearly described and interpreted.

      Weaknesses<br /> The previous weaknesses identified have been addressed.

    1. Reviewer #1 (Public Review):

      DMRT1 is essential in testis development in different species. While Dmrt1 is the testis-determining factor in chicken and deletion encompassing this gene lead to gonadal dysgenesis in human, the role of DMRT1 in testis development remains to be clarified. Despite an early expression of Dmrt1 in the mouse gonad and a potential function as a pioneer factor, DMRT1 is only required for the maintenance of the Sertoli cell identity in the postnatal testis. The use of a new animal model could provide new insights into the role of this factor in humans. Here the authors have generated a knockout model of DMRT1 in rabbits. They show that the XY mutant gonads differentiate as ovary indicating that DMRT1 is required for testis differentiation in rabbits. In addition, most of the germ cells remain pluripotent as evidenced by the maintenance of POU5F1 in both XY and XX mutant gonads. These are very important results potentially explaining gonadal dysgenesis associated with the DMRT1 locus in disorders of sex development in humans.

      The experiments are meticulous and convincing. I find the arguments of the authors about the role of DMRT1 in germ cells in addition to its function in Sertoli cell differentiation, both comprehensible and compelling. Clearly, this is an important insight in sex determination and gametogenesis.

    1. Reviewer #1 (Public Review):

      This study assesses the volatile profiles from the hair and bodies of 64 vertebrate species to compare odor constituents across taxa. Compared to a similar data set for floral volatiles, the study suggests that vertebrate odors are significantly less diverse and show little phylogenetic relationship regarding profile similarity. Human odors were particularly unique from other species. It is concluded that this may influence the odor coding of organisms (like vectors) who respond to these odors compared to plant-feeding organisms like most other insects. While the study is compelling, several methodological issues leave the conclusions less convincing. It is suggested that the paper be tempered accordingly with these issues mentioned.<br /> The study makes several assumptions about the methodology to be considered when interpreting the results:

      Major Concerns:

      1) Body hair as a proxy for animals. "Hair odour is likely a reasonable proxy for mammalian body odour, but may lose some volatile compounds during storage. Live-animal odour, on the other hand, can be contaminated with compounds from faeces or urine occasionally excreted during sampling." The study has addressed this by testing hair against the bodies of 4 humans, two rats, and one guinea pig (Figure S1). However, the results show that there are both quantitative and qualitative differences among all the samples. While the presence of waste accounts for some of this variation, this, too, is a natural response of the animal and could be present in natural settings. Also would not body heat in mammals have an impact on odors? The authors should support this. While this does not require reanalysis, the authors should address these differences, particularly when qualitative and quantitative differences are discussed heavily in the results.

      2) Sampling medium: Tenax TA was used to sample the vertebrate odors. Please note that any sorbent will exhibit specificity regarding selectivity and sensitivity to VOCs. See https://www.eva.mpg.de/documents/Elsevier/Marcillo_Comparison_JChromA_2017_2452774.pdf for one comparison. For example, it is not surprising that "Aldehydes, ketones, alcohols, aromatics, terpenes, and hydrocarbons dominated" the samples given that these types of compounds are well retained by the Tenax polymer:<br /> https://www.sisweb.com/index/referenc/tenaxta.htm<br /> Many chemical ecology studies will employ multiple polar and non-polar polymers to retain different VOCs for better profile comparison.<br /> By itself, this limitation must be noted. However, it becomes even more relevant when compared to the floral volatile study, which used a different sorbent (Poropak) which is less hydrophobic and may retain more polar compounds than Tenax: https://hero.epa.gov/hero/index.cfm/reference/details/reference_id/2859526<br /> Such differences must be considered when comparing these two datasets, particularly when the study makes conclusions about their differences. Alternatively, a small set of experiments with poropak and a few species (like for the hair vs. body control experiments) could clarify the effect of sorbent type on VOC retention.

      3) Sampling quantification: The methods note that " All extractions were run for 5-80 minutes depending on the expected odour concentration of the sample." What does this mean? Such differences in sampling timing in our lab have shown profound differences in the type and amount of volatiles collected. Generally, it is best to sample for as long as possible to ensure that the most volatiles are collected (up to 24 hours if possible). The compounds will eventually come into equilibrium with the sorbent. However, for quantification, the timing must be calibrated carefully, usually by using a representative set of likely compounds with different functional groups to determine the optimal length of sampling time. Was this done in this case? If not, how can one account for the significant variation in sampling time regarding quantification?<br /> A second issue with quantification is the need for an internal standard. Even with robotic assistance, slight variations in processing can significantly affect the quantity of volatile retained through detection at the MS. This is generally avoided by using an internal standard in the sampling arena. See this example with multiple sampling techniques (also employing TD-GCMS): https://www.frontiersin.org/articles/10.3389/fevo.2021.607555/full<br /> Without these methodological controls, it is unclear how effective quantification can be performed. It might be more prudent to confine the results to qualitative discussions.

    1. Reviewer #1 (Public Review):

      This study seeks to understand how selective mRNA translation informs cellular identity using the Drosophila brain as a model. Using drivers specific for either neurons or glia, the authors express a tagged large ribosomal subunit protein, which they then use as a handle for isolating total mRNA and ribosome footprints. Throughout the study, they compare these data sets to transcriptional and ribosome profiles from the whole fly head, which contains multiple cell types including fat tissue, pigment cells, and others, in addition to neurons and glia. Using GO term analyses, they demonstrate the specificity of their cell-type-based ribosome profiling: known glial mRNAs are efficiently translated in glia and likewise in neurons as well. In further examining their RNAseq data set, they find that "neuronal" mRNAs, such as ion channels, are expressed in both neurons and glia, but are translated at higher rates in neurons. Based on this, they hypothesize that neuronal mRNAs are actively suppressed in glia, and next seek to determine the underlying mechanism. By meta-analysis of all mapped ribosome footprints, they find that glia have higher ribosome occupancies in the 5' leader of neuronal mRNAs. This is corroborated by individual ribosome occupancy profiles for several neuronal mRNAs. In 5'leaders containing upstream AUG codons, they find that the glial data sets show enrichment of ribosomes at these upstream start sites. They thus conclude that 5' leaders containing upstream AUGs confer translational suppression in glia.

      Overall, the sequencing data sets generated in this study and their subsequent bioinformatic analyses seem robust and reliable. Their data echo the trends of cell-type specific translational profiles seen in previous studies (e.g. 27380875, 30650354), and making their data sets and analyses accessible to the broader scientific community would be quite helpful. The findings are presented in a logical and methodical manner, and the data are depicted clearly. The authors' results that 5' leaders facilitate translation suppression is well-supported in literature. However, they overinterpret their data by claiming that such suppression is key for maintaining glial/neuronal identity (it is even featured in their title), but do not present any evidence that loss of such regulation has any impact on cellular identity. In many places, the authors do not acknowledge possible biases in their analytical methods, or consider alternate explanations for their data. These weaken the manuscript in its current form, but many of these issues which I describe below, are rectifiable with modest effort.

      1. The authors' data in Fig. 2-S1A-B shows substantial cell-to-cell variation in RpL3::FLAG expression. The authors do not consider that this variation may cause certain neuronal/glial types to be overrepresented in their datasets. A related point is that the authors do not discuss whether RpL3::FLAG is only present in the cell body or if it is also trafficked to the neuronal/glial processes where localized translation is known to occur (reviewed in 31270476).

      2. The RNA-seq data set that they use to calculate translation efficiency (TE) only represents mRNAs associated with RpL3::FLAG, which is part of the large ribosome subunit. As the authors are likely aware, there are mRNAs on which the full ribosome moiety does not assemble and these are effectively excluded from this data set. Ideally, a more complete picture of the mRNA landscape can be obtained by 40S subunit profiling but I appreciate that this is technically very challenging. At a minimum, this caveat needs to be acknowledged.

      How does the TPM of differentially regulated transcripts (such as those in Fig. 2H) compare between whole heads, neurons, and glia? Since the whole head RNA-seq data was not from an enriched sample, this might serve as a decent proxy for showing that the neuron/glia RNA-seq data sets are representative of RNA abundance.

      3. The analysis in Fig. 2F shows that low abundance mRNAs in glia are further translationally suppressed, which the authors point out in lines 151-152. However, this data also shows that mRNAs with a 1:1 ratio in neuron:glia (which fall in the 0.5-1 and 1-2 bin) have a TE1; this suggests that on average, mRNAs that are equally abundant are translated equally efficiently. This is the opposite of the thesis presented in Fig. 2G-H where many mRNAs of equal abundance in neurons and glia are actually poorly translated in glia. How do the authors reconcile these observations?<br /> It is also unclear from the manuscript whether all mRNAs were considered for the analysis in Fig. 2F or if some cutoff was employed.

      4. Throughout the manuscript the authors favor a "translation suppression" model wherein glia (for example) actively suppress neuronal mRNAs, and this is substantiated in Fig. 3C showing higher ribosome occupancy on 5' leaders than in coding regions. However, they show no evidence that glial mRNAs (such as those indicated in Fig. 2B and 2-S2B) present a different pattern, say that of higher ribosome occupancy in CDS vs. 5' leaders. This type of positive control is a glaring omission from many of their analyses, including ribosome occupancy at upstream AUG codons (Fig. 4).<br /> In order to make a broad case (as they do in the title) that differential translation regulation specifies multiple cell types, it is necessary to show the corollary: that glial mRNAs (repo, bnb, pnt, etc) are suppressed in neurons. There is an inkling of this evidence in Fig. 3-S1 where fat body mRNAs in neurons are shown to have low ribosome occupancy in the CDS regions and enhanced occupancy in the 5' leader region. This data is not quantified, nor is a control neuron mRNA shown as a reference for what the ribosome occupancy profile of an actively translated mRNA looks like in a neuron.

      5. The cell-type specific ribosome profiling data sets in the manuscript are from mRNAs associated with 80s subunits that have been treated with cycloheximide during sample preparation. Cycloheximide, and many other translation inhibitors, are known to non-uniformly bias reads towards start codons (PMID: 22056041,22927429). This important caveat and its implications on the start-codon occupancy analysis in Fig. 4 are not acknowledged in the manuscript.<br /> Again, the ideal resolution would be a ribosome profiling data set from 40S footprinting or harringtonine-treated samples (PMIDs: 32589966, 27487212, 32589964) to show the true accumulation of ribosomes at AUG codons. In the absence of such a data set, a comparative meta-analysis of the ribosome distribution around upstream and initiation AUG codons of differentially translated transcripts from neurons would be a useful control.

      6. The authors chose Rhodopsin 1 (Rh1) as a model mRNA which is translated efficiently in neurons but suppressed in glia. Though the data in Fig. 2-S3B shows higher TE for Rh1 in neurons, the data in 5A show lower ribosome occupancy in the Rh1 CDS in neuron samples (at least in the fragment of the CDS visible). These data are somewhat contradictory.<br /> Further, given that the neuron data are from all nsyb-positive cells but that Rh1 is expressed only in R1-R6 photoreceptors, it is unclear what motivated them to choose Rh1 as opposed to an mRNA that is more broadly expressed in neurons.

      7. Similar to the heterogeneity in nsyb- and repo-GAL4 expression in Fig. 2-S1A-B, Fig. 5C shows substantial variation in the expression of the UAS-GFP reporter driven by tub-GAL4. This variable GAL4 activity makes the mRNA abundance data difficult to interpret. Also, since the authors presume that Rh1 mRNA is expressed in glia (it is not annotated in the RNA-seq analysis in Fig. 2-S2B), would Rh1-GAL4 not be a more apt driver?<br /> These issues are further compounded by the lack of a cellular compartment marker (repo marks glial nuclei) which makes it impossible to determine which cell the mRNA signal is in. There are also no negative controls presented for the mRNA probes.<br /> Most confoundingly though, the control reporter itself seems to show variable translation efficiencies from one cell to another, with high-GFP protein cells showing lower GFP mRNA and vice versa.<br /> The mRNA:protein ratio may be easier to examine by using repo-GAL4 to specifically drive the Rh1-reporter expression in glia (such as in Fig. 5-S1A) rather than simultaneous expression in both neurons and glia using tub-GAL4.

    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.

      The major weaknesses of this study are the inconsistent dissection of the targeted brain region and the loss of more mature excitatory neurons in samples from later developmental timepoint due to the use of single-cell RNA-seq. The authors mention that they could observe ventral progenitors and even midbrain neurons in their analyses. Ventral progenitors should not be present if the authors had properly dissected the parietal cortex. The fact that they obtained even midbrain cells point to an inadequate dissection or poor cell classification. If this is the result of poor classification, it could be easily fixed by using more markers with higher specificity. However, if it is the result of a poor dissection, some of the cells in other clusters could potentially be from midbrain as well. The loss of more mature excitatory neurons is also problematic because on top of hindering the analysis of these neurons in later developmental periods, it also affects the cell proportions the authors use to support some of their claims. The study could also benefit from the validation of some of the genes the authors uncovered to be specifically expressed in different populations of radial glia.

    1. Reviewer #1 (Public Review):

      The main goal of the study was to tease apart the associative and non-associative elements of cued fear conditioning that could influence which defensive behaviors are expressed. To do this, the authors compared groups conditioned with paired, unpaired, or shock only procedures followed by extinction of the cue. The cue used in the study was not typical; serial presentation of a tone followed by a white noise was used in order to assess switches in behavior across the transition from tone to white noise. Many defensive behaviors beyond the typical freezing assessments were measured, and both male and female mice were included throughout. The authors found changes in behavioral transitions from freezing to flight during conditioning as the tone transitioned into white noise, and a switch in freezing during extinction such that it became high during the white noise as flight behavior decreased. Overall, this was an interesting analysis of transitions in defensive behaviors to a serially presented cue consisting of two auditory stimuli during conditioning and then extinction. There are some concerns regarding the possibility that the white noise is more innately aversive than the tone, inducing more escape-like behaviors compared to a tone, especially since the shock only group also showed increased escape-like behaviors during the white noise versus tone. This issue would have been resolved by adding a control group where the order of the auditory stimuli was reversed (white noise->tone). While the more complete assessment of defensive behaviors beyond freezing is welcomed, the main conclusions in the discussion are overly focused on the paired group and the associative elements of conditioning, which would likely not be surprising to the field. If the goal, as indicated in the title, was to tease apart the associative and non-associative elements of conditioning and defensive behaviors, there needs to be a more emphasized discussion and explicit identification of the non-associative findings of their study, as this would be more impactful to the field.

    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 study, Nuria Martin-Flores, Marina Podpolny and colleagues investigate the role of Dickkopf-3 (DKK3), a Wnt antagonist in synaptic dysfunction in Alzheimer's disease. Loss of synapses is a feature of Alzheimer's and other forms of dementia such as frontotemporal dementia and linked amyotrophic lateral sclerosis (FTD). The authors utilise a broad range of experimental approaches. They show that DKK3 levels are increased in Alzheimer's disease and that this occurs early in disease. This is an important finding since early disease changes are believed to be the most important. They also show increases in DKK3 in transgenic mouse models of Alzheimer's disease and that DKK3 knockdown restores synapse number and memory in one such model. Finally, they link these DKK3 increases to loss of excitatory synapses via the blockade of the Wnt pathway and subsequent activation of GSK3B; GSK3B is strongly linked to both Alzheimer's disease and FTD. The quality of the data is good and the conclusions well supported by these data. There are no major weaknesses. The findings support studies that target the Wnt pathway as a potential therapeutic for Alzheimer's disease.

    1. Reviewer #1 (Public Review):

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

    1. Reviewer #1 (Public Review):

      This manuscript by He et al. explores the molecular basis of the different stinging behaviors of two related anemones. The freshwater Nematostella which only stings when a food stimulus is presented with mechanical stimulation and the saltwater Exaiptasia which stings in response to mechanical stimuli. The authors had previously shown that Nematostella stinging is calcium-dependent and mediated by a voltage-gated calcium channel (VGCC) with very pronounced voltage-dependent inactivation, which gets removed upon hyperpolarization produced by taste receptors.

      In this manuscript, they show that Exaiptacia and Nematostella differing stinging behavior is near optimal, according to their ecological niche, and conforms to predictions from a Markov decision model.

      It is also shown that Exaiptacia stinging is also calcium-dependent, but the calcium channel responsible is much less inactivated at resting potential and can readily induce nematocyte discharge only in the presence of mechanical stimulation. To this end, the authors record calcium currents from Exaipacia nematocysts and discover that the VGCCs in this anemone are not strongly inactivated and thus are easily activated by mechanical stimuli-induced depolarization accounting for the different stinging behavior between species. The authors further explore the role of the auxiliary beta subunit in the modulation of VGCC inactivation and show that different n-terminal splice variants in Exaiptacia produce strong and weak voltage-dependent inactivation.

      The manuscript is clear and well-written and the conclusions are in general supported by the experiments and analysis. The findings are very relevant to increase our understanding of the molecular basis of non-neural behavior and its evolutionary basis. This manuscript should be of general interest to biologists as well as to more specialized fields such as ion channel biophysics and physiology.

    1. Joint Public Review:

      The authors have previously established that activation of dopamine inputs to prefrontal cortex during adolescence can drive increases in mPFC DA bouton number and enhanced mPFC activity in WT mice. The current study was designed to test the hypothesis that neural circuit plasticity during adolescence can be targeted to restore cortical function under conditions of developmental disruptions that are relevant to psychiatric disorders.

      Specifically, the manuscript explores how transient adolescent stimulation of ventral midbrain neurons that project to the frontal cortex may help to improve performance on certain memory tasks. The authors used DREADDs to regulate the mesofrontal cortical dopamine system in two mouse models - one with a reporter replacing the Arc gene, and another with knockout of the schizophrenia-associated gene Disc1, both of which are thought to have reduced prefrontal cortical activity. The manuscript provides an interesting set of observations that DREADD-based activation over only 3 days during adolescence provides a fast-acting and long-lasting improvement in performance on Y-maze spontaneous alternation as well as aspects of neuronal function as assessed using in vivo imaging methods.

      A strength of this study is that the authors performed key manipulations using age and dose/intensity as dependent variables to show that the level of neural circuit activation during adolescence follows an inverted U-shape pattern, though the precise postsynaptic mechanisms underlying the positive impact of adolescent mesofrontal dopamine neuron stimulation were not addressed.

      One limitation discussed by the reviewers is that using TH-Cre mice (as compared with DAT-Cre) to drive transgene expression in VTA neurons could lead to expression outside the dopaminergic population of neurons, though in the revision the authors have provided additional lines of evidence to support their model of dopamine regulation of frontal cortex in this study.

      Collectively, this is a well-design study with many strengths and novel findings that are likely to positively impact a widespread of disciplines within the biological psychiatry and neuroscience field.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors present a comprehensive meta-analysis of Clostridioides difficile (CD) occurrence across 42,900 metagenomes from 253 public studies, largely representing stool samples from human adults, infants, and with a smaller fraction of samples from non-gut body sites and from environmental samples (e.g., non-human animals, wastewater, soil, etc.). In particular, the authors looked at adults who were healthy, diseased (but not with C. diff), and with diagnosed C. diff infection (CDI) and found that CD occurrence was fairly low: ~30% in adult CDI samples, ~2% in adult diseased samples, and ~1% in healthy samples. CD was much more prevalent in infants (15 and 40% in healthy and diseased infants, respectively). These findings, if they hold true, would be significant because they would suggest an over-diagnosis of CDI and an under-diagnosis of other putative enteric pathogens (also enriched in CDI samples) across the population. Furthermore, these results suggest that the asymptomatic carriage of CD in adults (~1-5%, depending on demographics) may be much lower than some prior estimates (some as high as ~30-40%).

      Strengths:

      The authors have done an admirable job pulling down an enormous data set for this CD-focused meta-analysis, which is a valuable service to the field. The results push against some common wisdom in the field, in terms of the prevalence of CD in CDI patients, which will be impactful if they hold up to further scrutiny. Furthermore, the identification of commensal bacteria that are positively or negatively associated with CD presence in both healthy and diseased people at different periods of the lifespan (infant, child, and adult), is a valuable synthesis with potential translational value. The manuscript is clearly written and the figures are presented well. The methodology is robust, although I have a few suggestions for improvement.

      Weaknesses:

      My main critique relates to detection limitations, both in terms of sequencing depth and read-mapping. Given that CD detection is the root of the main conclusions reported here, this deserves some additional care. The authors have already done some work to address this by including sequencing depth in their linear mixed effects model, which is great. Furthermore, they were conservative with how they labeled CD positive/negative individuals with multiple time points (i.e., if you had CD detected at any point, this sample was selected for the cross-sectional analysis, and that individual was labeled as CD positive). I have a few additional suggestions to explore this issue, which I outline in the recommendations for authors.

    1. Reviewer #1 (Public Review):

      The manuscript by Hussein et al. uses cryoEM structure, microscale thermophoresis (MST), and molecular dynamics simulations (conventional and CpHMD) to unravel the Zn2+ and proton role in the function of the Cation Diffusion Facilitator YiiP. First, they generate mutants that abolish each of the three Zn2+ models to study the role of each of them separately, both structurally and functionally. Next, they used a Monte Carlo approach refining the CpHMD data with the MST points to establish the Zn2+ or proton binding state depending on the pH. That predicted a stoichiometry of one Zn2+ to 2 or 3 protons (1:3 under lower pH values). Finally, they proposed a mechanism that involves first the binding of Zn2+ to one low-affinity site and then, after the Zn2+ migrates to the highest affinity site in the transmembrane portion of the protein. The lack of Zn2+ in the low-affinity site might induce occlusion of the transporter.

      The manuscript is well-written it is of interest to the field of Cation Facilitator Transporters. It is also an excellent example of a combination of different techniques to obtain relevant information on the mechanism of action of a transporter.

    1. Joint Public Review:

      This study provides evidence of the ability of sublethal imidacloprid doses to affect growth and development of honeybee larva. While checking the effect of doses that do not impact survival or food intake, the authors found changes in the expression of genes related to energy metabolism, antioxidant response, and metabolism of xenobiotics. The authors also identified cell death in the alimentary canal, and disturbances in levels of ROS markers, molting hormones, weight and growth ratio. The study strengths come from exploring different aspects and impacts of imidacloprid exposure on honeybee juvenile stages and for that it demonstrates potential for assessing the risks posed by pesticides. The study weaknesses come from the lack of in depth investigation and an incomplete methodological design. For instance, many of the study conclusions are based on RT-qPCR, which show only a partial snapshot of gene expression, which was performed at a single time point and using whole larvae. There is no understanding of how different organs/tissues might respond to exposure and how they change over time. That creates a problem to understand the mechanisms of damage caused by the pesticide in the situation studied here. There is no investigation of what happens after pupation. The authors show that the doses tested have no impact on survival, food consumption and time to pupation, and the growth index drops from ~0.96 to ~0.92 in exposed larvae, raising the question of its biological significance. The origin of ROS are not investigated, nor do the authors investigate if the larvae recover from the damage observed in the gut after pupation. That is important as it could affect the adult workers' health. One of the study's central claims is that the reduced growth index is due to the extra energy used to overexpress P450s and antioxidant enzymes, but that is based on RT-qPCR only. Other options are not well explored and whether the gut damage could be causing nutrient absorption problems, or the oxidative stress could be impairing mitochondrial energy production is not investigated. These alternatives may also affect the growth index. The authors also state that the honeybee larvae has 7 instars, which is an incorrect as Apis mellifera have 5 larval instars. It is not clear from methods which precise stage of larval development was used for gut preparations. That information is important because prior to pupation larvae defecate and undergo shedding of gut lining. That could profoundly affect some of the results in case gut preparations for microscopy were made close to this stage. A more in-depth investigation and more complete methodological design that investigates the mechanisms of damage and whether the exposures tested could affect adult bees may demonstrate the damage of low insecticide doses to a vital pollinator insect species.

    1. Reviewer #1 (Public Review):

      This manuscript deftly combines cryo-EM and electrophysiology to investigate the gating mechanisms of human CLC-2. Although another structure of CLC-2 was recently reported, this is the first structure to report density for the absolutely critical gating glutamate, and - an even more exciting result - the first structure to identify the N-terminal gating peptide that is the heart of this manuscript. There has been previous controversy over such a gating peptide in CLC-2, but the combined structural/functional approach appears to establish a role for this peptide in gating and sets up exciting future experiments to understand why its effects might change under different physiological scenarios. The experiments reported here are thoughtful and well-controlled and the data presentation is excellent. For the electrophysiology experiments, the use of inhibitor AK-42 (developed by the current senior author's lab) to establish a zero current level is a welcome advance and should become standard for electrophysiological studies of CLC-2.

    1. Joint Public Review:

      The manuscript by Aguirre et al. describes an elegant approach for developing selective inhibitors of inositol hexakisphosphate kinases (IP6Ks). There are 3 IP6K isozymes (IP6K1-3) in humans, which catalyze the synthesis of inositol pyrophosphates. The lack of isozyme-selective inhibitors has hampered efforts to understand their individual physiological roles. While several inhibitors of IP6Ks have been described, they either lack isozyme selectivity or inhibit other kinases. To address this gap, Aguirre et al. used an analog-sensitive approach, which involves the identification of a mutant that, in an ideal world, doesn't impact the activity of the enzyme but renders it sensitive to an inhibitor that is absolutely selective for the engineered (analog-sensitive) enzyme. Initially, they generated the canonical gatekeeper (Leu210 in IP6K1) mutations (glycine and alanine); unfortunately, these mutations had a deleterious effect on the enzymatic activity of IP6K1. Interestingly, mutation of Leu210 to a valine, a subtly smaller amino acid, didn't affect enzymatic activity. The authors then designed a clever high-throughput assay to identify compounds that show selectivity for L210V IP6K1 versus WT IP6K1. The assay monitors the reverse reaction catalyzed by IP6Ks, monitoring the formation of ATP using a luminescence-based readout. After validating the screen, the authors screened 54,912 compounds. After culling the list of compounds using several criteria, the authors focused on one particular compound, referred to as FMP-201300. FMP-201300 was ~10-fold more potent against L210V IP6K1 compared to WT IP6K1. This selectivity was maintained for IP6K2. Mechanistic studies showed that FMP-201300 is an allosteric inhibitor of IP6K1. The authors also did a small SAR campaign to identify key functional groups required for inhibition.

      Overall, this manuscript describes a unique and useful strategy for developing isozyme-selective inhibitors of IP6Ks. The serendipitous finding that subtle changes to the gatekeeper position can sensitize the IP6K1 mutant to allosteric inhibitors will undoubtedly inspire other analog-sensitive inhibitor studies. The manuscript is well-written and the experiments are generally well-controlled.

    1. Reviewer #1 (Public Review):

      In the "Drivers of species knowledge across the Tree of Life", Mammola and collaborators explore the determinants of scientific and societal interest across very wide taxonomic and spatial scales. Their work highlights our uneven knowledge of biodiversity and its potential causes.

    1. Joint Public Review:

      When the left-right asymmetry of an animal body is established, a barrier that prevents the mixing of signals or cells across the midline is essential. Such a midline barrier preventing the spreading of asymmetric Nodal signaling during early left-right patterning has been identified. However, midline barriers during later asymmetric organogenesis have remained largely unknown, except in the brain. In this study, the authors discovered an unexpected structure in the midline of the developing midgut in the chick. Using immunofluorescence, they convincingly show the chemical composition of this midline structure as a double basement membrane and its transient existence during the left-right patterning of the dorsal mesentery, which authors showed previously to be essential for forming the gut loop and guiding local vasculogenesis. Labelling experiments suggest a physical barrier function, to cell mixing and signal diffusion in the dorsal mesentery. Cell labelling and graft experiments rule out a cellular composition of the midline from dorsal mesenchyme or endoderm origin and rule out an inducing role by the notochord. Based on laminin expression pattern and Ntn4 resistance, the authors propose a model, whereby the midline basement membrane is progressively deposited by the descending endoderm.

      Laterality defects encompass severe malformations of visceral organs, with a heterogenous spectrum that remains poorly understood, by a lack of knowledge of the different players of left-right asymmetry. This fundamental work significantly advances our understanding of left-right asymmetric organogenesis, by identifying an organ-specific and stage-specific midline barrier. The complexities of basement membrane assembly, maintenance, and function are of importance in several other contexts, as for example in the kidney and brain. Thus, this original work is of broad interest.

      Overall, reviewers refer to a strong and elegant paper discovering a novel midline structure, combining classic but challenging techniques, to show the dynamics, chemical, and physical properties of the midline. However, reviewers also indicate that further work will be necessary to conclude on the origin and impact of the midline for asymmetric organogenesis. Three issues have been raised to strengthen the claims:

      1) The function of the midline as a physical barrier requires clarification. Dextran injection here seems to label cells and not the extracellular space. By counting the proportion of dextran-labeled cells rather than dextran intensity itself, the authors do not measure diffusion per se, but rather cell mixing.

      2) The descending endoderm zippering model for the formation of the midline lacks direct evidence. The claim of an endoderm origin is based on laminin expression, but the laminin observed in the midline with an antibody may not necessarily correspond to the same subtype assessed by in situ hybridization. The midline may be Ntn4 resistant until it is injected in the relevant source cells. Alternative origins could be considered, from the bilateral dorsal aortae or the paraxial mesoderm, which would explain the double layer as a meeting point of two lateral tissues.

      3) The title implies a role of the midline in left-right asymmetric gut development. However, the importance of the midline is currently inferred from previously published data and stage correlations and will require more direct evidence.

    1. Reviewer #1 (Public Review):

      This work is hugely significant in the context of the debates surrounding ancient Egyptian activity on the Red Sea and voyages to the Egyptian land of Punt. Using genetic studies to provenance baboon mummified baboons in Egypt with contemporary baboon populations in the Red Sea region, the study argues persuasively that Egyptians obtained Baboons from coastal Eritrea, and thus that Egyptian baboon trade involved this region. This of course brings up the larger issue of Egyptian trade with Punt and the southern Red Sea, a place known to have furnished Egyptians with baboons. The authors argue logically for the region of the ancient port-city of Adulis as being particularly important in this baboon trade, as the region around the harbour was said to have been a baboon habitat in the Graeco-Roman period.

      Along with other previous geoprovenancing scientific studies relating to baboon isotopes and obsidian trace elements, this study provides a solid foundation for considering coastal Sudan and especially Eritrea as part of the land of Punt.

    1. Reviewer #1 (Public Review):

      This manuscript describes conditions under which "Self-inactivating Rabies" (SiR) can be grown to limit mutations that would allow the virus to replicate in the absence of TEV protease. It is also shown that neurons directly infected with a non-mutated virus remain healthy and that the virus does not mutate in the brain in vivo. Remarkably there is nothing in the manuscript to address the obvious question that is raised by the observation that such mutations were occurring around the time of the initial description of circuit tracing with this virus. Can the transsynaptic tracing experiments in the absence of TEV expression (as described in their original Neuron paper) be replicated with SiR that is not mutated? This obvious omission suggests that the authors might have conducted such experiments and were unable to replicate their published results. It is imperative that the authors be forthcoming about whether they have conducted such experiments and what were the results. If they have not conducted such experiments, they should do them and include the results here. If they cannot replicate their results, then the reliability of the Neuron paper is in doubt.

      How do the results presented here relate to the results published in the Neuron paper and why are they not definitive with respect to the utility of SiR? The original publication in Neuron presents results that do not appear to be plausible and are best explained by the possibility that some experiments described in that manuscript were conducted using mutated SiR. This became most apparent when shortly after the Neuron publication, the Tripodi lab shared SiR as well as TEV expressing cell lines for propagation with other labs. Several of those groups observed that when they progagated the SiR received from the Tripodi lab, there was a mutation that removed the linkage of the PEST targeting sequence to N. This would be expected to allow the virus to replicate and spread without the need for TEV protease to remove the PEST sequence - precisely the phenotype observed in the trans-synaptic tracing experiments described in the Neuron paper. In the Neuron paper, culture experiments showed that the N-PEST (SiR) rabies could not replicate in the absence of TEV. And additional experiments showed that the virus is not toxic to neurons directly infected. These are the same experiments that are replicated in this submission. But then (in the Neuron paper) comes the unlikely report that this virus can spread trans-synaptically in vivo, in the absence of TEV expression. An alternative explanation would be that the virus used for those experiments was mutated and that is why TEV expression was not needed. There are no experiments in the original Neuron paper that address this possibility. Specifically, the experiments in Neuron describing cell survival during trans-synaptic tracing are not adequate to rule this out. This is because the two timepoints during which neurons were counted correspond to an early time when labeled neurons would be expected to still be accumulating and a later time that might be past the peak and represent a time when many neurons have died. To quantify proportions of neurons that survive, it is necessary to follow the same neurons over time, as has been done to demonstrate that only about half of neurons infected with G-deleted rabies die (half survive). Until tests are conducted testing whether TEV expression is required to obtain trans-synaptic labeling with an SiR that is known to not be mutated, it is irrelevant whether mutations can be prevented under particular culture conditions. The utility of this virus depends on whether it can be used for trans-synaptic tracing without toxicity and this manuscript presents no experiments to address that. Further, the omission of such experiments is glaring, as it is difficult to imagine that they have not been attempted.

      Other comments:

      "A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, represents the first tool for open-ended genetic manipulation of neural circuits."<br /> It is not clear what the authors intend to be claiming with respect to "open-ended genetic manipulation of neural circuits" but it is clear that this assertion is overblown. There are numerous tools that are available for genetic manipulation of neural circuits. This is not the first, won't be the last, and it is arguably not the best.

      "Interestingly, a fraction of tdTomato+ neurons survived in ΔG- Rab-CRE-injected brains, differing from what we observed when injecting ΔGRab-GFP, where no cells were detected at 3 weeks p.i. (Fig 3CD) (Ciabatti et al., 2017). " This is a known result (same as Chatterjee et al., 2018) with a known mechanism. GFP expression is not observed because the rabies virus transitions from transcription to replication resulting in the termination of GFP expression. But Cre-recombination of the genome permanently labels cells with TdTomato. This is how Chatterjee et al. demonstrated that half of the neurons infected with G-deleted rabies survive. They imaged cells and saw that the GFP disappeared but the cells marked by Cre-recombination and RFP expression remained healthy indefinitely. The consideration of this in the Introduction is strange. There is no reason to suppose that Cre expression would somehow protect cells from rabies infection and there is no need to propose any such mechanism to explain the observed results.

      "Here we show that revertant-free SiR-CRE efficiently traces neurons in vivo without toxicity in cortical and subcortical regions for several months p.i.."<br /> This wording is disingenuous and appears to be intentionally misleading. "Trace" implies that circuits were traced by transynaptic labeling, which they were not.

    1. Reviewer #1 (Public Review):

      Wang and colleagues show that tree shrews can detect optogenetic stimulation of the lateral geniculate nucleus (LGN) using an AAV2-CamKIIα-ChR2 construct after training detection of visual stimuli. Solid evidence links optogenetic stimulation to behavioural detection and neurophysiological responses in LGN and local field potentials in V1.

      The major strength is the carefully conducted optogenetic detection experiments showing that training of a visual detection task can be transferred to the the detection of focal optogenetic stimulation in the LGN. The optogenetic stimulation can evoke responses in LGN that can be transmitted to V1.

      However, the behavioural results are highly variable between individual animals and different optogenetic stimulation frequencies. The nature of this variability remains unclear. A weakness of this complex in vivo study lies in the underspecified description of some of the details and the links between the histology, the neurophysiology and optogenetic results, in order to understand this variability better. The neurophysiological results are clear and important, but the distribution of significant results across the different animals studied is missing. The expression patterns across layers of the optogenetic viruses appear to differ in the histology of three different animals shown, but it is unclear except for one animal from which experimental individuals these results stem. While the methods of the behavioural and neurophysiological results are well described, the methods section is incomplete with regards to the very nice histology presented (perfusion, sectioning, staining).

      The detection of optogenetic activation of LGN in this visual animal model suggests that LGN is a potential target for a neuroprosthetic device. This paper is potentially of interest to neuroscientists and clinicians working on the visual system and visual prostheses.

    1. Reviewer #1 (Public Review):

      Dolgova et al present a well-written manuscript focused on the mechanism of MEMO1 function in tumor cells. They use genome-wide analyses to predict function based on MEMO1 structure in yeast, identify MEMO1 expression in a screen of cancer cell lines, and demonstrate a correlation between MEMO1 expression and severity of disease in primary breast cancer cells. The authors focus on a breast cancer model as it overexpresses MEMO1 and melanoma as a control and uses CRISPR-Cas9 knockdown of MEMO1 in breast and melanoma cell lines and concurrently knockdown selected genes in the iron homeostasis pathway. In this data, MEMO1 appears to interact with elements involved in iron trafficking and sensing and its overexpression leads to possible hypersensitivity while knockout/knockdown leads to resistance to lipid oxidation. They also interrogate the effect of iron chelation on mitochondrial morphology and ferroptosis. In addition, they evaluate iron and copper binding loci and resolve MEMO1 structure. The work is of high quality. However, there are some inaccuracies regarding the known function of some iron-related elements. Furthermore, it is unresolved whether controlling iron per se (by modulating other importers and transporters or limiting iron availability in culture) recapitulates or ameliorates their findings, currently attributed specifically to the mechanism of action of MEMO1. In addition, the authors make claims that they have not substantiated about overexpression of MEMO1 by extrapolating from data about MEMO1 knockdown or knockout. Finally, the results show only indirect evidence for a central role for MEMO1 via regulation of iron trafficking and more targeted approaches are necessary to increase confidence in the claims.

    1. Joint Public Review:

      In this study, the authors investigate the biological function of the FK506-binding protein FKBP35 in the malaria-causing parasite Plasmodium falciparum. Like its homologs in other organisms, PfFKBP35 harbors peptidyl-prolyl isomerase and chaperoning activities, and has been considered a promising drug target due to its high affinity to the macrolide compound FK506. However, PfFKBP35 has not been validated as a drug target using reverse genetics, and the link between PfFKBP35-interacting drugs and their antimalarial activity remains elusive. The manuscript addresses the biological function of PfFKBP35 and the antimalarial activity of FK506.

      The authors combine conditional genome editing, proteomics and transcriptomics analysis to investigate the effects of FKBP35 depletion in P. falciparum. The work is very well performed and clearly described. The data provide conclusive evidence that FKBP35 is essential for P. falciparum blood stage growth. Conditional knockout of PfFKBP35 leads to a delayed death-like phenotype, associated with defects in ribosome maturation as detected by quantitative proteomics and stalling of protein synthesis in the parasite. The authors clearly demonstrate that FKBP35 is essential for parasite growth and that ribosome biogenesis is disrupted, but further insights into the pathway itself would be more convincing that this is a direct consequence rather than a secondary feature of parasite death.

      The knockdown of PfFKBP35 has no phenotypic consequence, showing that very low amounts of FKBP35 are sufficient for parasite survival and growth. In the absence of quantification of the protein during the course of the experiments, it remains unclear whether the delayed death-like phenotype in the knockout is due to the delayed depletion of the protein or to a delayed consequence of early protein depletion. This limitation also impacts the interpretation of the drug assays.

      The authors investigate the activity of FK506 on P. falciparum, and conclude that FK506 exerts its antimalarial effects independently of FKBP35, based on the observation that FK506 has the same activity on FKBP35 wild type and knock-out parasites, indicating that FK506 activity is independent of FKBP35 levels. Using cellular thermal shift assays, the authors confirm the interaction between FK506 and FKBP35, and further identify candidate proteins bound by the compound, albeit at lower affinity. Further work is needed to validate whether these putative targets contribute to the FKBP35-independent antimalarial activity of FK506.

    1. Reviewer #1 (Public Review):

      In this study, Jiamin Lin et al. investigated the potential positive feedback loop between ZEB2 and ACSL4, which regulates lipid metabolism and breast cancer metastasis. They reported a correlation between high expression of ZEB2 and ACSL4 and poor survival of breast cancer patients, and showed that depletion of ZEB2 or ACSL4 significantly reduced lipid droplets abundance and cell migration in vitro. The authors also claimed that ZEB2 activated ACSL4 expression by directly binding to its promoter, while ACSL4 in turn stabilized ZEB2 by blocking its ubiquitination. While the topic is interesting, there are several concerns with the study:

      1. My concern regarding the absence of appropriate thresholds or False Discovery Rate (FDR) adjustments for the RNA-seq analysis has not been addressed, leading to incorrect thresholds and erroneous identification of significant signals.

      2. In Figure 3B and C, it appears that the knockdown efficiency of ACSL4 is inadequate in these cells, which contradicts the Western blot results presented in Figure 2F.

      3. Regarding Figure 6, the discovery of consensus binding sequences (CACCT) for ZEB2 alone is insufficient evidence to support the direct binding of ZEB2 to the ACSL4 promoter.

      4. For Figure 7E, there are multiple bands present, and it appears that ZEB2-HA has been cropped, which should ideally be presented with unaltered raw data. Please provide the uncropped raw data.

      5. In Figure 7C, the author claimed to have used 293T cells for the ubiquitin assay, which are not breast cancer cells. Moreover, the efficiency of over-expression differs between ZEB2 and ACSL4 in 293T cell lines. Performing the experiment in an unrelated cell line to justify an important interaction is not acceptable.

    1. Reviewer #1 (Public Review):

      Summary:

      Tiemann et al. have undertaken an original study on the availability of molecular dynamics (MD) simulation datasets across the Internet. There is a widespread belief that extensive, well-curated MD datasets would enable the development of novel classes of AI models for structural biology. However, currently, there is no standard for sharing MD datasets. As generating MD datasets is energy-intensive, it is also important to facilitate the reuse of MD datasets to minimize energy consumption. Developing a universally accepted standard for depositing and curating MD datasets is a huge undertaking. The study by Tiemann et al. will be very valuable in informing policy developments toward this goal.

      Strengths:

      The study presents an original approach to addressing a growing concern in the field. It is clear that adopting a more collaborative approach could significantly enhance the impact of MD simulations in modern molecular sciences.

      The timing of the work is appropriate, given the current interest in developing AI models for describing biomolecular dynamics.

      Weaknesses:

      The study primarily focuses on one major MD engine (GROMACS), although this limitation is not significant considering the proof-of-concept nature of the study.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Khaitova and co-workers present here an analysis of centromere composition and function during elevated temperatures in the plant Arabidopsis. The work relates to the ongoing climate change during which spikes in high temperatures will be found. Hence, the paper addresses a timely subject.

      The authors start by confirming earlier studies that high temperatures reduce the fertility of Arabidopsis plants. Interestingly, a hypomorphic mutant of the centromeric histone variant CENH3 (CENP-A), which was previously described by the authors, sensitizes plants to heat and results in a drop in viable pollen and silique length. The drop in fertility coincides with the formation of micronuclei in meiosis and an extension of meiotic progression as revealed by live cell imaging. Based on this finding, the authors then show that at high temperatures, the fluorescence intensity of a YFP:CENH3 declines in meiosis but remarkably not in the surrounding cells (tapetum cells). In addition, the amount of BMF1 (a Bub1 homolog and part of the spindle assembly checkpoint) also appears to decline on the kinetochores of meiocytes as judged by BMF1 reporter line. However, whether this is dependent on a decline of CENH3 or represents a separate pathway is not clear. Finally, the authors measure the duration of the spindle checkpoint and find that it is extended under high temperatures from which they conclude that the attachment of spindle fibers to kinetochores is compromised under heat.

      Strengths:<br /> This is an interesting and important paper as it links centromere organization/function to heat stress in plants. A major conclusion of the authors is that weakened centromeres, presumably by heat, may be less effective in establishing productive interactions with spindle microtubules.

      Weaknesses:<br /> The paper does not explain the molecular reason why CENH3 levels in meiocyctes are reduced or why the attachment of spindle fibers to kinetochore is less efficient at high versus low temperatures.

    1. Reviewer #1 (Public Review):

      Su et al propose the existence of two mechanisms repressing SBF activity during entry into meiosis in budding yeast. First, a decrease in Swi4 protein levels by a LUTI-dependent mechanism where Ime1 would act closing a negative feedback loop. Second, the sustained presence of Whi5 would contribute to maintaining SBF inhibited under sporulation conditions. The article is clearly written and the experimental approaches used are adequate to the aims of this work. The results obtained are in line with the conclusions reached by the authors but, in my view, they could also be explained by the existing literature and, hence, would not represent a major advance in the field of meiosis regulation.

      Regarding the first mechanism, Fig 1 shows that Swi4 decreases very little after 1-2h in sporulation medium, whereas G1-cyclin expression is strongly repressed very rapidly under these conditions (panel D and work by others). This fact dampens the functional relevance of Swi4 downregulation as a causal agent of G1 cyclin repression. The authors use overexpression of Swi4 in Figs 2 and 3 to test the relevance of Swi4 downregulation but the pATG8-SWI4 construct produces levels much higher (4-5 fold) than the wild-type gene at time 0, which may likely introduce artifactual effects in the resulting observations. In addition, the LUTI-deficient SWI4 mutant does not cause any noticeable relief in CLN2 repression, arguing against the relevance of this mechanism in the repression of G1-cyclin transcription during entry into meiosis.

      The authors propose a second mechanism where Whi5 would maintain SBF inactive under sporulation conditions. The role of Whi5 as a negative regulator of the SBF regulon is well known. On the other hand, the double WHI5-AA SWI4-dLUTI mutant does not upregulate CLN2, the G1 cyclin with the strongest negative effect on sporulation, raising serious doubts on the functional relevance of this backup mechanism during entry into meiosis.

    1. Reviewer #1 (Public Review):

      Summary: The authors use the innovative CRISPRi method to uncover regulators of cell density and volume in neutrophils. The results show that cells require NHE activity during chemoattractant-driven cell migration. Before migration occurs, cells also undergo a rapid cell volume increase. These results indicate that water flux, driven by ion channels, appears to play a central role in neutrophil migration. The paper is very well written and clear. I suggest adding some discussion about the role of actin in the process, but this is not essential.

      Strengths: The novel use of CRIPSPi to uncover cell density regulators is very novel. Some of the uncovered molecules were known before, e.g. discussed in Li & Sun, Frontiers in Cell and Developmental Biology, 2021. Others are more interesting, for example PI3K-gamma. The use of caged fMLP is also nice.

      Weaknesses: One area of investigation that seems to be absent is mentioned in the introduction. I.e., actin is expected to play a role in regulating cell volume increase. Did the authors perform any experiments with LatA? What was seen there? Do cells still migrate with LatA, or is a different interplay seen? The role of PI3K is interesting, and maybe somewhat related to actin. But this may be a different line of inquiry for the future.

    1. Reviewer #1 (Public Review):

      In this work, Vezina et al. present Bactabolize, a rapid reconstruction tool for the generation of strain-specific metabolic models. Similar to other reconstruction pipelines such as CarveMe, Bactabolize builds a strain-specific draft reconstruction and subsequently gap-fills it. The model can afterwards be used to predict growth on carbon sources. The authors constructed a pan-model of the Klebsiella pneumoniae species complex (KpSC) and used it as input for Bactabolize to construct a genome-sale reconstruction of K. pneumoniae KPPR1. They compared the generated reconstruction with a reconstruction built through CarveMe as well as a manually curated reconstruction for the same strain. They then compared predictions of carbon, nitrogen, phosphor, and sulfur sources and found that the Bactabolize reconstruction had the overall highest accuracy. Finally, they built draft reconstructions for 10 clinical isolates of K. pneumoniae and evaluated their predictive performance. Overall, this is a useful tool, the data is well-presented, and the paper is well-written.

    1. Reviewer #1 (Public Review):

      Summary:

      Chartampila et al. describe the effect of early-life choline supplementation on cognitive functions and epileptic activity in a mouse model of Alzheimer's disease. The cognitive abilities are assessed by the novel object recognition test and the novel object location test, performed in the same cohort of mice at 3 months and 6 months of age. Neuronal loss was tested using NeuN immunoreactivity, and neuronal hyperexcitability was examined using FosB and video-EEG recordings.

      Strengths:

      The study was designed as a 6-month follow-up, with repeated behavioral and EEG measurements through disease development, providing valuable and interesting findings on AD progression and the effect of early-life choline supplantation. Moreover, the behavioral data that suggest an adverse effect of low choline in WT mice are interesting and important beyond the context of AD.

      Weaknesses:

      1. The multiple headings and subheadings, focusing on the experimental method rather than the narrative, reduce the readability.<br /> 2. Quantification of NeuN and FosB in WT littermates is needed to demonstrate rescue of neuronal death and hyperexcitability by high choline supplementation and also to gain further insights into the adverse effect of low choline on the performance of WT mice in the behavioral test.<br /> 3. Quantification of the discrimination ratio of the novel object and novel location tests can facilitate the comparison between the different genotypes and diets.<br /> 4. The longitudinal analyses enable the performance of multi-level correlations between the discrimination ratio in NOR and NOL, NeuN and Fos levels, multiple EEG parameters, and premature death. Such analysis can potentially identify biomarkers associated with AD progression. These can be interesting in different choline supplementation, but also in the standard choline diet.

    1. Reviewer #1 (Public Review):

      In this study, authors have investigated the effects of TMEM127 depletion on RET regulation and function that could potentially contribute to PCC pathogenesis. They have demonstrated that the loss of TMEM127 leads to cell surface accumulation and constitutive activation of RET due to membrane organization, leading to reduced efficiency of endocytosis, decreased internalization of RET, and a global impairment of membrane trafficking. TMEM127 depletion has contributed to increased RET half-life, constitutive RET-mediated signaling, increased membrane protein diffusibility, impaired normal membrane transitions, and inappropriate accumulation of actively signaling RET molecules at the cell membrane. Collectively, these findings have shown that the mis-localized RET is the pathogenic mechanism in TMEM127-mutant pheochromocytoma.

      Experimental design and mechanistic studies are thorough and sound. The methodological weakness lies in the lack of pheochromocytoma cell line utility to reproduce novel findings observed in generated cell lines. This may represent a significant challenge that could undermine the inferred value of these potentially paradigm-changing findings. 3-dimensional patient-derived pheochromocytoma organoid in vitro model and/or patient-derived organoid xenograft in vivo model may aid in reconciling these exciting new findings and factoring in that the pheochromocytoma is a hormonally active tumor.

      Fundamentally, the authors have successfully achieved all proposed aims supported by their conclusions.

      These findings carry potentially significant clinical impact and may offer new therapeutic venues in patients with pheochromocytoma.

    1. Reviewer #1 (Public Review):

      In this study, the authors aim to understand why decision formation during behavioural tasks is distributed across multiple brain areas. They hypothesize that multiple areas are used in order to implement an information bottleneck (IB). Using neural activity recorded from monkey DLPFC and PMd performing a 2-AFC task, they show that DLPFC represents various task variables (decision, color, target configuration), while downstream PMd primarily represents decision information. Since decision information is the only information needed to make a decision, the authors point out that PMd has a minimal sufficient representation (as expected from an IB). They then train 3-area RNNs on the same task and show that activity in the first and third areas resemble the neural representations of DLPFC and PMd, respectively. In order to propose a mechanism, they analyse the RNN and find that area 3 ends up with primarily decision information because feedforward connections between areas primarily propagate decision information.

      The paper addresses a deep, normative question, namely why task information is distributed across several areas.

      Overall, it reads well and the analysis is well done and mostly correct (see below for some comments). My major problem with the paper is that I do not see that it actually provides an answer to the question posed (why is information distributed across areas?). I find that the core problem is that the information bottleneck method, which is evoked throughout the paper, is simply a generic compression method. Being a generic compressor, the IB does not make any statements about how a particular compression should be distributed across brain areas - see major points (1) and (2).

      If I ignore the reference to the information bottleneck and the question of why pieces of information are distributed, I still see a more mechanistic study that proposes a neural mechanism of how decisions are formed, in the tradition of RNN-modelling of neural activity as in Mante et al 2013. Seen through this more limited sense, the present study succeeds at pointing out a good model-data match. I point out some suggestions for improvement below.

      Major points<br /> (1) It seems to me that the author's use of the IB is based on the reasoning that deep neural networks form decisions by passing task information through a series of transformations/layers/areas and that these deep nets have been shown to implement an IB. Furthermore, these transformations are also loosely motivated by the data processing inequality.

      However, assuming as a given that deep neural networks implement an IB does not mean that an IB can only be implemented through a deep neural network. In fact, IBs could be performed with a single transformation just as well. More formally, a task associates stimuli (X) with required responses (Y), and the IB principle states that X should be mapped to a representation Z, such that I(X;Z) is minimal and I(Y,Z) is maximal. Importantly, the form of the map Z=f(X) is not constrained by the IB. In other words, the IB does not impose that there needs to be a series of transformations. I therefore do not see how the IB by itself makes any statement about the distribution of information across various brain areas.

      A related problem is that the authors really only evoke the IB to explain the representation in PMd: Fig 2 shows that PMd is almost only showing decision information, and thus one can call this a minimal sufficient representation of the decision (although ignoring substantial condition independent activity). However, there is no IB prediction about what the representation of DLPFC should look like. Consequently, there is no IB prediction about how information should be distributed across DLPFC and PMd.

      (2) Now the authors could change their argument and state that what is really needed is an IB with the additional assumption that transformations go through a feedforward network. However, even in this case, I am not sure I understand the need for distributing information in this task. In fact, in both the data and the network model, there is a nice linear readout of the decision information in dPFC (data) or area 1 (network model). Accordingly, the decision readout could occur at this stage already, and there is absolutely no need to tag on another area (PMd, area 2+3).

      Similarly, I noticed that the authors consider 2,3, and 4-area models, but they do not consider a 1-area model. It is not clear why the 1-area model is not considered. Given that e.g. Mante et al, 2013, manage to fit a 1-area model to a task of similar complexity, I would a priori assume that a 1-area RNN would do just as well in solving this task.

      I think there are two more general problems with the author's approach. First, transformations or hierarchical representations are usually evoked to get information into the right format in a pure feedforward network. An RNN can be seen as an infinitely deep feedforward network, so even a single RNN has, at least in theory, and in contrast to feedforward layers, the power to do arbitrarily complex transformations. Second, the information coming into the network here (color + target) is a classical xor-task. While this task cannot be solved by a perceptron (=single neuron), it also is not that complex either, at least compared to, e.g., the task of distinguishing cats from dogs based on an incoming image in pixel format.

      (3) I am convinced of the author's argument that the RNN reproduces key features of the neural data. However, there are some points where the analysis should be improved.

      (a) It seems that dPCA was applied without regularization. Since dPCA can overfit the data, proper regularization is important, so that one can judge, e.g., whether the components of Fig.2g,h are significant, or whether the differences between DLPFC and PMd are significant.

      (b) I would have assumed that the analyses performed on the neural data were identical to the ones performed on the RNN data. However, it looked to me like that was not the case. For instance, dPCA of the neural data is done by restretching randomly timed trials to a median trial. It seemed that this restretching was not performed on the RNN. Maybe that is just an oversight, but it should be clarified. Moreover, the decoding analyses used SVC for the neural data, but a neural-net-based approach for the RNN data. Why the differences?

      (4) The RNN seems to fit the data quite nicely, so that is interesting. At the same time, the fit seems somewhat serendipitous, or at least, I did not get a good sense of what was needed to make the RNN fit the data. The authors did go to great lengths to fit various network models and turn several knobs on the fit. However, at least to me, there are a few (obvious) knobs that were not tested.

      First, as already mentioned above, why not try to fit a single-area model? I would expect that a single area model could also learn the task - after all, that is what Mante et al did in their 2013 paper and the author's task does not seem any more complex than the task by Mante and colleagues.

      Second, I noticed that the networks fitted are always feedforward-dominated. What happens when feedforward and feedback connections are on an equal footing? Do we still find that only the decision information propagates to the next area? Quite generally, when it comes to attenuating information that is fed into the network (e.g. color), then that is much easier done through feedforward connections (where it can be done in a single pass, through proper alignment or misalignment of the feedforward synapses) than through recurrent connections (where you need to actively cancel the incoming information). So it seems to me that the reason the attenuation occurs in the inter-area connections could simply be because the odds are a priori stacked against recurrent connections. In the real brain, of course, there is no clear evidence that feedforward connections dominate over feedback connections anatomically.

      More generally, it would be useful to clarify what exactly is sufficient:

      (a) the information distribution occurs in any RNN, i.e., also in one-area RNNs<br /> (b) the information distribution occurs when there are several, sparsely connected areas<br /> (c) the information distribution occurs when there are feedforward-dominated connections between areas

    1. Reviewer #1 (Public Review):

      Funabiki et al, performed a co-evolutionary analysis of Lsh/HELLS and CDCA7, two factors with links to DNA methylation pathways in mammals, amphibia and fish. The authors suggest that conserved roles for the two factors in DNA methylation maintenance pathways can be traced back to the last eukaryotic common ancestor. Overall, the findings are important and the results could be useful for researchers studying DNA methylation pathways in many different organisms.

    1. Reviewer #1 (Public Review):

      This paper focuses on the effects of a L114P mutation in the TALK-1 channel on islet function and diabetes. This mutation is clinically relevant and a cause of MODY diabetes. This work employs a mouse model with heterozygous and homozygous mutants. The homozygous mice are homozygous lethal from severe hyperglycemia. The work shows that the mutation increases K+ currents and inhibits insulin secretion. This is a very nice paper with mechanistic insight and clear clinical importance. It is generally well-written and the data is well-presented.

    1. Reviewer #1 (Public Review):

      In this manuscript, the authors are developing a new protocol that aims at expanding pancreatic progenitors derived from human pluripotent stem cells under GMP-compliant conditions. The strategy is based on hypothesis-driven experiments that come from knowledge derived from pancreatic developmental biology.

      The topic is of major interest in the view of the importance of amplifying human pancreatic progenitors (both for fundamental purposes and for future clinical applications). There is indeed currently a major lack of information on efficient conditions to reach this objective, despite major recurrent efforts by the scientific community.

      Using their approach that combines stimulation of specific mitogenic pathways and inhibition of retinoic acid and specific branches of the TGF-beta and Wnt pathways, the authors claim to be able, in a highly robust and reproducible manner) to amplify in 10 passages the number of pancreatic progenitors (PP) by 2,000 folds, which is really an impressive breakthrough.

      The work is globally well-performed and quite convincing. I have however some technical comments mainly related to the quantification of pancreatic progenitor amplification and to their differentiation into beta-like cells following amplification.

    1. Reviewer #1 (Public Review):

      This study provides insights into the early detection of malignancies with noninvasive methods. The study contained a large sample size with an external validation cohort, which raises the credibility and universality of this model. The new model achieved high levels of AUC in discriminating malignancies from healthy controls, as well as the ability to distinguish tumor of origin. Based on these findings, prospective studies are needed to further confirm its predictive capacity.

    1. Reviewer #1 (Public Review):

      Summary: The ciliary photoreceptor cells and its downstream neurons of larval annelid must be orchestrated in a specific pattern to promote downward swimming in response to long duration of UV exposure. The authors first conducted neuroanatomical examination of the circuit to identify NOS-expression neurons (INNOS) that are immediately downstream to the ciliary photoreceptor cells. The INNOS is activated by UV and produces NO. The NOS is required for UV avoidance by Platynereis larvae and neural dynamics of the photoreceptor cells and their downstream circuit. Following up the RNA-seq data with in situ hybridization experiments, the authors found that two unconventional guanylate cyclases, NIT-GC1 and NIT-GC2, are expressed and localized in different subcellular domain of the photoreceptor cells. Experiments using the culture cells and genetically encoded sensors demonstrated that NIT-GC1 can generate cGMP in response to nitric oxide. Finally, authors build a mathematical model that fit the live imaging data and used it to predict how the magnitude of the photoreceptor activation varied by intensity and duration of UV light.

      Strengths: The authors conducted comprehensive interrogations of the UV avoidance pathway at the molecular and circuit levels, and constructed a mathematical model. The main conclusions are supported by layers of evidence from different assays.

      Weaknesses: Statistics are missing in both figure legends and methods. The perturbations of genes and molecules were not cell-type-specific and therefore the observed behavioral defect could be attributed to the malfunction of the circuit elsewhere not examined in this study. I suggest adding more explanation about the functions of other NOS-expressing cells and conducting a control experiment to test behavioral response to a non-visual stimulus.

    1. Reviewer #1 (Public Review):

      Summary<br /> In this manuscript, the authors generate an AAV-deliverable tool that generates action potentials in response to red light, but not blue light, when expressed in neurons. To do this, they screen some red light-excitatory/blue light-inhibitory opsin pairs to find ones that are spectrally and temporally matched. They first show that this works with Chrimson and GtACR2, however, they expand their search after finding that the tau-off (inactivation after light cessation) kinetics of these two opsins are not well-matched. They directly examine a small set of options based on a literature search and settle on a variant of red light-excitatory Chrimson and blue light-inhibitory ZipACR. To more closely match the kinetics of this pair, the authors create a structure homology model of the ZipACR retinal binding pocket and use this to guide the generation of a small mutagenesis panel, leading to a more optimized ZipACR mutant. They then show that a bicistronically expressed fusion arrangement of these opsins, plus some functional peptides, can drive action potentials up to 20hz with red light and does not do so with blue light, in hippocampal cells transduced by AAV. They also show function in vivo, in a mouse, using a physiological readout. They conclude that their new tool may be useful for complex experimental designs requiring multiple optical channels for write-in/read-out.

      The major advantage claimed by the authors over existing tools is the temporal time-locking of their inhibitory opsin - this is driven by the contrast between the tau-off kinetics of their ZipACR variant compared to gtACR2, which is used by the leading competitor tool (BiPOLES).

      Big thoughts<br /> While the authors were carefully thoughtful about the potential influence of temporal kinetics on the efficiency of a tool such as this one, there were no experiments conducted that made use of the unique properties of this molecular strategy. To understand why they embarked on this engineering program, I was required to put on my neuroscientist hat and contemplate this question myself:

      First, experimental designs where I require multiple optical channels of control. This appears to be aligned with the author's thoughts, as they state, correctly, that opsins utilizing retinal as a light-sensing chromophore are universally activated by blue light (the so-called 'blue shoulder'). Therefore, their tool may be useful for stimulating multiple populations using a blue excitatory opsin in neuron A and their tool for red excitation of neuron B - or, in the author's own words, "A potential solution to the problem of cross-talk...". Yet, there are no data presented that showcases their new tool for this purpose (e.g. Vierock, Johannes, et al. "BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons." Nature Communications 12.1 (2021): 4527. Figure 4f-I; 6). The same set-up could be imagined for green GECI (or equivalent) imaging of cells in the same volume that their tool is being used in - for instance, interleaving red stimulation light and blue imaging light, (perhaps) without the typical concern of imaging light bleed-through activating the opsin itself.

      Second, for high-frequency temporal control over both excitation and inhibition in the same neuron. The red light turns the cell on, and blue light turns the cell off (see, for instance, Zhang, Feng, et al. "Multimodal fast optical interrogation of neural circuitry." Nature 446.7136 (2007): 633-639. Figure 2; Vierock as above, Figure 4a,b). Again, here the authors are long on theory ("The new system...can drive time-locked high-frequency action potentials in response to red pulses") and short on data. While they do show that red light = excitation and blue light = inhibition, they neither show 1) all-optical on/off modulation of the same cell; nor 2) high-frequency inhibition or excitation (max stim rate of 20hz, which is the same as the BiPOLES paper used for their LC stimulation paradigm; Vierock, as above, Figure 7a-d).

      Despite these major shortcomings, the further development and characterization of tandem opsins, such as this one, is of interest to the community. There is ongoing work by the BiPOLES team to create new iterations (e.g. Wahid, J., et al. "P-15 BiPOLES2 is a bidirectional optogenetic tool with a narrow activation spectrum and low red-light excitability." Clinical Neurophysiology 148 (2023): e16.). To make the case that the tool described in this manuscript is worth the effort that the authors are requesting the neuroscience community invest in trialing it in their own hands, they must revise the manuscript to show that their approach is both 1) different in some way when compared to BiPOLES (it is my understanding that they did not do this, as per the supplementary alignment of the BiPOLES sequence and the sequence of the BiPOLES-like construct that they did test) and 2) that the properties that the investigators specifically tailored their construct to have confer some sort of experimental advantage when compared to the existing standard.

      There are a number of additional concerns and clarifications that will strengthen the manuscript that are communicated directly to the authors through this peer-review process.