Reviewer #1 (Public Review):

Summary:

In this work, the authors present a novel, multi-layer computational model of motor control to produce realistic walking behaviour of a Drosophila model in the presence of external perturbations and under sensory and motor delays. The novelty of their model of motor control is that it is modular, with divisions inspired by the fly nervous system, with one component based on deep learning while the rest are based on control theory. They show that their model can produce realistic walking trajectories. Given the mostly reasonable assumptions of their model, they convincingly show that the sensory and motor delays present in the fly nervous system are the maximum allowable for robustness to unexpected perturbations.

Their fly model outputs torque at each joint in the leg, and their dynamics model translates these into movements, resulting in time-series trajectories of joint angles. Inspired by the anatomy of the fly nervous system, their fly model is a modular architecture that separates motor control at three levels of abstraction:<br /> (1) oscillator-based model of coupling of phase angles between legs,<br /> (2) generation of future joint-angle trajectories based on the current state and inputs for each leg (the trajectory generator), and<br /> (3) closed-loop control of the joint-angles using torques applied at every joint in the model (control and dynamics).

These three levels of abstraction ensure coordination between the legs, future predictions of desired joint angles, and corrections to deviations from desired joint-angle trajectories. The parameters of the model are tuned in the absence of external perturbations using experimental data of joint angles of a tethered fly. A notable disconnect from reality is that the dynamics model used does not model the movement of the body and ground contacts as is the case in natural walking, nor the movement of a ball for a tethered fly, but instead something like legs moving in the air for a tethered fly.

In order to validate the realism of the generated simulated walking trajectories, the authors compare various attributes of simulated to real tethered fly trajectories and show qualitative and quantitative similarities, including using a novel metric coined as Kinematic Similarity (KS). The KS score of a trajectory is a measure of the likelihood that the trajectory belongs to the distribution of real trajectories estimated from the experimental data. While such a metric is a useful tool to validate the quality of simulated data, there is some room for improvement in the actual computation of this score. For instance, the KS score is computed for any given time-window of walking simulation using a fraction of information from the joint-angle trajectories. It is unclear if the remaining information in joint-angle trajectories that are not used in the computation of the KS score can be ignored in the context of validating the realism of simulated walking trajectories.

The authors validate simulated walking trajectories generated by the trained model under a range of sensorimotor delays and external perturbations. The trained model is shown to generate realistic joint-angle trajectories in the presence of external perturbations as long as the sensorimotor delays are constrained within a certain range. This range of sensorimotor delays is shown to be comparable to experimental measurements of sensorimotor delays, leading to the conclusion that the fly nervous system is just fast enough to be robust to perturbations.

Strengths:

This work presents a novel framework to simulate Drosophila walking in the presence of external perturbations and sensorimotor delay. Although the model makes some simplifying assumptions, it has sufficient complexity to generate new, testable hypotheses regarding motor control in Drosophila. The authors provide evidence for realistic simulated walking trajectories by comparing simulated trajectories generated by their trained model with experimental data using a novel metric proposed by the authors. The model proposes a crucial role in future predictions to ensure robust walking trajectories against external perturbations and motor delay. Realistic simulations under a range of prediction intervals, perturbations, and motor delays generating realistic walking trajectories support this claim. The modular architecture of the framework provides opportunities to make testable predictions regarding motor control in Drosophila. The work can be of interest to the Drosophila community interested in digitally simulating realistic models of Drosophila locomotion behaviors, as well as to experimentalists in generating testable hypotheses for novel discoveries regarding neural control of locomotion in Drosophila. Moreover, the work can be of broad interest to neuroethologists, serving as a benchmark in modelling animal locomotion in general.

Weaknesses:

As the authors acknowledge in their work, the control and dynamics model makes some simplifying assumptions about Drosophila physics/physiology in the context of walking. For instance, the model does not incorporate ground contact forces and inertial effects of the fly's body. It is not clear how these simplifying assumptions would affect some of the quantitative results derived by the authors. The range of tolerable values of sensorimotor delays that generate realistic walking trajectories is shown to be comparable with sensorimotor delays inferred from physiological measurements. It is unclear if this comparison is meaningful in the context of the model's simplifying assumptions. The authors propose a novel metric coined as Kinematic Similarity (KS) to distinguish realistic walking trajectories from unrealistic walking trajectories. Defining such an objective metric to evaluate the model's predictions is a useful exercise, and could potentially be applied to benchmark other computational animal models that are proposed in the future. However, the KS score proposed in this work is calculated using only the first two PCA modes that cumulatively account for less than 50% of the variance in the joint angles. It is not obvious that the information in the remaining PCA modes may not change the log-likelihood that occurs in the real walking data.

Reviewer #1 (Public Review):

Summary:

This manuscript described a clinical trial to understand the different treatment durations of loperamide in preventing pyrotinib-induced diarrhea. The authors concluded that no significant differences were observed between 21-day and 42-day loperamide durations in preventing grade {greater than or equal to} grade 3 diarrhea. The authors suggested that considering the economic cost and patient compliance, 21-day loperamide prophylaxis might represent a more pragmatic and appropriate approach for clinical application.

Strengths:

It is essential to understand if loperamide for primary prevention of diarrhea helps or not for postoperative treatment with nab-paclitaxel and pyrotinib in HER2-positive patients. This clinical trial would answer this question eventually.

Weaknesses:

(1) There are no patients who have not received prophylactic treatment for diarrhea to serve as a control group. This limited the finding that if the loperamide for primary prevention of diarrhea benefits or not for postoperative treatment with nab-paclitaxel and pyrotinib in HER2-positive patients. This would not help much for the guidance of clinical use of the loperamide for primary prevention of diarrhea.

(2) The clinical trial needs double-blinding for evaluation of treatment. In this manuscript, the blinding was not employed.

Reviewer #1 (Public Review):

Summary:

Colomb et al have further explored the mechanisms of action of a family of three immunodulatory proteins produced by the murine gastrointestinal nematode parasite Heligmosomoides polygyrus bakeri. The family of HpARI proteins binds to the alarmin interleukin 33 and depending on family members, exhibits differential activities, either suppressive or enhancing. The present work extends previous studies by this group showing the binding of DNA by members of this family through a complement control protein (CCP1) domain. Moreover, they identify two members of the family that bind via this domain in a non-specific manner to the extracellular matrix molecule heparan sulphate through a basic charged patch in CCP1. The authors thus propose that binding to DNA or heparan sulphate extends the suppressive action of these two parasite molecules, whereas the third family member does not bind and consequently has a shorter half-life and may function via diffusion.

Strengths:

A strength of the work is the multifaceted approach to examining and testing their hypotheses, using a well-established and well-defined family of immunomodulatory molecules using multiple approaches including an in vivo setting.

Weaknesses:

There are a few weaknesses of the approach. Perhaps some discussion and speculation as to how these three family members might operate in concert during Heligmosomoides polygyrus bakeri infection would help place the biology of these molecules in context for the reader, e.g. when and where they are produced.

Reviewer #1 (Public Review):

Summary:

Yang. Hu et al. investigated the molecular mechanism that cause astrocyte activation and its implications for multiple sclerosis. This study focuses on the enzyme PKM2, known for its role in glycolysis, and its nuclear translocation in reactive astrocytes in a mouse model of multiple sclerosis (EAE). Preventing the nuclear translocation of PKM2 reduces astrocyte activation, proliferation, glycolysis, and inflammatory cytokine secretion. Importantly, the study reveals that TRIM21 controls PKM2's nuclear translocation through ubiquitination, promoting its nuclear import and enhancing its activity. Single-cell RNA sequencing and immunofluorescence confirm TRIM21 upregulation in EAE astrocytes, and alteration of TRIM21 levels affect PKM2-dependent glycolysis and proliferation. Their findings suggest that targeting the TRIM21-PKM2 axis could be a therapeutic strategy for treating neurological diseases involving astrocyte activation.

Strength:

This work provides a comprehensive exploration of PKM2's nuclear role and its interaction with TRIM21 in EAE, offering new insights for therapeutic strategies targeting metabolic reprogramming in astrocyte activation. The strength of the study is the use of advanced techniques such as single cell RNA sequencing, in vitro and in vivo knockdown techniques to support the data. With the addition of new data and explanations in the manuscript, the authors have rendered their claimed ideas more supportive.

Weakness:

The revisions and implementation of suggestions have greatly improved the overall quality of the manuscript. I would like to thank the authors for carefully evaluating all the suggestions and for providing extra explanations and response figures. However, there are still some points that need to be corrected and clarified.

Reviewer #1 (Public Review):

Summary:

Federer et al. tested AAVs designed to target GABAergic cells and parvalbumin-expressing cells in marmoset V1. Several new results were obtained. First, AAV-h56D targeted GABAergic cells with high specificity but in ways that varied across serotype and layer. Second, AAV-PHP.eB.S5E2 targeted parvalbumin-expressing neurons with similarly high specificity. Third, immunohistochemical GABA and PV signals were attenuated near viral injection sites.

A strength of this study is the analysis of marker gene expression at AAV injection sites. Some endogenous genes are difficult to detect following AAV injections, which is an important observation. A second contribution is the demonstration that AAV-S5E2 drives transgene expression selectively in parvalbumin-expressing neurons when vectors are delivered intraparenchymally (the study introducing AAV-S5E2 used intravenous injections).

A weakness of this study is that the data set is small. Which of the results would hold up had a larger number of injections been made into a larger number of marmosets remains unclear.

A major goal of this study was to quantify the specificity and coverage of AAV-h56D and AAV-S5E2 vectors in marmoset cortex. This goal was achieved. This report provides a valuable guide for other investigators using these tools. It also provides a rigorous survey of the laminar distributions of GABA+ and PV+ neurons in marmoset V1 which has value independent of the viral injections.

Reviewer #1 (Public Review):

Summary:

The authors of this manuscript characterize new anion conducting that is more red-shifted in its spectrum than prior variants called MsACR1. An additional mutant variant of MsACR1 that is renamed raACR has a 20 nm red-shifted spectral response with faster kinetics. Due to the spectral shift of these variants, the authors proposed that it is possible to inhibit expression of MsACR1 and raACR with lights at 635 nm in vivo and in vitro. The authors were able to demonstrate inhibition in vitro and in vivo with 635 nm light. Overall the new variants with unique properties should be able to suppress neuronal activities with red-shifted light stimulation.

Strengths:

The authors were able to identify a new class of anion conducting channelrhodopsin and have variants that respond strongly to lights with wavelength >550 nm. The authors were able to demonstrate this variant, MsACR1, can alter behavior in vivo with 635 nm light.

The second major strength of the study is the development of a red-shifted mutant of MsACR1 that has faster kinetics and 20 nm red-shifted from a single mutation.

Weaknesses:

There are many claims not supported by the evidence provided in the submitted version of the manuscript and would require further experiments to support such claims.

(1) From the data shown, the red-shifted raACR work much less efficiently than MsACR1 even with 635 nm light illumination both in vivo (Figure 4D) and in vitro (Figure 3E) despite the 20 nm red-shift. This is inconsistent with the benefits and effects of red-shifting the spectrum in raACR. The authors claimed that this is due to the faster kinetics of raACR which is plausible from the data shown in Fig 3E but this could be experimentally shown if more examples of continuous illumination and pulsed illumination (such as the one shown in Fig 3D) can be shown in supplemental figures. If this is truly due to the off-kinetics, the spikes would appear after the termination of the pulses but there is little difference in the cases of continuous illumination or during illumination. The fact that 635nm is equally effective as raACR suggests that there is an overall stronger effect of MsACR1 that compensates for the red-shift of raACR.

(2) There are limited comparisons to existing variants of ACRs under the same conditions in the manuscript overall. There should be more parallel comparison with gtACR1, ZipACR and RubyACR in identical conditions in cultured cell line, cultured neurons and in vivo. In terms of overall performance, efficiency, expression in identical conditions. Without this information, it is unclear whether the effects at 635 nm is due to the expression level which can compensate for the spectral shift (which may be the case for MsACR1). The authors stated they are saving this data for another manuscript, this is important data for the current manuscript which should be presented in the existing manuscript.

(3) Despite being able to activate the channelrhodopsin with 635 nm light, the main utility of the variant would be transcranial stimulation which were not demonstrated here.

(4) For the in vivo characterization, there is no mention of animal number and results from Fig 4 and 5 appear to come from multiple samples from a single animal. This is not sufficient scientific evidence to support the claims. Fig 4 and 5 should have statistical analysis from multiple animals and not multiple measurements from single animals in each of the conditions.

(5) As reviewer 2 also pointed out, there is a lack of proper controls (in addition to the low number of animals). The authors point out the current absence of technicians in the laboratory, this should not be a reason to not attempt or do the experiments.

Reviewer #1 (Public Review):

Summary:<br /> This study investigated spatial representations in deep feedforward neural network models (DDNs) that were often used in visual tasks. The authors create a three-dimensional virtual environment, and let a simulated agent randomly forage in a smaller two-dimensional square area. The agent "sees" images of the room within its field of view from different locations and heading directions. These images were processed by DDNs. Analyzing model neurons in DDNs, they found response properties similar to those of place cells, border cells and head direction cells in various layers of deep nets. A linear readout of network activity can recover key spatial variables. In addition, after removing neurons with strong place/border/head direction selectivity, one can still decode these spatial variables from the remaining neurons in the DNNs. Based on these results, the authors argue that that the notion of functional cell types in spatial cognition is misleading.

Strengths:<br /> This paper contains interesting and original ideas, and I enjoy reading it. Most previous studies (e.g., Banino, Nature, 2018; Cueva & Wei, ICLR, 2018; Whittington et al, Cell, 2020) using deep network models to investigate spatial cognition mainly relied on velocity/head rotation inputs, rather than vision (but see Franzius, Sprekeler, Wiskott, PLoS Computational Biology, 2007). Here, the authors find that, under certain settings, visual inputs alone may contain enough information about the agent's location, head direction and distance to the boundary, and such information can be extracted by DNNs. If confirmed, this is potentially an interesting and important observation.

Weaknesses:<br /> While the findings reported here are interesting, it is unclear whether they are the consequence of the specific model setting, and how well they would generalize. Furthermore, I feel the results are over-interpreted. There are major gaps between the results actually shown and the claim about the "superfluousness of cell types in spatial cognition". Evidence directly supporting the overall conclusion seems to be weak at the moment.

Major concerns:

(1) The authors reported that, in their model setting, most neurons throughout the different layers of CNNs show strong spatial selectivity. This is interesting and perhaps also surprising. It would be useful to test/assess this prediction directly based on existing experimental results. It is possible that the particular 2-d virtual environment used is special. The results will be strengthened if similar results hold for other testing environments.

In particular, examining the pictures shown in Fig. 1A, it seems that local walls of the 'box' contain strong oriented features that are distinct across different views. Perhaps the response of oriented visual filters can leverage these features to uniquely determine the spatial variable. This is concerning because this is a very specific setting that is unlikely to generalize.

(2) Previous experimental results suggest that various function cell types discovered in rodent navigation circuits persist in dark environments. If we take the modeling framework presented in this paper literally, the prediction would be that place cells/head direction cells should go away in darkness. This implies that key aspects of functional cell types in the spatial cognition are missing in the current modeling framework. This limitation needs to be addressed or explicitly discussed.

(3) Place cells/border cell/ head direction cells are mostly studied in the rodent's brain. For rodents, it is not clear whether standard DNNs would be good models of their visual systems. It is likely that rodent visual system would not be as powerful in processing visual inputs as the DNNs used in this study.

(4) The overall claim that those functional cell types defined in spatial cognition are superfluousness seems to be too strong based on the results reported here. The paper only studied a particular class of models, and arguably, the properties of these models have a major gap to those of real brains. Even though, in the DNN models simulated in this particular virtual environment, (i) most model neurons have strong spatial selectivity; (ii) removing model neurons with the strongest spatial selectivity still retain substantial spatial information, why this is relevant to the brain? The neural circuits may operate in a very different regime. Perhaps a more reasonable interpretation of the results would be: these results raise the possibility that those strongly selective neurons observed in the brain may not be essential for encoding certain features, as something like this is observed in certain models. It is difficult to draw definitive conclusions about the brain based on the results reported.

Reviewer #1 (Public Review):<br /> Summary:<br /> This interesting and well written article by Tuckowski et al. summarizes work connecting the flavin-containing monooxygenase FMO-4 with increased lifespan through a mechanism involving calcium signaling in the nematode Caenorhabditis elegans.

The authors have previously studied another fmo in worms, FMO-2, prompting them to look at additional members of this family of proteins. They show that fmo-4 is up in dietary restricted worms and necessary for the increased lifespan of these animals as well as of rsks-1 (s6 kinase) knockdown animals. They then show that overexpression of fmo-4 is sufficient to significantly increase lifespan, as well as healthspan and paraquat resistance. Further, they demonstrate that overexpression of fmo-4 solely in the hypodermis of the animal recapitulates the entire effect of fmo-4 OE.

In terms of interactions between fmo-2 and fmo-4 they show that fmo-4 is necessary for the previously reported effects of fmo-2 on lifespan, while the effects of fmo-4 do not depend on fmo-2.

Next the authors use RNASeq to compare fmo-4 OE animals to wild type. Their analyses suggested the possibility that FMO-4 was modulating calcium signaling, and through additional experiments specifically identified the calcium signaling genes crt-1, itr-1, and mcu-1 as important fmo-4 interactors<br /> in this context. As previously published work has shown that loss of the worm transcription factor atf-6 can extend lifespan through crt-1, itr-1 and mcu-1, the authors asked about interactions between fmo-4 and atf-6. They showed that fmo-4 is necessary for both lifespan extension and increased paraquat resistance upon RNAi knockdown of atf-6.

Overall this clearly written manuscript summarizes interesting and novel findings of great interest in the biology of aging and suggests promising avenues for future work in this area.

Strengths:<br /> This paper contains a large number of careful, well executed and analysed experiments in support of its existing conclusions, and which also point toward significant future directions for this work. In addition it is clear and very well written.

Weaknesses:<br /> Within the scope of the current work there are no major weaknesses. That said, the authors themselves note pressing questions beyond the scope of this study that remain unanswered. For instance, the mechanistic nature of the interactions between FMO-4 and the other players in this story, for example in terms of direct protein-protein interactions, is not at all understood yet. Further, powerful tools such as GCaMP expressing animals will enable a much more detailed understanding of what exactly is happening to calcium levels, and where and when it is happening, in these animals.

Reviewer #1 (Public Review):

This manuscript by Kleinman & Foster investigates the dependence of hippocampal replay on VTA activity. They recorded neural activity from the dorsal CA1 region of the hippocampus while chemogenetically silencing VTA dopamine neurons as rats completed laps on a linear track with reward delivery at each end. Reward amount changed across task epochs within a session on one end of the track. The authors report that VTA activity is necessary for an increase in sharp-wave rate to remain localized to the feeder that undergoes a change in reward magnitude, an effect that was especially pronounced in a novel environment. They follow up on this result with a second experiment in which reward magnitude varies unpredictably at one end of the linear track and report that changes in sharp-wave rate at the variable location reflect both the amount of reward rats just received there, in addition to a smaller modulation that is reminiscent of reward prediction error coding, in which the previous reward rats received at the variable location affects the magnitude of the subsequent change in sharp-wave rate that occurs on the present visit.

This work is technically innovative, combining neural recordings with chemogenetic inactivation. The question of how VTA activity affects replay in the hippocampus is interesting and important given that much of the work implicating hippocampal replay in memory consolidation and planning comes from reward-motivated behavioral tasks. Enthusiasm for the manuscript is dampened by some technical considerations about the chemogenetic portion of the experiments. Additionally, there are some interpretational issues related to whether changes in reward magnitude affected sharp-wave rate directly, or whether the reported changes in sharp-wave rate alter behavior and these behavioral changes affect sharp-wave rate.

Major issues:

Chemogenetics validation

Little validation is provided for the chemogenetic manipulations. The authors report that animals were excluded due to lack of expression but do not quantify/document the extent of expression in the animals that were included in the study. There's no independent verification that VTA was actually inhibited by the chemogenetic manipulation besides the experimental effects of interest.

The authors report a range of CNO doses. What determined the dose that each rat received? Was it constant for an individual rat? If not, how was the dose determined? The authors may wish to examine whether any of their CNO effects were dependent on dose.

The authors tested the same animal multiple times per day with relatively little time between recording sessions. Can they be certain that the effect of CNO wore off between sessions? Might successive CNO injections in the same day have impacted neural activity in the VTA differently? Could the chemogenetic manipulation have grown stronger with each successive injection (or maybe weaker due to something like receptor desensitization)? The authors could test statistically whether the effects of CNO that they report do not depend on the number of CNO injections a rat received over a short period of time.

Motivational considerations

In a similar vein, running multiple sessions per day raises the possibility that rats' motivation was not constant across all data collection time points. The authors could test whether any measures of motivation (laps completed, running speed) changed across the sessions conducted within the same day. This is a particularly tricky issue, because my read of the methods is that saline sessions were only conducted as the first session of any recording day, which means there's a session order/time of day and potential motivational confound in comparing saline to CNO sessions.

Statistics, statistical power, and effect sizes

Throughout the manuscript, the authors employ a mixture of t-tests, ANOVAs, and mixed-effects models. Only the mixed effects models appropriately account for the fact that all of this data involves repeated measurements from the same subject. The t-tests are frequently doubly inappropriate because they both treat repeated measures as independent and are not corrected for multiple comparisons.

The number of animals in these studies is on the lower end for this sort of work, raising questions about whether all of these results are statistically reliable and likely to generalize. This is particularly pronounced in the reward volatility experiment, where the number of rats in the experimental group is halved to just two. The results of this experiment are potentially very exciting, but the sample size makes this feel more like pilot data than a finished product.

The effect sizes of the various manipulations appear to be relatively modest, and I wonder if the authors could help readers by contextualizing the magnitude of these results further. For instance, when VTA inactivation increases mis-localization of SWRs to the unchanged end of the track, roughly how many misplaced sharp-waves are occurring within a session, and what would their consequence be? On this particular behavioral task, it's not clear that the animals are doing worse in any way despite the mislocalization of sharp-waves. And it seems like the absolute number of extra sharp-waves that occur in some of these conditions would be quite small over the course of a session, so it would be helpful if the authors could speculate on how these differences might translate to meaningful changes in processes like consolidation, for instance.

How directly is reward affecting sharp-wave rate?

Changes in reward magnitude on the authors' task cause rats to reallocate how much time they spent at each end. Coincident with this behavioral change, the authors identify changes in the sharp-wave rate, and the assumption is that changing reward is altering the sharp-wave rate. But it also seems possible that by inducing longer pauses, increased reward magnitude is affecting the hippocampal network state and creating an occasion for more sharp-waves to occur. It's possible that any manipulation so altering rats' behavior would similarly affect the sharp-wave rate.

For instance, in the volatility experiment, on trials when no reward is given sharp-wave rate looks like it is effectively zero. But this rate is somewhat hard to interpret. If rats hardly stopped moving on trials when no reward was given, and the hippocampus remained in a strong theta network state for the full duration of the rat's visit to the feeder, the lack of sharp-waves might not reflect something about reward processing so much as the fact that the rat's hippocampus didn't have the occasion to emit a sharp-wave. A better way to compute the sharp-wave rate might be to use not the entire visit duration in the denominator, but rather the total amount of time the hippocampus spends in a non-theta state during each visit. Another approach might be to include visit duration as a covariate with reward magnitude in some of the analyses. Increasing reward magnitude seems to increase visit duration, but these probably aren't perfectly correlated, so the authors might gain some leverage by showing that on the rare long visit to a low-reward end sharp-wave rate remains reliably low. This would help exclude the explanation that sharp-wave rate follows increases in reward magnitude simply because longer pauses allow a greater opportunity for the hippocampus to settle into a non-theta state.

The authors seem to acknowledge this issue to some extent, as a few analyses have the moments just after the rat's arrival at a feeder and just before departure trimmed out of consideration. But that assumes these sorts of non-theta states are only occurring at the very beginning and very end of visits when in fact rats might be doing all sorts of other things during visits that could affect the hippocampus network state and the propensity to observe sharp-waves.

Minor issues

The title/abstract should reflect that only male animals were used in this study.

The title refers to hippocampal replay, but for much of the paper the authors are measuring sharp-wave rate and not replay directly, so I would favor a more nuanced title.

Relatedly, the interpretation of the mislocalization of sharp-waves following VTA inactivation suggests that the hippocampus is perhaps representing information inappropriately/incorrectly for consolidation, as the increased rate is observed both for a location that has undergone a change in reward and one that has not. However, the authors are measuring replay rate, not replay content. It's entirely possible that the "mislocalized" replays at the unchanged end are, in fact, replaying information about the changed end of the track. A bit more nuance in the discussion of this effect would be helpful.

The authors use decoding accuracy during movement to determine which sessions should be included for decoding of replay direction. Details on cross-validation are omitted and would be appreciated. Also, the authors assume that sessions failed to meet inclusion criteria because of ensemble size, but this information is not reported anywhere directly. More info on the ensemble size of included/excluded sessions would be helpful.

For most of the paper, the authors detect sharp-waves using ripple power in the LFP, but for the analysis of replay direction, they use a different detection procedure based on the population firing rate of recorded neurons. Was there a reason for this switch? It's somewhat difficult to compare reported sharpwave/replay rates of the analyses given that different approaches were used.

Reviewer #1 (Public Review):

Summary:

In this study, the authors re-analyzed Experiment 1 of a public dataset (Rademaker et al, 2019, Nature Neuroscience) which includes fMRI and behavioral data recorded while participants held an oriented grating in visual working memory (WM) and performed a delayed recall task at the end of an extended delay period. In that experiment, participants were pre-cued on each trial as to whether there would be a distracting visual stimulus presented during the delay period (filtered noise or randomly oriented grating). In this manuscript, the authors focused on identifying whether the neural code in the retinotopic cortex for remembered orientation was 'stable' over the delay period, such that the format of the code remained the same, or whether the code was dynamic, such that information was present, but encoded in an alternative format. They identify some time points - especially towards the beginning/end of the delay - where the multivariate activation pattern fails to generalize to other time points and interpret this as evidence for a dynamic code. Additionally, the authors compare the representational format of remembered orientation in the presence vs absence of a distracting stimulus, averaged over the delay period. This analysis suggested a 'rotation' of the representational subspace between distracting orientations and remembered orientations, which may help preserve simultaneous representations of both remembered and viewed stimuli.

Strengths:

(1) Direct comparisons of coding subspaces/manifolds between time points and task conditions is an innovative and useful approach for understanding how neural representations are transformed to support cognition.

(2) Re-use of existing datasets substantially goes beyond the authors' previous findings by comparing the geometry of representational spaces between conditions and time points, and by looking explicitly for dynamic neural representations

Weaknesses:

(1) Only Experiment 1 of Rademaker et al (2019) is reanalyzed. The previous study included another experiment (Expt 2) using different types of distractors which did result in distractor-related costs to neural and behavioral measures of working memory. The Rademaker et al (2019) study uses these two results to conclude that neural WM representations are protected from distraction when distraction does not impact behavior, but conditions that do impact behavior also impact neural WM representations. Considering this previous result is critical for relating the present manuscript's results to the previous findings, it seems necessary to address Experimentt 2's data in the present work

(2) Primary evidence for 'dynamic coding', especially in the early visual cortex, appears to be related to the transition between encoding/maintenance and maintenance/recall, but the delay period representations seem overall stable, consistent with previous findings

(3) Dynamicism index used in Figure 1f quantifies the proportion of off-diagonal cells with significant differences in decoding performance from the diagonal cell. It's unclear why the proportion of time points is the best metric, rather than something like a change in decoding accuracy. This is addressed in the subsequent analysis considering coding subspaces, but the utility of the Figure 1f analysis remains weakly justified.

(4) There is no report of how much total variance is explained by the two PCs defining the subspaces of interest in each condition, and timepoint. It could be the case that the first two principal components in one condition (e.g., sensory distractor) explain less variance than the first two principal components of another condition.

(5) Converting a continuous decoding metric (angular error) to "% decoding accuracy" serves to obfuscate the units of the actual results. Decoding precision (e.g., sd of decoding error histogram) would be more interpretable and better related to both the previous study and behavioral measures of WM performance.

(6) This report does not make use of behavioral performance data in the Rademaker et al (2019) dataset.

(7) Given there were observed differences between individual retinotopic ROIs in the temporal cross-decoding analyses shown in Figure 1, the lack of data presented for the subspace analyses for the corresponding individual ROIs is a weakness

Reviewer #1 (Public Review):

In this work Jeong and colleagues focus on exploring the role of the acyltransferase ZDHHC9 in myelinating OLs in particular in the palmitoylation of several myelin proteins. After confirming the specific enrichment of the Zdhhc9 transcript in mouse and human OLs, the authors examine the subcellular localization of the protein in vitro and observed that in comparison with other isoforms, ZDHHC9 localizes at OLs cell bodies and at discrete puncta in the processes. These observations (Figures 1 and 2) led the authors to hypothesize that ZDHHC9 plays an important role in myelination. No gross changes were detected in OL development in Zdhhc9 KO mice and analyses from P28 Zdhhc9 KO mice crossed with Mobp-EGFP reporter mice did not show changes in EGFP+ OL differentiation (Figure 3). However, and given the observed subcellular localization of ZDHHC9 in OL processes (Figure 2) and the observation that the percentage of unmyelinated axons is increased in Zdhhc9 KO (Figure 6), early time points to examine the differentiated pools of OLs and their capacity to extend processes/contact axons need to be considered.

Maturation of OL in Zdhhc9 KO was examined by crossing Zdhhc9 KO with Pdgfra-CreER; R26- EGFP and following the newly EGFP-labelled OPCs following tamoxifen administration. No changes in the numbers of EGFP+ OL were detected. The authors concluded that the loss of ZDHHC9 does not alter oligodendrogenesis in either the young or mature CNS. The authors observed defects in Zdhhc9 KO OL protrusions that they attributed to abnormal OL membrane expansion (Fig 4 and 5). Can they show evidence for this?

The authors report that Zdhhc9 KO primary and secondary branches in OL were longer, some contained spheroid-like swellings and the OL protrusion complexity was higher. However, these data is partially contradictory to what they show in OL differentiation experiments in vitro (Fig 7). There is also no evidence for increased membrane expansion in Zdhhc9 knockdown myelin forming cells in culture. How to reconcile this?

Reviewer #1 (Public Review):

Summary:

In the paper, Yan and her colleagues investigate at which stage of development different categorical signals can be detected with EEG using a steady-state visual evoked potential paradigm. The study reports the development trajectory of selective responses to five categories (i.e., faces, limbs, corridors, characters, and cars) over the first 1.5 years of life. It reveals that while responses to faces show significant early development, responses to other categories (i.e., characters and limbs) develop more gradually and emerge later in infancy. The paper is well-written and enjoyable, and the content is well-motivated and solid.

Strengths:

(1) This study contains a rich dataset with a substantial amount of effort. It covers a large sample of infants across ages (N=45) and asks an interesting question about when visual category representations emerge during the first year of life.

(2) The chosen category stimuli are appropriate and well-controlled. These categories are classic and important for situating the study within a well-established theoretical framework.

(3) The brain measurements are solid. Visual periodicity allows for the dissociation of selective responses to image categories within the same rapid image stream, which appears at different intervals. This is important for the infant field, as it provides a robust measure of ERPs with good interpretability.

Weaknesses:

The study would benefit from a more detailed explanation of analysis choices, limitations, and broader interpretations of the findings. This includes:<br /> a) improving the treatment of bias from specific categories (e.g., faces) towards others;<br /> b) justifying the specific experimental and data analysis choices;<br /> c) expanding the interpretation and discussion of the results.

I believe that giving more attention to these aspects would improve the study and contribute positively to the field.

Reviewer #1 (Public Review):

This work presents a replicable difference in predictive processing between subjects with and without tinnitus. In two independent MEG studies and using a passive listening paradigm, the authors identify an enhanced prediction score in tinnitus subjects compared to control subjects. In the second study, individuals with and without tinnitus were carefully matched for hearing levels (next to age and sex), increasing the probability that the identified differences could truly be attributed to the presence of tinnitus. Results from the first study could successfully be replicated in the second, although the effect size was notably smaller.

Throughout the manuscript, the authors provide a thoughtful interpretation of their key findings and offer several interesting directions for future studies. Their conclusions are fully supported by their findings. Moreover, the authors are sufficiently aware of the inherent limitations of cross-sectional studies.

Strengths:

The robustness of the identified differences in prediction scores between individuals with and without tinnitus is remarkable, especially as successful replication studies are rare in the tinnitus field. Moreover, the authors provide several plausible explanations for the decline of the effect size observed in the second study.

The rigorous matching for hearing loss, in addition to age and sex, in the second study is an important strength. This ensures that the identified differences cannot be attributed to differences in hearing levels between the groups.

The used methodology is explained clearly and in detail, ensuring that the used paradigms may be employed by other researchers in future studies. Moreover, the registering of the data collection and analysis methods for Study 2 as a Registered Report should be commended, as the authors have clearly adhered to the methods as registered.

Weaknesses:

Although the authors have been careful to match their experimental groups for age, sex, and hearing loss, there are other factors that may confound the current results. For example, subjects with tinnitus might present with psychological comorbidities such as anxiety and depression. The authors' exclusion of distress as a candidate for explaining the found effects is based solely on an assessment of tinnitus-related distress, while it is currently not possible to exclude the effects of elevated anxiety or depression levels on the results. Additionally, as the authors address in the discussion, the presence of hyperacusis may also play a role in predictive processing in this population.

The authors write that sound intensity was individually determined by presenting a short audio sequence to the participants and adjusting the loudness according to an individual pleasant volume. Neural measurements made during listening paradigms might be influenced by sound intensity levels. The intensity levels chosen by the participants might therefore also have an effect on the outcomes. The authors currently do not provide information on the sound intensity levels in the experimental groups, making it impossible to assess whether sound intensity levels might have played a role.

Reviewer #1 (Public Review):

Summary:

The manuscript by Ozcan et al., presents compelling evidence demonstrating the latent potential of glial precursors of the adult cerebral cortex for neuronal reprogramming. The findings substantially advance our understanding of the potential of endogenous cells in the adult brain to be reprogrammed. Moreover, they describe a molecular cocktail that directs reprogramming toward corticospinal neurons (CSN).

Strengths:

Experimentally, the work is compelling and beautifully designed, with no major caveats. The main conclusions are fully supported by the experiments. The work provides a characterization of endogenous progenitors, genetic strategies to isolate them, and proof of concept of exploiting these progenitors' potential to produce a specific desired neuronal type with "a la carte" combination of transcription factors.

Weaknesses:

Some issues need to be addressed or clarified before publication. The manuscript requires editing. It is dense and rich in details while in other parts there are a few mistakes.

Reviewer #1 (Public Review):

Summary:

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

Reviewer #1 (Public Review):

The manuscript by Feng et al. reported that the Endothelin B receptor (ETBR) expressed by the satellite glial cells (SGCs) in the dorsal root ganglions (DRG) acted to inhibit sensory axon regeneration in both adult and aged mice. Thus, pharmacological inhibition of ETBR with specific inhibitors resulted in enhanced sensory axon regeneration in vitro and in vivo. In addition, sensory axon regeneration significantly reduces in aged mice and inhibition of ETBR could restore such defect in aged mice. Moreover, the study provided some evidence that the reduced level of gap junction protein connexin 43 might act downstream of ETBR to suppress axon regeneration in aged mice. Overall, the study revealed an interesting SGC-derived signal in the DRG microenvironment to regulate sensory axon regeneration. It provided additional evidence that non-neuronal cell types in the microenvironment function to regulate axon regeneration via cell-cell interaction.

However, the molecular mechanisms by which ETBR regulates axon regeneration are unclear, and the manuscript's structure is not well organized, especially in the last section. Some discussion and explanation about the data interpretation are needed to improve the manuscript.

(1) The result showed that the level of ETBR did not change after the peripheral nerve injury. Does this mean that its endogenous function is to limit spontaneous sensory axon regeneration? In other words, the results suggest that SGCs expressing ETBR or vascular endothelial cells expressing its ligand ET-1 act to suppress sensory axon regeneration. Some explanation or discussion about this is necessary. Moreover, does the protein level of ETBR or its ligand change during aging?

(2) In ex vivo experiments, NGF was added to the culture medium. Previous studies have shown that adult sensory neurons could initiate fast axon growth in response to NGF within 24 hours. In addition, dissociated sensory neurons could also initiate spontaneous regenerative axon growth without NGF after 48 hours. Some discussion or rationale is needed to explain the difference between NGF-induced or spontaneous axon growth of culture adult sensory neurons and the roles of ETBR and SGCs.

(3) In cultured dissociated sensory neurons, inhibiting ETBR also enhanced axon growth, which meant the presence of SGCs surrounding the sensory neurons. Some direct evidence is needed to show the cellular relationship between them in culture.

(4) In Figure 3, the in vivo regeneration experiments first showed enhanced axon regeneration either 1 day or 3 days after the nerve injury. The study then showed that inhibiting ETBR could enhance sensory axon growth in vitro from uninjured naïve neurons or conditioning lesioned neurons. To my knowledge, in vivo sensory axon regeneration is relatively slow during the first 2 days after the nerve injury and then enters the fast regeneration mode on the 3rd day, representing the conditioning lesion effect in vivo. Some discussion is needed to compare the in vitro and the in vivo model of axon regeneration.

(5) In Figure 5, the study showed that the level of connexin 43 increased after ETBR inhibition in either adult or aged mice, proposing an important role of connexin 43 in mediating the enhancing effect of ETBR inhibition on axon regeneration. However, in the study, there was no direct evidence supporting that ETBR directly regulates connexin 43 expression in SGCs. Moreover, there was no functional evidence that connexin 43 acted downstream of ETBR to regulate axon regeneration.

Reviewer #1 (Public Review):

Summary:

This work sets out to elucidate mechanistic intricacies in inflammatory responses in pneumonia in the context of the aging process (Terc deficiency - telomerase functionality).

Strengths:

Very interesting, conceptually speaking, approach that is by all means worth pursuing. An overall proper approach to the posited aim.

Weaknesses:

The work is heavily underpowered and may have statistical deficits. This precludes it in its current state from drawing unequivocal conclusions.

Reviewer #1 (Public Review):

Summary:

The report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. Repressive binding of K3 to the kinase prevents phosphorylation of its recognised substrate, EIF2α (the α subunit of the Eukaryotic Initiation Factor 2). The interaction of K3 is probed by saturation mutation within four regions of PKR chosen by modelling the molecules' interaction. They identify K3-resistant PKR variants that recognise that the K3/EIF2α-binding surface of the kinase is malleable. This is reasonably interpreted as indicating the potential adaptability of this antiviral protein to combat viral virulence factors.

Strengths:

This is a well-conducted study that probes the versatility of the antiviral response to escape a viral inhibitor. The experimentation is very diligent, generating and screening a large number of variants to recognise the malleability of residues at the interface between PKR and K3.

Weaknesses:

These are minor. The protein interaction between PKR and K3 has been previously well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays. Accordingly, these findings largely reinforce what had been established rather than making major discoveries.

There are some presumptions:

It isn't established that the different PKR constructs are expressed equivalently so there is the contingency that this could account for some of the functional differences.

Details about the confirmation of PKR used to model the interaction aren't given so it isn't clear how accurately the model captures the active kinase state. This is important for the interaction with K3/EIF2α.

Not all regions identified to form the interface between PKR and K3 were assessed in the experimentation. It isn't clear why residues between positions 332-358 weren't examined, particularly as this would have made this report more complete than preceding studies of this protein interaction.

Reviewer #1 (Public Review):

Summary:

Torsekar et al. use a leaf litter decomposition experiment across seasons, and in an aridity gradient, to provide a careful test of the role of different-sized soil invertebrates in shaping the rates of leaf litter decomposition. The authors found that large-sized invertebrates are more active in the summer and small-sized invertebrates in the winter. The summed effects of all invets then translated into similar levels of decomposition across seasons. The system breaks down in hyper-arid sites.

Reviewer #1 (Public Review):

Summary:

Wang and colleagues identify biallelic variants of DNAH3 in four unrelated Han Chinese infertile men through whole-exome sequencing, which contributes to abnormal sperm flagellar morphology and ultrastructure. To investigate the importance of DNAH3 in male infertility, the authors generated crispant Dnah3 knockout (KO) male mice. They observed that KO mice are also infertile, showing a severe reduction in sperm movement with abnormal IDA (inner dynein arms) and mitochondrion structure. Moreover, nonfunctional DNAH3 expression decreased the expression of IDA-associated proteins in the spermatozoa of patients and KO mice, which are involved in the disruption of sperm motility. Interestingly, the infertility of patients and KO mice is rescued by intracytoplasmic sperm injection (ICSI). Taken together, the authors propose that DNAH3 is a novel pathogenic gene for asthenoterozoospermia and male infertility.

Strengths:

This work investigates the role of DNAH3 in sperm mobility and male infertility. By using gold-standard molecular biology techniques, the authors demonstrate with exquisite resolution the importance of DNAH3 in sperm morphology, showing strong evidence of its role in male infertility. Overall, this is a very interesting, well-written, and appealing article. All aspects of the study design and methods are well described and appropriate to address the main question of the manuscript. The conclusions drawn are consistent with the analyses conducted and supported by the data.

Weaknesses:

The paper is solid, and in its current form, I have not detected relevant weaknesses.

Reviewer #1 (Public Review):

Summary:

This study uses an online cognitive task to assess how reward and effort are integrated in a motivated decision-making task. In particular the authors were looking to explore how neuropsychiatric symptoms, in particular, apathy and anhedonia, and circadian rhythms affect behavior in this task. Amongst many results, they found that choice bias (the degree to which integrated reward and effort affect decisions) is reduced in individuals with greater neuropsychiatric symptoms, and late chronotypes (being an 'evening person').

Strengths:

The authors recruited participants to perform the cognitive task both in and out of sync with their chronotypes, allowing for the important insight that individuals with late chronotypes show a more reduced choice bias when tested in the morning.<br /> Overall, this is a well-designed and controlled online experimental study. The modelling approach is robust, with care being taken to both perform and explain to the readers the various tests used to ensure the models allow the authors to sufficiently test their hypotheses.

Weaknesses:

This study was not designed to test the interactions of neuropsychiatric symptoms and chronotypes on decision making, and thus can only make preliminary suggestions regarding how symptoms, chronotypes and time-of-assessment interact.

Reviewer #1 (Public Review):

Summary:

The work in the manuscript utilized patch-clamp techniques to explore the electrophysiological characteristics of VIP interneurons in the early stages of AD using the 3xTg mouse model. The study revealed that VIP interneurons exhibited prolonged action potentials and reduced firing rates. These changes could not be attributed to modifications in input signals or morphological transformations. The authors attributed aberrant VIP activity to the accumulation of beta-amyloid in those interneurons.

The decreased frequency of VIP inhibitory events were associated with no observed changes in excitatory drive to these interneurons. Consequently, heightened activity in the general population of CA1 interneurons was observed during a decision-making task and an object recognition test. In light of these findings, the authors concluded that the altered firing patterns of VIP interneurons may initiate early-stage dysfunction in hippocampal CA1 circuits, potentially influencing the progression of AD pathology.

Strengths:

Overall the work is novel and moves the field of Alzheimer's disease forward in a significant way. The manuscript reports a novel concept of aberrant activity in VIP interneurons during the early stages of AD thus contributing to dysfunctions of the CA1 microcircuit. This results in enhancement of the inhibitory tone on the primary cells of CA1. Thus, the disinhibition by VIP interneurons of Principal Cells is dampened. The manuscript was skillfully composed, the study was of strong scientific rigor featuring well-designed experiments. Necessary controls were present. Both sexes were included.

Major limitations were not adequately addressed in the revised manuscript

(1) The authors attributed aberrant circuit activity to accumulation of "Abeta intracellularly" inside IS-3 cells. That is problematic. 6E10 antibody recognizes amyloid plaques in addition to Amyloid Precursor Protein (APP) as well as the C99 fragment. There are no plaques at the ages 3xTg mice were examined. Lack of plaques was addressed in revised manuscript. The staining shown in Fig. 1a is of APP/C99 inside neurons, not abeta accumulations in neurons. At the ages of 3-6 months, 3xTg mice start producing and releasing extracellular abeta oligomers and potentially tau oligomers as well (Takeda et al., 2013 PMID: 23640054; Takeda et al., 2015 PMID: 26458742 and others). Emerging literature suggests that extracellular not intracellular abeta and tau oligomers disrupt circuit function. Thus, a more likely explanation of extracellular abeta and tau oligomers disrupting the activity of VIP neurons is plausible. Presence of intracellular abeta is currently controversial in the field and needs to be discussed as such. Some of the references added in the revised version of the manuscript are erroneously cited. The authors provide no original data in support of "intracellular" abeta.

(2) Authors suggest that their animals do not exhibit loss of synaptic connections and show Fig. 3d in support of that suggestion. However, imaging with confocal microscopy of 70 micron thick sections would not allow resolution of pre- and post-synaptic terminals. More sensitive measures such as electron microscopy or array tomography are the appropriate techniques to pursue. It is important for the authors to either remove that data from the manuscript or address/discuss the limitations of their technique in the discussion section. There is a possibility of loss of synaptic connections in their mouse model at the ages examined. Discussion of that possibility and of the limitations of the methodology used is missing.

Reviewer #1 (Public Review):

In this study, the authors address a fundamental unresolved question in cerebellar physiology: do synapses between granule cells (GCs) and Purkinje cells (PCs) made by the ascending part of the axon (AA) have different synaptic properties to those made by parallel fibers? This is an important question because GCs integrate sensorimotor information from many brain areas with a precise and complex topography.

The authors argue that GCs located close to the PCs essentially contact PC dendrites through the ascending part of their axon. They demonstrate that high-frequency (100 Hz) joint stimulation of distant parallel fibers and local GCs potentiates AA-PC synapses, while parallel fiber-PC synapses are depressed. On the basis of paired pulse ratio analysis, they concluded that evoked plasticity was postsynaptic. When individual pathways are stimulated alone, no LTP is observed. This associative plasticity appears to be sensitive to timing, as stimulation of parallel fibers first results in depression, while stimulation of the AA pathway has no effect. NMDA, mGluR1 and GABAA receptors are involved in this plasticity.

Overall, associative modulation of synaptic transmission is convincing, and the experiments carried out support this conclusion.

One of its weaknesses is that it contradicts the numerous experiments conducted by many groups that have studied plasticity at this connection (e.g. Bouvier et al 2016, Piochon et al 2016, Binda et al, 2016, Schonewille et al 2021). According to the literature, high-frequency stimulation of parallel fibers leads to postsynaptic potentiation under many different experimental conditions (blocked or unblocked inhibition, stimulation protocols, internal solution composition). This discrepancy was not investigated experimentally.

Another weakness is the lack of evidence that AAs have been stimulated. Indeed, without filling the PC with fluorescent dye or biocytin during the experiment, and without reconstructing the anatomical organization, it is difficult to assess whether the stimulating pipette is actually positioned in the GC cluster that potentially contacts the PC with AAs. Although the idea that AAs repeatedly contact the same Purkinje cell has been propagated, to the reviewer's knowledge, no direct demonstration of this hypothesis has yet been published. In fact, what has been demonstrated (Walter et al 2009; Spaeth et al 2022) is that GCs have a higher probability of being connected to nearby PCs, but not necessarily associated with AAs.

Reviewer #1 (Public Review):

Summary:

Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head restrained mice running down a virtual linear path. Mice were trained to collect water reward at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile, and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

The revised manuscript included additional evidence of increased (but transient) signal in LC axons after a transition to a novel environment during periods of immobility, and also that a change from dark to familiar environment induces a peak in LC axon activity, showing that LC input to dCA1 may not solely signal novelty.

Strengths:

The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis at the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

Weaknesses:

Aspects of the methodology, data analysis, and interpretation diminish the overall significance of the findings, as detailed below.

The LC axonal recordings are well powered, but the DA axonal recordings are severely underpowered, with recordings taken from a mere 7 axons (compare to 87 LC axons). Additionally, 2 different calcium indicators with differential kinetics and sensitivity to calcium changes (GCaMP6S and GCaMP7b) were used (n=3, n=4 respectively) and the data pooled. This makes it very challenging to draw any valid conclusions from the data, particularly in the novelty experiment. The surprising lack of novelty-induced DA axon activity may be a false negative. Indeed, at least 1 axon (axon 2) appears to be showing novelty-induced rise in activity in Figure 3C. Changes in activity in 4/7 axons are also referred to as a 'majority' occurrence in the manuscript, which again is not an accurate representation of the observed data

The authors conducted analysis on recording data exclusively from periods of running in the novelty experiment to isolate the effects of novelty from novelty-induced changes in behavior. However, if the goal is to distinguish between changes in locus coeruleus (LC) axon activity induced by novelty and those induced by motion, analyzing LC axon activity during periods of immobility would enhance the robustness of the results.

The authors attribute the ramping activity of the DA axons to the encoding of the animals' position relative to reward. However, given the extensive data implicating the dorsal CA1 in timing, and the remarkable periodicity of the behavior, the fact that DA axons could be signalling temporal information should be considered.

The authors should explain and justify the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments.

AFTER REVISIONS:

The authors have addressed my concerns in a thorough manner. The reviewer also appreciates the increased transparency of reporting in the revised manuscript.

Listed below are some remaining comments.<br /> The increase in LC activity with any change in environment (from familiar to novel or from dark to familiar) suggests that LC input acts not solely as a novelty signal, but as a general arousal or salience signal in response to environmental changes. Based on this, I have a couple of questions:

• Is the overall claim that LC input to the dHC signals novelty still valid based on observed findings - as claimed throughout the manuscript?<br /> • Would the omission of a reward be considered a salient change in the environment that activates LC signals, or is the LC not involved with processing reward-related information? Has the activity of LC and VTA axons been analysed in the seconds following reward presentation and/or omission?

Reviewer #1 (Public Review):

Summary:<br /> Du et al. report 16 new well-preserved specimens of atiopodan arthropods from the Chengjiang biota, which demonstrate both dosal and vental anatomies of a pothential new taxon of atiopodans that are closely related to trolobites. Authors assigned their specimens to Acanthomeridion serratum, and proposed A. anacanthus as a junior subjective synonym of Acanthomeridion serratum. Critially, the presence of ventral plates (interpreted as cephalic liberigenae), together with phylogenic results, lead authors to conclude that the cephalic sutures originated multiple times within the Artiopoda.

Strengths:<br /> New specimens are highly qualified and informative. The morphology of dorsal exoskeleton, except for the supposed free cheek, were well illustrated and described in detail, which provide a wealth of information for taxonmic and phylogenic analyses.

Weaknesses:<br /> The weaknesses of this work is obvious in a number of aspects. Technically, ventral morphlogy is less well revealed and is poorly illustrated. Additional diagrams are necessary to show the trunk appendages and suture lines. Taxonomically, I am not convinced by authors' placement. The specimens are markedly different from either Acanthomeridion serratum Hou et al. 1989 or A. anacanthus Hou et al. 2017. The ontogenetic description is extremely weak and the morpholical continuity is not established. Geometric and morphomitric analyses might be helpful to resolve the taxonomic and ontogenic uncertainties. I am confused by author's description of free cheek (libragena) and ventral plate. Are they the same object? How do they connect with other parts of cephalic shield, e.g. hypostome and fixgena. Critically, homology of cephalic slits (eye slits, eye notch, doral suture, facial suture) not extensivlely discussed either morphologically or functionally. Finally, authors claimed that phylogenic results support two separate origins rather than a deep origin. However, the results in Figure 4 can be explain a deep homology of cephalic suture in molecular level and multiple co-options within the Atiopoda.

Comments on the revised version:

I have seen the extensive revision of the manuscript. The main point "Multiple origins of dorsal ecdysial sutures in atiopoans" is now partially supported by results presented by the authors. I am still unsatisfied with descriptions and interpretations of critical features newly revealed by authors. The following points might be useful for the author to make further revisions.

(1) The antennae were well illustrated in a couple of specimens, while it was described in a short sentence.<br /> (2) There are also imprecise descriptions of features.<br /> (3) Ontogeny of the cephalon was not described.<br /> (3) The critical head element is the so called "ventral plate". How this element connects with the cephalic shield is not adequately revealed. The authors claimed that the suture is along the cephalic margin. However, the lateral margin of cephalon is not rounded but exhibit two notches (e.g. Fig 3C) . This gives an indication that the supposed ventral plates have a dorsal extension to fit the notches. Alternatively, the "ventral plate" can be interpreted as a small free cheek with a large ventral extension, providing evidence for librigenal hypothesis.

Reviewer #1 (Public Review):

In this paper the authors provide a characterisation of auditory responses (tones, noise, and amplitude modulated sounds) and bimodal (somatosensory-auditory) responses and interactions in the higher order lateral cortex (LC) of the inferior colliculus (IC) and compare these characteristic with the higher order dorsal cortex (DC) of the IC - in awake and anaesthetised mice. Dan Llano's group have previously identified gaba'ergic patches (modules) in the LC distinctly receiving inputs from somatosensory structures, surrounded by matrix regions receiving inputs from auditory cortex. They here use 2P calcium imaging combined with an implanted prism to - for the first time - get functional optical access to these subregions (modules and matrix) in the lateral cortex of IC in vivo, in order to also characterise the functional difference in these subparts of LC. They find that both DC and LC of both awake and anaesthetised appears to be more responsive to more complex sounds (amplitude modulated noise) compared to pure tones and that under anesthesia the matrix of LC is more modulated by specific frequency and temporal content compared to the gaba'ergic modules in LC. However, while both LC and DC appears to have low frequency preferences, this preference for low frequencies is more pronounced in DC. Furthermore, in both awake and anesthetized mice somatosensory inputs are capable of driving responses on its own in the modules of LC, but very little in the matrix. The authors now compare bimodal interactions under anaesthesia and awake states and find that effects are different in some cases under awake and anesthesia - particularly related to bimodal suppression and enhancement in the modules.

The paper provides new information about how subregions with different inputs and neurochemical profiles in the higher order auditory midbrain process auditory and multisensory information, and is useful for the auditory and multisensory circuits neuroscience community.

Reviewer #2 (Public Review):

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

Comments on revised version:

Please add proper references to indicate the invasion of ETEC into organoids after 1 h of infection.

Reviewer #1 (Public Review):

Summary:

The authors originally investigated the function of p53 isoforms with an alternative C-terminus encoded by the Alternatively Spliced (AS) exon in place of exon 11 encoding the canonical "α" C-terminal domain. For this purpose, the authors create a mouse model with a specific deletion of the AS exon.

Strengths:

Interestingly, wt or p53ΔAS/ΔAS mouse embryonic fibroblasts did not differ in cell cycle control, expression of well-known p53 target genes, proliferation under hyperoxic conditions, or the growth of tumor xenografts. However, p53-AS isoforms were shown to confer male-specific protection against lymphomagenesis in Eμ-Myc transgenic mice, prone to highly penetrant B-cell lymphomas. In fact, p53ΔAS/ΔAS Eμ-Myc mice were less protected from developing B-cell lymphomas compared to WT counterparts. The important difference that the authors find between WT and p53ΔAS/ΔAS Eμ-Myc males is a higher number of immature B cells in p53ΔAS/ΔAS vs WT mice. Higher expression of Ackr4 and lower expression of Mt2 was found in p53+/+ Eμ-Myc males compared to p53ΔAS/ΔAS counterparts, suggesting that these two transcripts are in part regulators of B-cell lymphomagenesis and enrichment for immature B cells.

The manuscript integrates an elegant genetic approach with in vivo analyses providing a robust set of data which strengthens the role of p53 isoforms in leukemogenesis.

Reviewer #1 (Public Review):

Summary

This article delves into the role of Ecdysone in regulating female sexual receptivity in Drosophila. The researchers discovered that PTTH, a positive regulator of Ecdysone production, hurts the receptivity of adult virgin females. Specifically, the researchers found that losing larval PTTH before metamorphosis significantly increases female receptivity immediately after adult eclosion. In addition, Ecdysone, through its receptor EcR-A, is necessary during metamorphic neurodevelopment for the proper development of P1 neurons, as its silencing leads to morphological changes associated with reduced adult female receptivity. Furthermore, Torso enhances receptivity in the adult stage. The molecular mechanisms linking each molecule to female receptivity have yet to be fully understood; therefore, the involvement of the juvenile-to-adult hormonal pathway (PTTH/Torso/ecdysone) in female receptivity is not proven.

Strengths

(1) Robust Methodology and Experimental Design: The study employs a comprehensive and well-structured experimental approach, combining genetic manipulations, behavioral assays, and molecular analyses. This multi-faceted methodology allows for a thorough investigation of the role of PTTH and Ecdysone in regulating female sexual receptivity in Drosophila. The use of specific gene knockouts, RNA interference, and overexpression techniques provides strong evidence supporting the findings.<br /> (2) Clear and Substantial Findings: The authors provide compelling data showing that PTTH negatively regulates female receptivity during the larval stage, which is rescued by Ecdysone feeding. Instead, metamorphic Ecdysone has a positive role during neurodevelopment. The experiments demonstrate this dual and temporally distinct role of PTTH/Ecdysone, shedding light on a complex hormonal regulation mechanism.<br /> (3) Clarification of Experimental Details: In response to the initial review, the authors have clarified important experimental details, such as the precise timing of genetic manipulations and the specific developmental stages examined. This clarification enhances the reproducibility and understanding of the study.

Weaknesses

(1) Unresolved Contradictions and Complexity in Results: Despite the detailed responses, the paper still presents complex and somewhat contradictory findings regarding the roles of PTTH, Torso, and Ecdysone. The observed increase in EcR-A expression in PTTH mutants and the nuanced explanation regarding the feedforward relationship, while insightful, do not fully resolve the initial confusion about the differing effects of PTTH and Ecdysone manipulations on female receptivity. This required more exploration.<br /> (2) Insufficient Exploration of Mechanistic Pathways: The potential mechanisms underlying the role of PTTH/Torso-Ecdysone across different developmental stages remain underexplored. While the authors suggest a feedforward relationship and possible interaction with other neurons, these hypotheses are not thoroughly tested or elaborated upon, leaving gaps in the mechanistic understanding.<br /> (3) Limited Scope of Validation Experiments: While the authors addressed some reviewer concerns about validation, the scope remains somewhat limited. The lack of existing PTTH mutants and the challenges in manipulating PTTH expression without affecting receptivity suggests that further work is needed to validate these pathways robustly. The inability to fully replicate the PTTHdelete phenotype through other means leaves some questions unanswered.<br /> (4). Complexity in Interpretation of dsx-Positive Neurons: The relevance of dsx-positive neurons in the context of PTTH's effects on female receptivity remains ambiguous. Although the authors provide some context, the biological significance of these observations is not fully clarified.

Conclusion<br /> The manuscript presents a well-conceived study with significant findings that advance the understanding of hormonal regulation of female receptivity in Drosophila. However, complexities in the data and unresolved mechanistic questions suggest that further work is needed to clarify the exact pathways and interactions involved. The authors' responses to feedback have strengthened the paper, but additional experiments and more thorough mechanistic exploration would enhance the robustness and clarity of the conclusions.

Reviewer #1 (Public Review):

Summary:

Willems and colleagues test whether unexpected shock omissions are associated with reward-related prediction errors by using an axiomatic approach to investigate brain activation in response to unexpected shock omission. Using an elegant design that parametrically varies shock expectancy through verbal instructions, they see a variety of responses in reward-related networks, only some of which adhere to the axioms necessary for prediction error. In addition, there were associations between omission-related responses and subjective relief. They also use machine learning to predict relief-related pleasantness and find that none of the a priori "reward" regions were predictive of relief, which is an interesting finding that can be validated and pursued in future work.

Strengths:

The authors pre-registered their approach and the analyses are sound. In particular, the axiomatic approach tests whether a given region can truly be called a reward prediction error. Although several a priori regions of interest satisfied a subset of axioms, no ROI satisfied all three axioms, and the authors were candid about this. A second strength was their use of machine learning to identify a relief-related classifier. Interestingly, none of the ROIs that have been traditionally implicated in reward prediction error reliably predicted relief, which opens important questions for future research.

Weaknesses:

The authors have done many analyses to address weaknesses in response to reviews. I will still note that given that one third of participants (n=10) did not show parametric SCR in response to instructions, it seems like some learning did occur. As prediction error is so important to such learning, a weakness of the paper is that conclusions about prediction error might differ if dynamic learning were taken into account using quantitative models.

Reviewer #1 (Public Review):

Summary:

Winged seeds or ovules from the Devonian are crucial to understanding the origin and early evolutionary history of wind dispersal strategy. Based on exceptionally well-preserved fossil specimens, the present manuscript documented a new fossil plant taxon (new genus and new species) from the Famennian Series of Upper Devonian in eastern China and demonstrated that three-winged seeds are more adapted to wind dispersal than one-, two- and four-winged seeds by using mathematical analysis.

Strengths:

The manuscript is well organised and well presented, with superb illustrations. The methods used in the manuscript are appropriate.

Weaknesses:

I would only like to suggest moving the "Mathematical analysis of wind dispersal of ovules with 1-4 wings" section from the supplementary information to the main text, leaving the supplementary figures as supplementary materials.

Reviewer #1 (Public Review):

Summary:<br /> Medina-Feliciano et al. investigated the single cell transcriptomic profile of holoturian regenerating intestine following evisceration, a process used to expel their viscera in response to predation. Using single cell RNA-sequencing and standard analysis such as "Find cluster markers", "Enrichment analysis of Gene Ontology" and "RNA velocity", they identify 13 cell clusters and potential identity. Based merely on bioinformatic analysis they identified potentially proliferating clusters and potential trajectories of cell differentiation. This manuscript represents a useful dataset that can provide candidate cell types and cell markers for more in-depth functional analysis for gaining a better understanding of the holoturian intestine regeneration. The conclusions of this paper are supported only by bioinformatic analyses, since the in vivo validation through HCR does not sufficiently support them.

Strengths:<br /> - The Authors are providing a single cell dataset obtained from sea cucumber regenerating their intestine. This represents a first fundamental step to an unbiased approach to better understand this regeneration process and the cellular dynamics taking part in it.<br /> - The Authors run all the standard analyses providing the reader with a well digested set of information about cell clusters, potential cell types, potential functions and potential cell differentiation trajectories.

Weaknesses:<br /> - The entire study is based on only 2 adult animals, that were used for both the single cell dataset and the HCR. Additionally, the animals were caught from the ocean preventing information about their age or their life history. This makes the n extremely small and reduces the confidence of the conclusions.<br /> - All the fluorescent pictures present in this manuscript present red nuclei and green signals being not color-blind friendly. Additionally, many of the images lack sufficient quality to determine if the signal is real. Additional images of a control animal (not eviscerated) and of a negative control would help data interpretation. Finally, in many occasions a zoomed out image would help the reader to provide context and have a better understanding of where the signal is localized.<br /> - The Authors frequently report the percentage of cells with a specific feature (either labelled or expressing a certain gene or belonging to a certain cluster). This number can be misleading since that is calculated after cell dissociation and additional procedures (such as staining or sequencing and dataset cleanup) that can heavily bias the ratio between cell types. Similarly, the Authors cannot compare cell percentage between anlage and mesentery samples since that can be affected by technical aspects related to cell dissociation, tissue composition and sequencing depth.<br /> - The Authors decided to validate only a few clusters and in many cases there are no positive controls (such as specific localization, specific function, changes between control and regenerating animals, co-stain) that could actually validate the cluster identity and the specificity of the selected marker. There is no validation of the trajectory analysis and there is no validation of the proliferating cluster with H3P or BrdU stainings.<br /> - It is not clear what is already known about holothurian intestine regeneration and what are the new findings in this manuscript. The Authors reference several papers throughout the whole result sectioning mentioning how the steps of regeneration, the proliferating cells, some of the markers and some of the cell composition of mesenteries and anlages was already known.

Reviewer #1 (Public Review):

Summary:

Dalal and Haddad investigated how neurons in the olfactory bulb are synchronized in oscillatory rhythms at gamma frequency. Temporal coordination of action potentials fired by projection neurons can facilitate information transmission to downstream areas. In a previous paper (Dalal and Haddad 2022, https://doi.org/10.1016/j.celrep.2022.110693), the authors showed that gamma frequency synchronization of mitral/tufted cells (MTCs) in the olfactory bulb enhances the response in the piriform cortex. The present study builds on these findings and takes a closer look at how gamma synchronization is restricted to a specific subset of MTCs in the olfactory bulb. They combined odor and optogenetic stimulations in anesthetized mice with extracellular recordings.<br /> The main findings are that lateral synchronization of MTCs at gamma frequency is mediated by granule cells (GCs), independent of the spatial distance, and strongest for MTCs with firing rates close to 40 Hz. The authors conclude that this reveals a simple mechanism by which spatially distributed neurons can form a synchronized ensemble. In contrast to lateral synchronization, they found no evidence for the involvement of GCs in lateral inhibition of nearby MTCs.

Strengths:

Investigating the mechanisms of rhythmic synchronization in vivo is difficult because of experimental limitations for the readout and manipulation of neuronal populations at fast timescales. Using spatially patterned light stimulation of opsin-expressing neurons in combination with extracellular recordings is a nice approach. The paper provides evidence for an activity-dependent synchronization of MTCs in gamma frequency that is mediated by GCs.

Weaknesses:

An important weakness of the study is the lack of direct evidence for the main conclusion - the synchronization of MTCs in gamma frequency. The data shows that paired optogenetic stimulation of MTCs in different parts of the olfactory bulb increases the rhythmicity of individual MTCs (Figure 1) and that combined odor stimulation and GC stimulation increases rhythmicity and gamma phase locking of individual MTCs (Figure 4). However, a direct comparison of the firing of different MTCs is missing. This could be addressed with extracellular recordings at two different locations in the olfactory bulb. The minimum requirement to support this conclusion would be to show that the MTCs lock to the same phase of the gamma cycle. Also, showing the evoked gamma oscillations would help to interpret the data.

Another weakness is that all experiments are performed under anesthesia with ketamine/medetomidine. Ketamine is an antagonist of NMDA receptors and NMDA receptors are critically involved in the interactions of MTCs and GCs at the reciprocal synapses (see for example Lage-Rupprecht et al. 2020, https://doi.org/10.7554/eLife.63737; Egger and Kuner 2021, https://doi.org/10.1007/s00441-020-03402-7). This should be considered for the interpretation of the presented data.

Furthermore, the direct effect of optogenetic stimulation on GCs activity is not shown. This is particularly important because they use Gad2-cre mice with virus injection in the olfactory bulb and expression might not be restricted to granule cells and might not target all subtypes of granule cells (Wachowiak et al., 2013, https://doi.org/10.1523/JNEUROSCI.4824-12.2013). This should be considered for the interpretation of the data, particularly for the absence of an effect of GC stimulation on lateral inhibition.

Several conclusions are only supported by data from example neurons. The paper would benefit from a more detailed description of the analysis and the display of some additional analysis at the population level:

- What were the criteria based on which the spots for light-activation were chosen from the receptive field map?

- The absence of an effect on firing rate for paired stimulations is only shown for one example (Figure 1c). A quantification of the population level would be interesting.

- Only one example neuron is shown to support the conclusion that "two different neural circuits mediate suppression and entrainment" in Figure 3. A population analysis would provide more evidence.

- Only one example neuron is shown to illustrate the effect of GC stimulation on gamma rhythmicity of MTCs in Figures 4 f,g.

- In Figure 5 and the corresponding text, "proximal" and "distal" GC activation are not clearly defined.

Reviewer #1 (Public Review):

Kainov et al investigated the prevalence of mutations in 3'UTR that affect gene expression in cancer to identify noncoding cancer drivers.

The authors used data from normal controls (1000 genome data) and compared it to cancer data (PCAWG). They found that in cancer 3'UTR mutations had a stronger effect on cleavage than the normal population. These mutations are negatively selected in the normal population and positively selected in cancers. The authors used PCAWG data set to identify such mutations and found that the mutations that lead to a reduction of gene expression are enriched in tumor suppressor genes and those that are increased in gene expression are enriched for oncogenes. 3'UTR mutations that reduce gene expression or occur in TSGs co-occur with non-synonymous mutations. The authors then validate the effect of 3'UTR mutations experimentally using a luciferase reporter assay. These data identify a novel class of noncoding driver genes with mutations in 3'UTR that impact polyadenylation and thus gene expression.

This is an elegant study with fundamental insight into identifying cancer driver genes. The conclusions of this paper are mostly well supported by data, but some aspects of data analysis need to be extended.

(1) It would be important for the authors to show if the findings of this study hold for metastatic cancers since most deaths occur due to metastasis and tumor heterogeneity changes when cancer progresses to metastasis. The authors should use the Hartwig data and show if metastatic cancers are enriched for 3'UTR mutations.

(2) Figure 2 should show the distribution of 3'UTR mutations by cancer type especially since authors go on to use colorectal cancer only for validations. It would be helpful to bring Figures S3A and S3C to this panel since these findings make the connections to cancer biology. Are any molecular functions enriched in addition to biological processes? Are kinases, phosphatases, etc more or less affected by 3'UTR mutations?

(3) Figure 3 looks at the co-occurrence of 3'UTR mutations with non-synonymous mutations but what about copy number change? You would expect the loss of the other allele to be enriched. Along the same line, are these data phased? Do you know that the non-synonymous mutations are in the other allele or in the same allele that shows 3'UTR mutation?

Reviewer #1 (Public Review):

Summary:

This study investigated the mechanism underlying Congenital NAD Deficiency Disorder (CNDD) using a mouse model with loss of function of the HAAO enzyme which mediates a key step in the NAD de novo synthesis pathway. This study builds on the observation that the kynurenine pathway is required in the conceptus, as HAAO null embryos are sensitive to maternal deficiency of NAD precursors (vitamin B3) and tryptophan, and narrows the window of sensitivity to a 3-day period.

An important finding is that de novo NAD synthesis occurs in an extra-embryonic tissue, the visceral yolk sac, before the liver develops in the embryo. It is suggested that lack of this yolk sac activity leads to impaired NAD supply in the embryo leading to structural abnormalities found later in development.

Strengths:

Previous studies show a requirement for HAAO activity for the normal development of embryos. Abnormalities develop under conditions of maternal vitamin B3 deficiency, indicating a requirement for NAD synthesis in the conceptus. Analysis of scRNA-seq datasets combined with metabolite analysis of yolk sac tissue shows that the NAD synthesis pathway is expressed and functional in the yolk sac from E10.5 onwards (prior to liver development).

HAAO enzyme assay enabled quantification of enzyme activity in relevant tissues including the liver (from E12.5), placenta, and yolk sac (from E11.5).

Comprehensive metabolite analysis of the NAD synthesis pathway supports the predicted effects of Haao knockout and provides analysis of the yolk sac, placenta, and embryo at a series of stages.

The dietary study (with lower vitamin B3 in maternal diet from E7.5-10.5) is an incremental addition to previous studies that imposed similar restrictions from E7.5-12.5.

Nevertheless, this emphasises the importance of the synthesis pathway on the conceptus at stages before the liver activity is prominent.

Weaknesses:

The current dietary study narrows the period when deficiency can cause malformations (analysed at E18.5), and altered metabolite profiles (eg, increased 3HAA, lower NAD) are detected in the yolk sac and embryo at E10.5. However, without analysis of embryos at later stages in this experiment it is not known how long is needed for NAD synthesis to be recovered - and therefore until when the period of exposure to insufficient NAD lasts. This information would inform the understanding of the developmental origin of the observed defects.

More importantly, there is still a question of whether in addition to the yolk sac, there is HAAO activity within the embryo itself prior to E12.5 (when it has first been assayed in the liver - Figure 1C). The prediction is that within the conceptus (embryo, chorioallantoic placenta, and visceral yok sac) the embryo is unlikely to be the site of NAD synthesis prior to liver development. Reanalysis of scRNA-seq (Fig 1B) shows expression of all the enzymes of the kynurenine pathway from E9.5 onwards. However, the expression of another available dataset at E10.5 (Fig S3) suggested that expression is 'negligible'. While the expression in Figure 1B, Figure S1 is weak this creates a lack of clarity about the possible expression of HAAO in the hepatocyte lineage, or especially elsewhere in the embryo prior to E10.5 (corresponding to the period when the authors have demonstrated that de novo NAD synthesis in the conceptus is needed). Given these questions, a direct analysis of RNA and/or protein expression in the embryos at E7.5-10.5 would be helpful.

Reviewer #1 (Public Review):

Summary:

The authors aim to measure the apoptotic fraction of motorneurons in developing zebrafish spinal cord to assess the extent of neuronal apoptosis during the development of a vertebrate embryo in an in vivo context.

Strengths:

The transgenic fish line tg (mnx1:sensor C3) appears to be a good reagent for motorneuron apoptosis studies, while further validation of its motorneuron specificity should be performed.

Weaknesses:

The results do not support the conclusions. The main "selling point" as summarized in the title is that the apoptotic rate of zebrafish motorneurons during development is strikingly low (~2% ) as compared to the much higher estimate (~50%) by previous studies in other systems. The results used to support the conclusion are that only a small percentage (under 2%) of apoptotic cells were found over a large population at a variety of stages 24-120hpf. This is fundamentally flawed logic, as a short-time window measure of percentage cannot represent the percentage in the long term. For example, at any year under 1% of the human population dies, but over 100 years >99% of the starting group will have died. To find the real percentage of motorneurons that died, the motorneurons born at different times must be tracked over the long term or the new motorneuron birth rate must be estimated.

A similar argument can be applied to the macrophage results. Here the authors probably want to discuss well-established mechanisms of apoptotic neuron clearance such as by glia and microglia cells.

The conclusion regarding the timing of axon and cell body caspase activation and apoptosis timing also has clear issues. The ~minutes measurement is too long as compared to the transport/diffusion timescale between the cell body and the axon, caspase activity could have been activated in the cell body, and either caspase or the cleaved sensor moves to the axon in several seconds. The authors' results are not high-frequency enough to resolve these dynamics

Many statements suggest oversight of literature, for example, in the abstract "However, there is still no real-time observation showing this dying process in live animals.".

Many statements should use more scholarly terms and descriptions from the spinal cord or motor neuron, neuromuscular development fields, such as line 87 "their axons converged into one bundle to extend into individual somite, which serves as a functional unit for the development and contraction of muscle cells"

The transgenic line is perhaps the most meaningful contribution to the field as the work stands. However, the mnx1 promoter is well known for its non-specific activation - while the images suggest the authors' line is good, motor neuron markers should be used to validate the line. This is especially important for assessing this population later as mnx1 may be turned off in mature neurons.

Overall, this work does not substantiate its biological conclusions and therefore does not advance the field. The transgenic line has the potential to address the questions raised but requires different sets of experiments. The line and the data as reported are useful on their own by providing a short-term rate of apoptosis of the motorneuron population.

Reviewer #1 (Public Review):

The authors report the results of a randomized clinical trial of taVNS as a neuromodulation technique in SAH patients. They found that taVNS appears to be safe without inducing bradycardia or QT prolongation. taVNS also increased parasympathetic activity, as assessed by heart rate variability measures. Acute elevation in heart rate might be a biomarker to identify SAH patients who are likely to respond favorably to taVNS treatment. The latter is very important in light of the need for acute biomarkers of response to neuromodulation treatments.

Comments:

(1) Frequency domain heart rate variability measures should be analyzed and reported. Given the short duration of the ECG recording, the frequency domain may more accurately reflect autonomic tone.

(2) How was the "dose" chosen (20 minutes twice daily)?

(3) The use of an acute biomarker of response is very important. A bimodal response to taVNS has been previously shown in patients with atrial fibrillation (Kulkarni et al. JAHA 2021).

Reviewer #1 (Public Review):

Summary:

Gekko, Nomura et al., show that Drp1 elimination in zygotes using the Trim-Away technique leads to mitochondrial clustering and uneven mitochondrial partitioning during the first embryonic cleavage, resulting in embryonic arrest. They monitor organellar localization and partitioning using specific targeted fluorophores. They also describe the effects of mitochondrial clustering in spindle formation and the detrimental effect of uneven mitochondrial partitioning to daughter cells.

Strengths:

The authors have gathered solid evidence for the uneven segregation of mitochondria upon Drp1 depletion through different means: mitochondrial labelling, ATP labelling and mtDNA copy number assessment in each daughter cell. Authors have also characterised the defects in cleavage mitotic spindles upon Drp1 loss

Weaknesses:

While this study convincingly describes the phenotype seen upon Drp1 loss, my major concern is that the mechanism underlying these defects in zygotes remains unclear. The authors refer to mitochondrial fragmentation as the mechanism ensuring organelle positioning and partitioning into functional daughters during the first embryonic cleavage. However, could Drp1 have a role beyond mitochondrial fission in zygotes? I raise these concerns because, as opposed to other Drp1 KO models (including those in oocytes) which lead to hyperfused/tubular mitochondria, Drp1 loss in zygotes appears to generate enlarged yet not tubular mitochondria. Lastly, while the authors discard the role of mitochondrial transport in the clustering observed, more refined experiments should be performed to reach that conclusion.

Reviewer #2 (Public Review):

This paper examines the recruitment of the inflammasome seeding pattern recognition receptor NLRP3 to the Golgi. Previously, electrostatic interactions between the polybasic region of NLRP3 and negatively charged lipids were implicated in membrane association. The current study concludes that reversible S-acylation of the conserved Cys-130 residue, in conjunction with upstream hydrophobic residues plus the polybasic region, act together to promote Golgi localization of NLRP3, although additional parts of the protein are needed for full Golgi localization. Treatment with the bacterial ionophore nigericin inhibits membrane traffic and apparently prevents Golgi-associated thioesterases from removing the acyl chain, causing NLRP3 to become immobilized at the Golgi. This mechanism is put forth as an explanation for how NLRP3 is activated in response to nigericin.

The experiments are generally well presented. It seems likely that Cys-130 does indeed play a previously unappreciated role in Golgi association of NLRP3. However, the evidence for S-acylation at Cys-130 is largely indirect, and the process by which nigericin enhances membrane association is not yet fully understood. Therefore, this interesting study points the way for further analysis.

Reviewer #1 (Public Review):

Summary:

The authors address cellular mechanisms underlying the early stages of Sjogren's syndrome, using a mouse model in which 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA) is applied to stimulate the interferon gene (STING) pathway. They show that in this model salivary secretion in response to neural stimulation is greatly reduced, even though calcium responses of individual secretory cells was enhanced. They attribute the secretion defect to reduced activation of Ca2+ -activated Cl- channels (TMEM16a), due to an increased distance between Ca2+ release channels (IP3 receptors) and TMEM16a which is expected to reduce the [Ca2+] sensed by TMEM16a. A variety of disruptions in mitochondria were also observed after DMXAA treatment, including reduced abundance, altered morphology, depolarization and reduced oxygen consumption rate. The results of this study shed new light on some of the early events leading to the loss of secretory function in Sjogren's syndrome, at a time before inflammatory responses cause the death of secretory cells.

Strengths:

Two-photon microscopy enabled Ca2+ measurements in the salivary glands of intact animals in response to physiological stimuli (nerve stimulation. This approach has been shown previously by the authors as necessary to preserve the normal spatiotemporal organization of calcium signals that lead to secretion under physiological conditions.

Superresolution (STED) microscopy allowed precise measurements of the spacing of IP3R and TMEM16a and the cell membranes that would otherwise be prevented by the diffraction limit. The measured increase of distance (from 84 to 155 nm) would be expected to reduce [Ca2+] at the TMEM16a channel.

The authors effectively ruled out a variety of alternative explanations for reduced secretion, including changes in AQP5 expression, and TMEM16a expression, localization and Ca2+ sensitivity as indicated by Cl- current in response to defined levels of Ca2+. Suppression of Cl- currents by a fast buffer (BAPTA) but not a slow one (EGTA) supports the idea that increased distance between IP3R and TMEM16A contributes to the secretory defect in DMXAA-treated cells.

Weaknesses:

While the Ca2+ distribution in the cells was less restricted to the apical region in DMXAA-treated cells, it is not clear that this is relevant to the reduced activation of TMEM16a or to pathophysiological changes associated with Sjogren's syndrome.

Despite the decreased level of secretion, Ca2+ signal amplitudes were higher in the treated cells, raising the question of how much this might compensate for the increased distance between IP3R and TMEM16a. The authors assume that the increased separation of IP3R and TMEM16a (and the resulting decrease in local [Ca2+]) outweighed the effect of higher global [Ca2+], but this point was not addressed directly.

The description of mitochondrial changes in abundance, morphology, membrane potential, and oxygen consumption rate were not well integrated into the rest of the paper. While they may be a facet of the multiple effects of STING activation and may occur during Sjogren's syndrome, their possible role in reducing secretion was not examined. As it stands, the mitochondrial results are largely descriptive and more studies are needed to connect them to the secretory deficits in SJogren's syndrome.

Reviewer #1 (Public Review):

In this manuscript, Leikina et al. investigate the role of redox changes in the ubiquitous protein La in promotion of osteoclast fusion. In a recently published manuscript, the investigators found that osteoclast multinucleation and resorptive activity are regulated by a de-phosphorylated and proteolytically cleaved form of the La protein that is present on the cell surface of differentiating osteoclasts. In the present work, the authors build upon these findings to determine the physiologic signals that regulate La trafficking to the cell membrane and ultimately, the ability of this protein to promote fusion. Building upon other published studies that show 1) that intracellular redox signaling can elicit changes in the confirmation and localization of La, and 2) that osteoclast formation is dependent on ROS signaling, the authors hypothesize that oxidation of La in response to intracellular ROS underlies the re-localization of La to the cell membrane and that this is necessary for its pro-fusion activity. The authors test this hypothesis in a rigorous manner using antioxidant treatments, recombinant La protein, and modification of cysteine residues predicted to be key sites of oxidation. Osteoclast fusion is then monitored in each condition using fluorescence microscopy. These data strongly support the conclusion that oxidized La is de-phosphorylated, increases in abundance at the cell surface of differentiating osteoclasts, and promotes cell-cell fusion. A strength of this manuscript is the use of multiple complementary approaches to test the hypothesis, especially the use of Cys mutant forms of La to directly tie the observed phenotypes to changes in residues that are key targets for oxidation. The manuscript is also well written and describes a clearly articulated hypothesis based on a precise summation of the existing literature. The findings of this manuscript will be of interest to researchers in the field of bone biology, but also more generally to cell biologists. The data in this manuscript may also lead to future studies that target La for bone diseases in which there is increased osteoclast activity. Weaknesses of the first version of the manuscript were minor and predominantly related to data presentation choices and some statistical analyses. These weaknesses were comprehensively addressed in the revised manuscript, and therefore the study has increased clarity and rigor.

Reviewer #1 (Public Review):

Summary:

In this manuscript the authors investigate the contributions of the long noncoding RNA snhg3 in liver metabolism and MAFLD. The authors conclude that liver-specific loss or overexpression of Snhg3 impacts hepatic lipid content and obesity through epigenetic mechanisms. More specifically, the authors invoke that nuclear activity of Snhg3 aggravates hepatic steatosis by altering the balance of activating and repressive chromatin marks at the Pparg gene locus. This regulatory circuit is dependent on a transcriptional regulator SNG1.

Strengths:

The authors developed a tissue specific lncRNA knockout and KI models. This effort is certainly appreciated as few lncRNA knockouts have been generated in the context of metabolism. Furthermore, lncRNA effects can be compensated in a whole organism or show subtle effects in acute versus chronic perturbation, rendering the focus on in vivo function important and highly relevant. In addition, Snhg3 was identified through a screening strategy and as a general rule the authors the authors attempt to follow unbiased approaches to decipher the mechanisms of Snhg3.

Weaknesses:

Despite efforts at generating a liver-specific knockout, the phenotypic characterization is not focused on the key readouts. Notably missing are rigorous lipid flux studies and targeted gene expression/protein measurement that would underpin why loss of Snhg3 protects from lipid accumulation. Along those lines, claims linking the Snhg3 to MAFLD would be better supported with careful interrogation of markers of fibrosis and advanced liver disease. In other areas, significance is limited since the presented data is either not clear or rigorous enough. Finally, there is an important conceptual limitation to the work since PPARG is not established to play a major role in the liver.

Reviewer #1 (Public Review):

This study presents a valuable finding on the expression levels of circHMGCS1 regulating arginase-1 by sponging miR-4521observed in diabetes-induced vascular endothelial dysfunction, leading to decrease in vascular nitric oxide secretion and inhibition of endothelial nitric oxide synthase activity. Further, increase in the expression of adhesion molecules and generation of cellular reactive oxygen species reduced vasodilation and accelerated the impairment of vascular endothelial function.<br /> Modulating circHMGCS1/miR-4521/ARG1 axis could serve as a potential strategy to prevent diabetes-associated cardiovascular diseases.

Comments on revised version:

The authors answered all questions satisfactorily.

Reviewer #1 (Public Review):

Summary:

In this work, the authors continue their investigations on the key role of glycosylation to modulate the function of a therapeutic antibody. As a follow-up to their previous demonstration on how ADCC was heavily affected by the glycans at the Fc gamma receptor (FcγR)IIIa, they now dissect the contributions of the different glycans that decorate the diverse glycosylation sites. Using a well-designed mutation strategy, accompanied by exhaustive biophysical measurements, with extensive use of NMR, using both standard and newly developed methodologies, they demonstrate that there is one specific locus, N162, which is heavily involved in the stabilization of (FcγR)IIIa and that the concomitant NK function is regulated by the glycan at this site.

Strengths:

The methodological aspects are carried out at the maximum level.

Weaknesses:

The exact (or the best possible assessment) of the glycan composition at the N162 site is not defined.

Reviewer #1 (Public Review):

Summary:

The authors report an inability to reproduce a transgenerational memory of avoidance of the pathogen PA14 in C. elegans. Instead, the authors demonstrate intergenerational inheritance for a single F1 generation, in embryos of mothers exposed to OP50 and PA14, where embryos isolated from these mothers by bleaching are capable of remembering to avoid PA14 in a manner that is dependent on systemic RNAi proteins sid-1 and sid-2. This could reflect systemic sRNAs generated by neuronal daf-7 signaling that are transmitted to F1 embryos. The authors note that transgenerational memory of PA14 was reported by the Murphy group at Princeton, but that environmental or strain variation (worms or bacteria) might explain the single generation of inheritance observed at Harvard. The Hunter group tried different bacterial growth conditions and different worm growth temperatures for independent PA14 strains, which they showed to be strongly pathogenic. However, the authors could not reproduce a transgenerational effect at Harvard. This important data will allow members of the scientific community to focus on the robust and reproducible inheritance of PA14 avoidance transmitted to F1 embryos of mothers exposed to PA14, which the authors demonstrate depends on small RNAs in a manner that is downstream of or in parallel to daf-7. This paper honestly and importantly alters expectations and questions the model that avoidance of PA14 is mediated by a bacterial ncRNA whose siRNAs target a C. elegans gene. Instead, endogenous C. elegans sRNAs that affect pathogen response may be the culprit that explains sRNA-mediated avoidance.

Overall, this is an important paper that demonstrates that one model for transgenerational inheritance in C. elegans is not reproducible. This is important because it is not clear how many of the reported models of transgenerational inheritance reported in C. elegans are reproducible. The authors do demonstrate a memory for F1 embryos that could be a maternal effect, and the authors confirm that this is mediated by a systemic small RNA response. There are several points in the manuscript where a more positive tone might be helpful.

Strengths:

The authors note that the high copy number daf-7::GFP transgene used by the Murphy group displayed variable expression and evidence for somatic silencing or transgene breakdown in the Hunter lab, as confirmed by the Murphy group. The authors nicely use single copy daf-7::GFP to show that neuronal daf-7::GFP is elevated in F1 but not F2 progeny with regards to the memory of PA14 avoidance, speaking to an intergenerational phenotype.

The authors nicely confirm that sid-1 and sid-2 are generally required for intergenerational avoidance of F1 embryos of moms exposed to PA14. However, these small RNA proteins did not affect daf-7::GFP elevation in the F1 progeny. This result is unexpected given previous reports that single copy daf-7::GFP is not elevated in F1 progeny of sid mutants. Because the Murphy group reported that daf-7 mutation abolishes avoidance for F1 progeny, this means that the sid genes function downstream of daf-7 or in parallel, rather than upstream as previously suggested.

The authors studied antisense small RNAs that change in Murphy data sets, identifying 116 mRNAs that might be regulated by sRNAs in response to PA14. Importantly, the authors show that the maco-1 gene, putatively targeted by piRNAs according to the Kaletsky 2020 paper, displays few siRNAs that change in response to PA14. The authors conclude that the P11 ncRNA of PA14, which was proposed to promote interkingdom RNA communication by the Murphy group, is unlikely to affect maco-1 expression by generating sRNAs that target maco-1 in C. elegans. The authors define 8 genes based on their analysis of sRNAs and mRNAs that might promote resistance to PA14, but they do not further characterize these genes' role in pathogen avoidance. The Murphy group might wish to consider following up on these genes and their possible relationship with P11.

Weaknesses:

This very thorough and interesting manuscript is at times pugnacious.

Please explain more clearly what is High Growth media for E. coli in the text and methods, conveying why it was used by the Murphy lab, and if Normal Growth or High Growth is better for intergenerational heritability assays.

Reviewer #3 (Public Review):

Summary:

In this work, the authors plate different type of cells on circular micropatterns and question how the organization and dynamics of the actin cytoskeleton correlate with particular actin chiral properties and rotational direction of the nucleus. The observe that cell spreading on large patterns correlates with the emergence of anti-clockwise rotations (ACW), while spreading on small patterns leads preferentially to clockwise rotations (CW). ACW originate, as previously demonstrated, from the polymerization of radial fibers, while clockwise rotations (CW) are observed when radial fibers are disorganized or absent and when transverse arcs take over to power CW rotations. These data are supported by a large number of observations and use of multiple drugs lead to observations that are consistent with the proposed model.

Strengths:

This is a beautiful work in which the authors rely on a large number of high-quality microscopic observations and use a full arsenal of drugs to test their model as thoroughly as possible.<br /> This study examines the influence of multiple actin networks. This is a challenging task in that the assembly and dynamics of different actin networks are interdependent, making it difficult to unambiguously analyze the importance of any specific network.

Reviewer #1 (Public Review):

Summary:

This paper uses single-molecule FRET to investigate the molecular basis for the distinct activation mechanisms between 2 GPCR responding to the chemokine CXCL12 : CXCR4, that couples to G-proteins, and ACKR3, which is G-protein independent and displays a higher basal activity.

Strengths:

It nicely combines the state-of-the-art techniques used in the studies of the structural dynamics of GPCR. The receptors are produced from eukaryotic cells, mutated, and labeled with single molecule compatible fluorescent dyes. They are reconstituted in nanodiscs, which maintain an environment as close as possible to the cell membrane, and immobilized through the nanodisc MSP protein, to avoid perturbing the receptor's structural dynamics by the use of an antibody for example.

The smFRET data are analysed using the HHMI technique, and the number of states to be taken into account is evaluated using a Bayesian Information Criterion, which constitutes the state-of-the-art for this task.

The data show convincingly that the activation of the CXCR4 and ACKR3 by an agonist leads to a shift from an ensemble of high FRET states to an ensemble of lower FRET states, consistent with an increase in distance between the TM4 and TM6. The two receptors also appear to explore a different conformational space. A wider distribution of states is observed for ACKR3 as compared to CXCR4, and it shifts in the presence of agonists toward the active states, which correlates well with ACKR3's tendency to be constitutively active. This interpretation is confirmed by the use of the mutation of Y254 to leucine (the corresponding residue in CXCR4), which leads to a conformational distribution that resembles the one observed with CXCR4. It is correlated with a decrease in constitutive activity of ACKR3.

Weaknesses:

Although the data overall support the claims of the authors, there are however some details in the data analysis and interpretation that should be modified, clarified, or discussed in my opinion.

Concerning the amplitude of the changes in FRET efficiency: the authors do not provide any structural information on the amplitude of the FRET changes that are expected. To me, it looks like a FRET change from ~0.9 to ~0.1 is very important, for a distance change that is expected to be only a few angstroms concerning the movement of the TM6. Can the authors give an explanation for that? How does this FRET change relate to those observed with other GPCRs modified at the same or equivalent positions on TM4 and TM6?

Concerning the intermediate states: the authors observe several intermediate states.

(1) First I am surprised, looking at the time traces, by the dwell times of the transitions between the states, which often last several seconds. Is such a long transition time compatible with what is known about the kinetic activation of these receptors?

(2) Second is it possible that these « intermediate » states correspond to differences in FRET efficiencies, that arise from different photophysical states of the dyes? Alexa555 and Cy5 are Cyanines, that are known to be very sensitive to their local environment. This could lead to different quantum yields and therefore different FRET efficiencies for a similar distance. In addition, the authors use statistical labeling of two cysteines, and have therefore in their experiment a mixture of receptors where the donor and acceptor are switched, and can therefore experience different environments. The authors do not speculate structurally on what these intermediate states could be, which is appreciated, but I think they should nevertheless discuss the potential issue of fluorophore photophysics effects.

(3) It would also have been nice to discuss whether these types of intermediate states have been observed in other studies by smFRET on GPCR labeled at similar positions.

On line 239: the authors talk about the R↔R' transitions that are more probable. In fact it is more striking that the R'↔R* transition appears in the plot. This transition is a signature of the behaviour observed in the presence of an agonist, although IT1t is supposed to be an inverse agonist. This observation is consistent with the unexpected (for an inverse agonist) shift in the FRET histogram distribution. In fact, it appears that all CXCR4 antagonists or inverse agonists have a similar (although smaller) effect than the agonist. Is this related to the fact that these (antagonist or inverse agonist) ligands lead to a conformation that is similar to the agonists, but cannot interact with the G-protein ?? Maybe a very interesting experiment would be here to repeat these measurements in the presence of purified G-protein. G-protein has been shown to lead to a shift of the conformational space explored by GPCR toward the active state (using smFRET on class A and class C GPCR). It would be interesting to explore its role on CXCR4 in the presence of these various ligands. Although I am aware that this experiment might go beyond the scope of this study, I think this point should be discussed nevertheless.

The authors also mentioned in Figure 6 that the energetic landscape of the receptors is relatively flat ... I do not really agree with this statement. For me, a flat conformational landscape would be one where the receptors are able to switch very rapidly between the states (typically in the submillisecond timescale, which is the timescale of protein domain dynamics). Here, the authors observed that the transition between states is in the second timescale, which for me implies that the transition barrier between the states is relatively high to preclude the fast transitions.

Reviewer #1 (Public Review):

Summary:

In this paper by Zhang, the authors build a physical framework to probe the mechanisms that underlie exchange of molecules between coexisting dense and dilute liquid-like phases of condensates. They first propose a continuum model, in the context of a FRAP-like experiment where the fluorescently labeled molecules inside the condensate are bleached at t=0 and the recovery of fluorescence is measured. Through this model, they identify how the key timescales of internal molecular mixing, replenishment from dilute phase, and interface transfer contribute to molecular exchange timescale. Motivated by a recent experiment reported by some of the co-authors previously (Brangwynne et al. in 2019) finding strong interfacial resistance in in vitro protein droplets of LAF-1, they seek to understand the microscopic features contributing to the interfacial conductance (inversely proportional to the resistance). To check, they perform coarse-grained MD-simulations of sticker-spacer self-associative polymers and report how conductance varies significantly even across the few explored sequences. Further, by looking at individual trajectories, they postulate the "bouncing" i.e., molecules that approach the interface but are not successfully absorbed is a strong contributor to this mass transfer limitation. Consistent with their predictions, sequences that have more free unbound stickers (i.e., for example through imbalance sequence sticker stoichiometries) have higher conductances and they show a simple linear scaling between number of unbound stickers and conductance. Finally, they predict that an droplet-size dependent transition in recovery time behavior.

Strengths:

(1) This paper is overall well-written and clear to understand.<br /> (2) By combining coarse-grained simulations, continuum modeling, and comparison to published data, the authors provide a solid picture of how their proposed framework relates to molecular exchange mechanisms that are dominated by interface resistance and LAF-1 droplets.<br /> (3) The choice of different ways to estimate conductance from simulation and reported data are thoughtful and convincing on their near-agreement (although a little discussion of why and when they differ would be merited as well).

Updated re-review:

This revised update by Zhang et al. is improved and addresses many of the concerns raised by myself and the other reviewer, especially with the expanded discussion, contextualized text in model description, and the addition of a nice example case-study in revised Fig. 4. I believe the paper provides solid evidence of how "bouncing" may contribute to interfacial resistance/exchange dynamics in biomolecular condensates and is a useful study for the community.

Note:<br /> In their response, the authors bring up an important point in references for LAF1 mutant FRAP data. While I found a few papers, for example https://www.pnas.org/doi/abs/10.1073/pnas.2000223117 and https://www.cell.com/biophysj/fulltext/S0006-3495(23)00464-2 , these are likely to be not whole droplet bleaches. I wonder whether it may be possible to approximately predict the conductance from other parameters (such as from effective expressions in eq 14) to roughly estimate what the effect maybe since LAF-1 has fairly "known" stickers and spacers. Note that this is not required at all, but I just bring this up in case it may be of interest to authors!

Reviewer #1 (Public Review):

Summary:

In this study, the authors describe the construction of an extremely large-scale anatomical model of juvenile rat somatosensory cortex (excluding the barrel region), which extends earlier iterations of these models by expanding across multiple interconnected cortical areas. The models are constructed in such a way as to maintain biological detail from a granular scale - for example, individual cell morphologies are maintained, and synaptic connectivity is founded on anatomical contacts. The authors use this model to investigate a variety of properties, from cell-type specific targeting (where the model results are compared to findings from recent large-scale electron microscopy studies) to network metrics. The model is also intended to serve as a platform and resource for the community by being a foundation for simulations of neuronal circuit activity and for additional anatomical studies that rely on the detailed knowledge of cellular identity and connectivity.

Strengths:

As the authors point out, the combination of scale and granularity of their model is what makes this study valuable and unique. The comparisons with recent electron microscopy findings are some of the most compelling results presented in the study, showing that certain connectivity patterns can arise directly from the anatomical configuration, while other discrepancies highlight where more selective targeting rules (perhaps based on molecular cues) are likely employed. They also describe intriguing effects of cortical thickness and curvature on circuit connectivity and characterize the magnitude of those effects on different cortical layers.

The detailed construction of the model is drawn on a wide range of data sources (cellular and synaptic density measures, neuronal morphologies, cellular composition measures, brain geometry, etc.) that are integrated together; other data sources are used for comparison and validation. This consolidation and comparison also represent a valuable contribution to the overall understanding of the modeled system.

Weaknesses:

The scale of the model, which is a primary strength, also can carry some drawbacks. In order to integrate all the diverse data sources together, many specific decisions must be made about, for example, translating findings from different species or regions to the modeled system, or deciding which aspects of the system can be assumed to be the same and which should vary. All these decisions will have effects on the predicted results from the model, which could limit the types of conclusions that can be made (both by the others and by others in the community who may wish to use the model for their own work).

As an example, while it is interesting that broad brain geometry has effects on network structure (Figure 7), it is not clear how those effects are actually manifested. I am not sure if some of the effects could be due to the way the model is constructed - perhaps there may be limited sets of morphologies that fit into columns of particular thicknesses, and those morphologies may have certain idiosyncrasies that could produce different statistics of connectivities where they are heavily used. That may be true to biology, but it may also be somewhat artifactual if, for example, the only neurons in the library that fit into that particular part of the cortex differ from the typical neurons that are actually found in that region (but may not have been part of the morphological sampling). I also wonder how much the assumption that the layers have the same relative thicknesses everywhere in the cortex affects these findings, since layer thicknesses do in fact vary across the cortex.

In addition, the complexity of the model means that some complicated analyses and decisions are only presented in this manuscript with perhaps a single panel and not much textual explanation. I find, for example, that the panels of Figure S2 seem to abstract or simplify many details to the point where I am not clear about what they are actually illustrating - how does Figure S2D represent the results of "the process illustrated in B"? Why are there abrupt changes in connectivity at region borders (shown as discontinuous colors), when dendrites and axons span those borders and so would imply interconnectivity across the borders? What do the histograms in E1 and E2 portray, and how are they related to each other?

Overall, the model presented in this study represents an enormous amount of work and stands as a unique resource for the community, but also is made somewhat unwieldy for the community to employ due to the weight of its manifold specific construction decisions, size, and complexity.

Reviewer #1 (Public Review):

Summary:

The authors propose that the energy landscape of animals can be thought of in the same way as the fundamental versus realized niche concept in ecology. Namely, animals will use a subset of the fundamental energy landscape due to a variety of factors. The authors then show that the realized energy landscape of eagles increases with age as the animals are better able to use the energy landscape.

Strengths:

This is a very interesting idea and that adds significantly to the energy landscape framework. They provide convincing evidence that the available regions used by birds increase with size.

Review of revised version:

The authors have addressed all my comments and concerns. This is a really nice and important manuscript. I have one minor suggestion: Line 74-85: when discussing the effect of ontogeny, the authors give examples of how these may change due to improved cognition and memory. I would recommend they also give examples of how these may change with morphology (e.g. change in wing or fin relative area, buoyancy in sharks etc) should also be included. Most growth in fish for example is allometric so the relative measures of area of fins to body size should also change.

This is of course up to the authors but it would highlight how their study is applicable to many other systems beyond just birds (even though morphology is of little importance for their eagles).

Reviewer #1 (Public Review):

Summary:

Ren et al developed a novel computational method to investigate cell evolutionary trajectory for scRNA-seq samples. This method, MGPfact, estimates pseudotime and potential branches in the evolutionary path by explicitly modeling the bifurcations in a Gaussian process. They benchmarked this method using synthetic as well as real-world samples and showed superior performance for some of the tasks in cell trajectory analysis. They further demonstrated the utilities of MGPfact using single-cell RNA-seq samples derived from microglia or T cells and showed that it can accurately identify the differentiation timepoint and uncover biologically relevant gene signatures.

Strengths:

Overall I think this is a useful new tool that could deliver novel insights for the large body of scRNA-seq data generated in the public domain. The manuscript is written in a logical way and most parts of the method are well described.

Weaknesses:

Some parts of the methods are not clear.

It should be outlined in detail how pseudo time T is updated in Methods. It is currently unclear either in the description or Algorithm 1.

There should be a brief description in the main text of how synthetic data were generated, under what hypothesis, and specifically how bifurcation is embedded in the simulation.

Please explain what the abbreviations mean at their first occurrence.

In the benchmark analysis (Figures 2/3), it would be helpful to include a few trajectory plots of the real-world data to visualize the results and to evaluate the accuracy.

It is not clear how this method selects important genes/features at bifurcation. This should be elaborated on in the main text.

It is not clear how survival analysis was performed in Figure 5. Specifically, were critical confounders, such as age, clinical stage, and tumor purity controlled?

I recommend that the authors perform some sort of 'robustness' analysis for the consensus tree built from the bifurcation Gaussian process. For example, subsample 80% of the cells to see if the bifurcations are similar between each bootstrap.

Reviewer #1 (Public Review):

The authors proposed a framework to estimate the posterior distribution of parameters in biophysical models. The framework has two modules: the first MLP module is used to reduce data dimensionality and the second NPE module is used to approximate the desired posterior distribution. The results show that the MLP module can capture additional information compared to manually defined summary statistics. By using the NPE module, the repetitive evaluation of the forward model is avoided, thus making the framework computationally efficient. The results show the framework has promise in identifying degeneracy. This is an interesting work.

Reviewer #1 (Public Review):

In their manuscript, Gan and colleagues identified a functional critical residue, Tyr404, which when mutated to W or A results in GOF and LOF of TRPML1 activity, respectively. In addition, the authors provide a high-resolution structure of TRPML1 with PI(4,5)P2 inhibitor. This high-resolution structure also revealed a bound phospholipid likely sphingomyelin at the agonist/antagonist site, providing a plausible explanation for sphingomyelin inhibition of TRPML1.

This is an interesting study, revealing valuable additional information on TRPML1 gating mechanisms including effects on endogenous phospholipids on channel activity. The provided data are convincing. Some major open questions remain. The work will be of interest to a wide audience including industry researchers occupied with TRPML1 exploration as a drug target.

Reviewer #1 (Public Review):

Summary:

Sun et al. generated germline-specific cKO mice for the Znhit1 gene and examined its effect on male meiosis. The authors found that the loss of Znhit1 affects the transcriptional activation of pachytene. Znhit1 is a subunit of the SRCAP chromatin remodeling complex and a depositor of H2AZ, and in cKO spermatocytes, H2AZ is not deposited into the gene region. The authors claim that this is why the PGA was not activated. These findings provide important insights into the mechanisms of transcriptional regulation during the meiotic prophase.

Strengths:

The authors used samples from their original mouse model, analyzing both the epigenome and the transcriptome in detail using diverse NGS analyses to gain new insights into PGA. The quality of the results appeared excellent.

Weaknesses:

Overall, the data is inconsistent with the authors' claims and does not support their final conclusions. In addition, the sample used may not be the most suitable for the analysis, but a more suitable sample would dramatically improve the overall quality of the paper.

Reviewer #1 (Public Review):

Summary:

This work introduces a new imaging tool for profiling tumor microenvironments through glucose conversion kinetics. Using GL261 and CT2A intracranial mouse models, the authors demonstrated that tumor lactate turnover mimicked the glioblastoma phenotype, and differences in peritumoral glutamate-glutamine recycling correlated with tumor invasion capacity, aligning with histopathological characterization. This paper presents a novel method to image and quantify glucose metabolites, reducing background noise and improving the predictability of multiple tumor features. It is, therefore, a valuable tool for studying glioblastoma in mouse models and enhances the understanding of the metabolic heterogeneity of glioblastoma.

Strengths:

By combining novel spectroscopic imaging modalities and recent advances in noise attenuation, Simões et al. improve upon their previously published Dynamic Glucose-Enhanced deuterium metabolic imaging (DGE-DMI) method to resolve spatiotemporal glucose flux rates in two commonly used syngeneic GBM mouse models, CT2A and GL261. This method can be standardized and further enhanced by using tensor PCA for spectral denoising, which improves kinetic modeling performance. It enables the glioblastoma mouse model to be assessed and quantified with higher accuracy using imaging methods.

The study also demonstrated the potential of DGE-DMI by providing spectroscopic imaging of glucose metabolic fluxes in both the tumor and tumor border regions. By comparing these results with histopathological characterization, the authors showed that DGE-DMI could be a powerful tool for analyzing multiple aspects of mouse glioblastoma, such as cell density and proliferation, peritumoral infiltration, and distant migration.

Weaknesses:

Although the paper provides clear evidence that DGE-DMI is a potentially powerful tool for the mouse glioblastoma model, it fails to use this new method to discover novel features of tumors. The data presented mainly confirm tumor features that have been previously reported. While this demonstrates that DGE-DMI is a reliable imaging tool in such circumstances, it also diminishes the novelty of the study.

When using DGE-DMI to quantitatively map glycolysis and mitochondrial oxidation fluxes, there is no comparison with other methods to directly identify the changes. This makes it difficult to assess how sensitive DGE-DMI is in detecting differences in glycolysis and mitochondrial oxidation fluxes, which undermines the claim of its potential for in vivo GBM phenotyping.

The study only used intracranial injections of two mouse glioblastoma cell lines, which limits the application of DGE-DMI in detecting and characterizing de novo glioblastomas. A de novo mouse model can show tumor growth progression and is more heterogeneous than a cell line injection model. Demonstrating that DGE-DMI performs well in a more clinically relevant model would better support its claimed potential usage in patients.

Reviewer #1 (Public Review):

Summary:

Previous work has shown that the evolutionarily-conserved division-orienting protein LGN/Pins (vertebrates/flies) participates in division orientation across a variety of cell types, perhaps most importantly those that undergo asymmetric divisions. Micromere formation in echinoids relies on asymmetric cell division at the 16-cell stage, and these authors previously demonstrated a role for the LGN/Pins homolog AGS in that ACD process. Here they extend that work by investigating and exploiting the question of why echinoids but not other echinoderms form micromeres. Starting with a phylogenetics approach, they determine that much of the difference in ACD and micromere formation in echinoids can be attributed to differences in the AGS C-terminus, in particular a GoLoco domain (GL1) that is missing in most other echinoderms.

Strengths:

There is a lot to like about this paper. It represents a superlative match of the problem with the model system and the findings it reports are a valuable addition to the literature. It is also an impressively thorough study; the authors should be commended for using a combination of experimental approaches (and consequently generating a mountain of data).

Weaknesses:

There is an intriguing finding described in Figure 1. AGS in sea cucumbers looks identical to AGS in the pencil urchin, at least at the C terminus (including the GL1 domain). Nevertheless, there are no micromeres in sea cucumbers. Therefore another mechanism besides GL motif organization has arisen to support micromere formation. It is a consequential finding and an important consideration in interpreting the data, but I could not find any mention of it in the text. That is a missed opportunity and should be remedied, ideally not only through discussion but also experimentation. Specifically: does sea cucumber AGS (SbAGS) ever localize to the vegetal cortex in sea cucumbers? Can it do so in echinoids? Will that support micromere formation?

The authors point out that AGS-PmGL demonstrates enrichment at the vegetal cortex (arrow in 5G, quantifications in 5H), unlike PmAGS. AGS-PmGL does not however support ACD. They interpret this result to indicate "that other elements of SpAGS outside of its C-terminus can drive its vegetal cortical localization but not function." This is a critical finding and deserves more attention. Put succinctly: Vegetal cortical localization of AGS is insufficient to promote ACD, even in echinoids. Why should this be?

The authors did perform experiments to address this problem, hypothesizing that the difference might be explained by the linker region, which includes a conserved phosphorylation site that mediates binding to Dlg. They write "To test if this serine is essential for SpAGS localization, we mutated it to alanine (AGS-S389A in Fig. S3A). Compared to the Full AGS control, the mutant AGS-S389A showed reduced vegetal cortical localization (Fig. S3B-C) and function (Fig. S3D-E). Furthermore, we replaced the linker region of PmAGS with that of SpAGS (PmAGS-SpLinker in Fig. S4A-B). However, this mutant did not show any cortical localization nor proper function in ACD (Fig. S4C-F). Therefore, the SpAGS C-terminus is the primary element that drives ACD, while the linker region serves as the secondary element to help cortical localization of AGS."

The experiments performed only make sense if the AGS-PmGL chimeric protein used in Figure 5 starts the PmGL sequence only after the Sp linker, or at least after the Sp phosphorylation site. I can't tell from the paper (Figure S3 indicates that it does, whereas S5 suggests otherwise), but it's a critical piece of information for the argument. Another piece of missing information is whether the PmAGS can be phosphorylated at its own conserved phosphorylation site. The authors don't test this, which they could at least try using a phosphosite prediction algorithm, but they do show that the candidate phosphorylation site has a slightly different sequence in Pm than in Et and Sp (Fig. S4A). With impressive rigor, the authors go on to mutate the PmAGS phosphorylation site to make it identical to Sp. Nothing happens. Vegetal cortical localization does not increase over AGS-PmGL alone. Micromere formation is unrescued.

There is therefore a logic problem in the text, or at least in the way the text is written. The paragraph begins "Additionally, AGS-PmGL unexpectedly showed cortical localization (Figure 5G), while PmAGS showed no cortical localization (Figure 5B)." We want to understand why this is true, but the explanation provided in the remainder of the paragraph doesn't match the question: according to quite a bit of their own data, the phosphorylation site in the linker does not explain the difference. It might explain why AGS-PmGL fails to promote micromere formation, but only if the AGS-PmGL chimeric protein uses the Pm linker domain (see above).

Another concern that is potentially related is the measurement of cortical signal. For example, in the control panel of Figure 5C, there is certainly a substantial amount of "non-cortical" signal that I believe is nuclear. I did not see a discussion of this signal or its implications. My impression of the pictures generally is that the nuclear signal and cortical signal are inversely correlated, which makes sense if they are derived from the same pool of total protein at different points of the cell cycle. If that's the case (and it might not be) I would expect some quantifications to be impacted. For example, the authors show in Figure S3B that AGS-S389A mutant does not localize to the cortex. However, this mutant shows a radically different localization pattern to the accompanying control picture (AGS), namely strong enrichment in what I assume to be the nucleus. Is the S389 mutant preventing AGS from making it to the cortex? Or are these pictures instead temporally distinct, meaning that AGS hasn't yet made it out of the nucleus? Notably, the work of Johnston et al. (Cell 2009), cited in the text, does not show or claim that the linker domain impacts Pins localization. Their model is rather that Pins is anchored at the cortex by Gαi, not Dlg, and that is the same model described in this manuscript. In agreement with that model and the results of Johnston et al., a later study (Neville et al. EMBO Reports 2023) failed to find a role for Dlg or the conserved phosphorylation site in Pins localization.

Reviewer #1 (Public Review):

Summary:

The authors identified nanobodies that were specific for the trypanosomal enzyme pyruvate kinase in previous work seeking diagnostic tools. They have shown that a site involved in the allosteric regulation of the enzyme is targeted by the nanobody and using elegant structural approaches to pinpoint where binding occurs, opening the way to the design of small molecules that could also target this site.

Strengths:

The structural work shows the binding of a nanobody to a specific site on Trypanosoma congolense pyruvate kinase and provides a good explanation as to how binding inhibits enzyme activity. The authors go on to show that by expressing the nanobodies within the parasites they can get some inhibition of growth, which albeit rather weak, they provide a case on how this could point to targeting the same site with small molecules as potential trypanocidal drugs.

Weaknesses:

The impact on growth is rather marginal. Although explanations are offered on the reasons for that, including the high turnover rate of the expressed nanobody and the difficulty in achieving the high levels of inhibition of pyruvate kinase required to impact energy production sufficiently to kill parasites, this aspect of the work doesn't offer great support to developing small molecule inhibitors of the same site.

DOI: 10.1007/978-1-4939-8772-6_3

Resource: Bloomington Stock Center

Curator: @maulamb

SciCrunch record: Bloomington Stock Center

What is this?

El mapa mental debería tener ramas más detalladas que permitan recuperar y justificar información incluso meses después de lo que estamos haciendo

Me gustaría saber como lograste procesar el documento de audio, ¿estaba descargado previamente en el computador o copiaste el link?

El ejercicio esta muy bien hecho, quizá lo que falta es interiorizar mejor algunos conceptos. Pero la manera en la que desarrolla el ejercicio y como lo explica deja claro la manera en la que Felipe se relaciona con la herramienta

Sería conveniente agregar el hecho de que las respuestas dicen que apelan a más fuentes de las que realmente usan. Para ello es bueno tener la respuesta textual fuera de la captura de pantalla

Buena presentación visual acompañada por capturas de pantalla. Sugeriría colocar respuestas textuales también, transcritas desde NucliaDB de manera que se puedan revisar en detalle fuera de la captura.

Reviewer #1 (Public Review):

In the manuscript entitled "SARS-CoV-2 NSP13 interacts with TEAD to suppress Hippo-YAP signaling", Meng et al. report that SARS-CoV-2 infection disrupts YAP downstream gene transcription in both patient lung samples and the iPSC-cardiomyocytes. Among the tested SARS-CoV-2 proteins, the helicase nonstructural protein 13 (NSP13) was identified to target YAP transcriptional activity both in vitro and in vivo, independent of the Hippo pathway. Mechanistically, NSP13 inhibits YAP transcriptional activity through its interaction with TEAD4 and a group of nuclear repressor proteins, a process that requires its helicase activity. Overall, this study uncovers a novel regulation of the YAP/TEAD complex by SARS-CoV-2 infection, highlighting its impact on cellular signaling events. The manuscript is well-written and easy to follow. Here are some suggestions for the authors to further improve their work.

Major points

(1) The authors discovered a novel regulation of the Hippo-YAP pathway by SARS-CoV-2 infection but did not address the pathological significance of this finding. It remains unclear why YAP downstream gene transcription needs to be inhibited in response to SARS-CoV-2 infection. Is this inhibition crucial for the innate immune response to SARS-CoV-2? The authors should re-analyze their snRNA-seq and bulk RNA-seq data described in Figure 1 to determine whether any of the affected YAP downstream genes are involved in this process.

(2) The authors concluded that helicase activity is required for NSP13-induced inhibition of YAP transcriptional activity based on mutation studies (Figure 3B). This finding is somewhat confusing, as K131, K345/K347, and R567 are all essential residues for NSP13 helicase activity while mutating K131 did not affect NSP13's ability to inhibit YAP (Figure 3B). Additionally, there are no data showing exactly how NSP13 inhibits the YAP/TEAD complex through its helicase function. This point was also not reflected in their proposed working model (Figure 4H).

(3) The proposed model that NSP13 binds TEAD4 to recruit repressor proteins and inhibits YAP/TEAD downstream gene transcription (Figure 4H) needs further characterization. First, it is notable that the provided NSP13 IP-MS data did not reveal any TEAD family members as binding proteins for NSP13 (Supplement Figure 4C and the tables), suggesting that NSP13 may modulate the YAP/TEAD complex through other mechanisms, possibly involving other binding proteins. Second, NSP13 is a DNA-binding protein, and its nucleic acid-binding mutant K345A/K347A failed to inhibit YAP transcriptional activity (Figure 3B). The authors should investigate whether NSP13 could bind to the TEAD binding sequence or the nearby sequence on the genome to modulate TEAD's DNA binding ability. Third, regarding the identified nuclear repressors, the authors should validate the interaction of NSP13 with the ones whose loss activates YAP transcriptional activity (Figure 4G). Lastly, why can't NSP13 bind TEAD4 in the cytoplasmic fractionation if both NSP13 and TEAD4 are detected there (Figure 3B)? This finding indicates their interaction is not a direct protein-protein interaction but is mediated by something in the nucleus, such as genomic DNA.

Reviewer #1 (Public Review):

Yun et al. examined the molecular and neuronal underpinnings of changes in Drosophila female reproductive behaviors in response to social cues. Specifically, the authors measure the ejaculate-holding period, which is the amount of time females retain male ejaculate after mating (typically 90 min in flies). They find that female fruit flies, Drosophila melanogaster, display shorter holding periods in the presence of a native male or male-associated cues, including 2-Methyltetracosane (2MC) and 7-Tricosene (7-T). They further show that 2MC functions through Or47b olfactory receptor neurons (ORNs) and the Or47b channel, while 7-T functions through ppk23 expressing neurons. Interestingly, their data also indicates that two other olfactory ligands for Or47b (methyl laurate and palmitoleic acid) do not have the same effects on the ejaculate-holding period. By performing a series of behavioral and imaging experiments, the authors reveal that an increase in cAMP activity in pC1 neurons is required for this shortening of the ejaculate-holding period and may be involved in the likelihood of remating. This work lays the foundation for future studies on sexual plasticity in female Drosophila.

The conclusions of this paper are supported by the data and the authors have revised the manuscript in accordance with comments of the reviewers. This revised version also contains the expression pattern of the lines used for modulating individual pC1 subtypes. These data and reagents open interesting avenues for future studies on female receptivity and mate choice.

Reviewer #1 (Public Review):

The process of EMT is a major contributor of metastasis and chemoresistance in breast cancer. By using a modified PyMT model that allows identification of cells undergoing EMT and their decedents via S100A4-Cre mediated recombination of the mTmG allele, Ban et al. tackle a very important question of how tumor metastasis and therapy resistance by EMT can be blocked. They identified that pathways associated with ribosome biogenesis (RiBi) are activated during transition cell states. This finding represents a promising therapeutic target to block any transition from E to M (activated during cell dissemination and invasion) as well as from M to E (activated during metastatic colonization). Inhibition of RiBi-blocked EMT also reduced the establishment of chemoresistance that is associated with an EMT phenotype. Hence, RiBi blockage together with standard chemotherapy showed synergistic effects, resulting in impaired colonization/metastatic outgrowth in an animal model. The study is of great interest and of high clinical relevance as the authors show that blocking the transition from E to M or vice versa targets both aspects of metastasis, dissemination form the primary tumor and colonization in distant organs.

The study is done with high skill using state of the art technology and the conclusions are convincing and solid, but some aspects require some additional experimental support and clarification. It remains elusive whether blocking of EMT/MET is necessary for the synergistic effect of standard chemotherapy together with RiBi blockage or whether a general growth disadvantage of RiBi treated cells independent of blocking transition is responsible. How can specific effect on state transition by RiBI block be seperated from global effects attributed to overall reduced protein biosynthesis, proliferation etc.? Some other aspects are misleading or need extension:

In the revised version, the authors appropriately addressed all my comments. I'd like to congratulate the authors for this wonderful work!

Reviewer #2 (Public Review):

This study reports a physical interaction between the kinase DYRK1A and the Tuberous Sclerosis Complex (TSC) protein complex (TSC1, TSC2, TBC1D7). Furthermore, this study demonstrates that DYRK1A, upon interaction with the TSC proteins, regulates mTORC1 activity and cell size. Additionally, this study identifies T1462 on TSC2 as a phosphorylation target of DYRK1A. Finally, the authors demonstrate that DYRK1A impacts cell size using human, mouse and Drosophila cells.

The interaction described here is highly impactful to the field of mTORC1-regulated cell growth and uncovers a previously unrecognized TSC-associated interacting protein. DYRK1A and its regulation of mTORC1 activation may have an impact for multiple diseases in which mTORC1 is hyperactivated.

Reviewer #1 (Public Review):

Summary:

Working memory is imperfect - memories accrue error over time and are biased towards certain identities. For example, previous work has shown memory for orientation is more accurate near the cardinal directions (i.e., variance in responses is smaller for horizontal and vertical stimuli) while being biased towards diagonal orientations (i.e., there is a repulsive bias away from horizontal and vertical stimuli). The magnitude of errors and biases increase the longer an item is held in working memory and when more items are held in working memory (i.e., working memory load is higher). Previous work has argued that biases and errors could be explained by increased perceptual acuity at cardinal directions. However, these models are constrained to sensory perception and do not explain how biases and errors increase over time in memory. The current manuscript builds on this work to show how a two-layer neural network could integrate errors and biases over a memory delay. In brief, the model includes a 'sensory' layer with heterogenous connections that lead to the repulsive bias and decreased error at the cardinal directions. This layer is then reciprocally connected with a classic ring attractor layer. Through their reciprocal interactions, the biases in the sensory layer are constantly integrated into the representation in memory. In this way, the model captures the distribution of biases and errors for different orientations that has been seen in behavior and their increasing magnitude with time. The authors compare the two-layer network to a simpler one-network model, showing that the one model network is harder to tune and shows an attractive bias for memories that have lower error (which is incompatible with empirical results).

Strengths:

The manuscript provides a nice review of the dynamics of items in working memory, showing how errors and biases differ across stimulus space. The two-layer neural network model is able to capture the behavioral effects as well as relate to neurophysiological observations that memory representations are distributed across sensory cortex and prefrontal cortex.

The authors use multiple approaches to understand how the network produces the observed results. For example, analyzing the dynamics of memories in the low-dimensional representational space of the networks provides the reader with an intuition for the observed effects.

As a point of comparison with the two-layer network, the authors construct a heterogenous one-layer network (analogous to a single memory network with embedded biases). They argue that such a network is incapable of capturing the observed behavioral effects but could potentially explain biases and noise levels in other sensory domains where attractive biases have lower errors (e.g., color).

The authors show how changes in the strength of Hebbian learning of excitatory and inhibitory synapses can change network behavior. This argues for relatively stronger learning in inhibitory synapses, an interesting prediction.

The manuscript is well-written. In particular, the figures are well done and nicely schematize the model and the results.

Weaknesses:

Despite its strengths, the manuscript does have some weaknesses. These weaknesses are adequately discussed in the manuscript and motivate future research.

One weakness is that the model is not directly fit to behavioral data, but rather compared to a schematic of behavioral data. As noted above, the model provides insight into the general phenomenon of biases in working memory. However, because the models are not fit directly to data, they may miss some aspects of the data.

In addition, directly fitting the models to behavioral data could allow for a broader exploration of parameter space for both the one-layer and two-layer models (and their alternatives). Such an approach would provide stronger support for the papers claims (such as "....these evolving errors...require network interaction between two distinct modules."). That being said, the manuscript does explore several alternative models and also acknowledges the limitation of not directly fitting behavior, due to difficulties in fitting complex neural network models to data.

One important behavioral observation is that both diffusive noise and biases increase with the number of items in working memory. The current model does not capture these effects and it isn't clear how the model architecture could be extended to capture these effects. That being said, the authors note this limitation in the Discussion and present it as a future direction.

Overall:

Overall, the manuscript was successful in building a model that captured the biases and noise observed in working memory. This work complements previous studies that have viewed these effects through the lens of optimal coding, extending these models to explain the effects of time in memory. In addition, the two-layer network architecture extends previous work with similar architectures, adding further support to the distributed nature of working memory representations.

Reviewer #1 (Public Review):

This work by Stauber et al., is focused on understanding the signaling mechanisms that are associated with tendinopathy development, and by screening a panel of human tendinopathy samples, identified IL-6/JAK/STAT as a potential mediator of this pathology. Using an innovate explant model they delineated the requirement for IL-6 in the main body of the tendon to alter the dynamics of extrinsic fibroblasts. These studies are complemented by in vivo studies that include a Scx-GFP reporter. This approach facilitates examination of the effects of IL6-/- on Scx+ cells, and the differences observed between ex vivo and in vivo contexts.

The use of a publicly available existing dataset is considered a strength, since this dataset includes expression data from several different human tendons experiencing tendinopathy. The revised analysis that includes only non-sheathed tendons facilitates the identification of potentially conserved regulators of the tendinopathy phenotype, with immunostaining for CD90, IL-6R, and IL-6 expression in human tendinopathy samples providing important validation of the transcriptomic studies.

Reviewer #1 (Public Review):

Summary:

In their paper, Hou and co-workers explored the use of a FRET sensor for endogenous g-sec activity in vivo in the mouse brain. They used AAV to deliver the sensor to the brain for neuron specific expression and applied NIR in cranial windows to assess FRET activity; optimizing as well an imaging and segmentation protocol. In brief they observe clustered g-sec activity in neighboring cells arguing for a cell non-autonomous regulation of endogenous g-sec activity in vivo.

Strengths:

Mone.

Weaknesses:

Overall the authors provide a very limited data set and in fact only a proof of concept that their sensor can be applied in vivo. This is not really a research paper, but a technical note. With respect to their observation of clustered activity, they now provide an overview image, next to zoomed details. However, from these images one cannot conclude 'by eye' any clustering event. This aligns with the very low r values. All neurons in the field show variable activity and a clustering is not really evident from these examples. Even within a cluster, there is variability. The authors now confirm that expression levels are indeed variable but are independent from the ratio measurements. Further, they controlled for specificity by including DAPT treatments, but opposite to their own in vitro data (in primary neurons) the ratios increased. The authors argue that both distance and orientation can either decrease or increase ratios and that the use of this biosensor should be explored model-by-model. This doesn't really confer high confidence and may hinder other groups in using this sensor reliably.

Secondly, there is still no physiological relevance for this observation. The experiments are performed in wild-type mice, but it would be more relevant to compare this with a fadPSEN1 KI or a PSEN1cKO model to investigate the contribution of a gain of toxic function or LOF to the claimed cell non-autonomous activations. The authors acknowledge this shortcoming but argue that this is for a follow-up study.

For instance, they only monitor activity in cell bodies, and miss all info on g-sec activity in neurites and synapses: what is the relevance of the cell body associated g-sec and can it be used as a proxy for neuronal g-sec activity? If cells 'communicate' g-sec activities, I would expect to see hot spots of activity at synapses between neurons.

Without some more validation and physiologically relevant studies, it remains a single observation and rather a technical note paper, instead of a true research paper.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

The paper is well written. The biological motivation and the need for such methods are formulated extraordinarily well. The battery of experimental models is impressive.

Weaknesses:

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

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

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

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

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

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

Reviewer #1 (Public Review):

The manuscript by Wang et al is, like its companion paper, very unusual in the opinion of this reviewer. It builds off of the companion theory paper's exploration of the "Wright-Fisher Haldane" model but applies it to the specific problem of diversity in ribosomal RNA arrays. The authors argue that polymorphism and divergence among rRNA arrays are inconsistent with neutral evolution, primarily stating that the amount of polymorphism suggests a high effective size and thus a slow fixation rate, while we, in fact, observe relatively fast fixation between species, even in putatively non-functional regions. They frame this as a paradox in need of solving, and invoke the WFH model.

The same critiques apply to this paper as to the presentation of the WFH model and the lack of engagement with the literature, particularly concerning Cannings models and non-diffusive limits. However, I have additional concerns about this manuscript, which I found particularly difficult to follow.

My first, and most major, concern is that I can never tell when the authors are referring to diversity in a single copy of an rRNA gene compared to when they are discussing diversity across the entire array of rRNA genes. I admit that I am not at all an expert in studies of rRNA diversity, so perhaps this is a standard understanding in the field, but in order for this manuscript to be read and understood by a larger number of people, these issues must be clarified.

The authors frame the number of rRNA genes as roughly equivalent to expanding the population size, but this seems to be wrong: the way that a mutation can spread among rRNA gene copies is fundamentally different than how mutations spread within a single copy gene. In particular, a mutation in a single copy gene can spread through vertical transmission, but a mutation spreading from one copy to another is fundamentally horizontal: it has to occur because some molecular mechanism, such as slippage, gene conversion, or recombination resulted in its spread to another copy. Moreover, by collapsing diversity across genes in an rRNA array, the authors are massively increasing the mutational target size.

For example, it's difficult for me to tell if the discussion of heterozygosity at rRNA genes in mice starting on line 277 is collapsed or not. The authors point out that Hs per kb is ~5x larger in rRNA than the rest of the genome, but I can't tell based on the authors' description if this is diversity per single copy locus or after collapsing loci together. If it's the first one, I have concerns about diversity estimation in highly repetitive regions that would need to be addressed, and if it's the second one, an elevated rate of polymorphism is not surprising, because the mutational target size is in fact significantly larger.

Even if these issues were sorted out, I'm not sure that the authors framing, in terms of variance in reproductive success is a useful way to understand what is going on in rRNA arrays. The authors explicitly highlight homogenizing forces such as gene conversion and replication slippage but then seem to just want to incorporate those as accounting for variance in reproductive success. However, don't we usually want to dissect these things in terms of their underlying mechanism? Why build a model based on variance in reproductive success when you could instead explicitly model these homogenizing processes? That seems more informative about the mechanism, and it would also serve significantly better as a null model, since the parameters would be able to be related to in vitro or in vivo measurements of the rates of slippage, gene conversion, etc.

In the end, I find the paper in its current state somewhat difficult to review in more detail, because I have a hard time understanding some of the more technical aspects of the manuscript while so confused about high-level features of the manuscript. I think that a revision would need to be substantially clarified in the ways I highlighted above.

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 neuros encode cue value/sucrose probability and lick vigor. Direct comparisons of single unit responses between the two regions now utilize linear mixed effects models with random effects for subject,

Weaknesses:

The manuscript still includes mention of differences in effect size or differing "levels" of significance between VP and OT D1 neurons without reports of a direct comparisons between the two populations. This is somewhat mitigated by the comprehensive statistical reporting in the supplemental information, but interpretation of some of these results is clouded by the inclusion of OT D2 neurons in these analyses, and the limited description or contextualization in the main text.

Reviewer #1 (Public Review):

Summary:

This work explored intra and interspecific niche partitioning along spatial, temporal, and dietary niche partitioning between apex carnivores and mesocarnivores in the Qilian Mountain National Park of China, using camera trapping data and DNA metabarcoding sequencing data. They conclude that spatial niche partitioning plays a key role in facilitating the coexistence of apex carnivore species, spatial and temporal niche partitioning facilitate the coexistence of mesocarnivore species, and spatial and dietary niche partitioning facilitate the coexistence between apex and mesocarnivore species. The information presented in this study is important for wildlife conservation and will contribute substantially to the current understanding of carnivore guilds and effective conservation management in fragile alpine ecosystems.

Strengths:

Extensive fieldwork is evident in the study. Aiming to cover a large percentage of the Qilian Mountain National Park, the study area was subdivided into squares, as a geographical reference to distribute the sampling points where the camera traps were placed and the excreta samples were collected.

They were able to obtain many records in their camera traps and collected many samples of excreta. This diversity of data allowed them to conduct robust analyses. The data analyses carried out were adequate to obtain clear and meaningful results that enabled them to answer the research questions posed. The conclusions of this paper are mostly well supported by data.

The study has demonstrated the coexistence of carnivore species in the landscapes of the Qilian Mountains National Park, complementing the findings of previous studies. The information presented in this study is important for wildlife conservation and will contribute substantially to the current understanding of carnivore guilds and effective conservation management in fragile alpine ecosystems.

Weaknesses:

It is necessary to better explain the methodology because it is not clear what is the total sampling effort. In methodology, they only claim to have used 280 camera traps, and in the results, they mention that there are 319 sampling sites. However, the total sampling effort (e.g. total time of active camera traps) carried out in the study and at each site is not specified.

Reviewer #1 (Public Review):

Kainate receptors play various important roles in synaptic transmission. The receptors can be divided into low affinity kainate receptors (GluK1-3) and high affinity kainate receptos (GluK4-5). The receptors can assemble as homomers (GluK1-3) or low-high affinity heteromers (GluK4-5). The functional diversity is further increased by RNA splicing. Previous studies have investigated C-terminal splice variants of GluK1, but GluK1 N-terminal (exon 9) insertions have not been previously characterized. In this study Dhingra et al investigate the functional implications of a GluK1 splice variant that inserts a 15 amino acid segment into the extracellular N-terminal region of the protein using whole-cell and excised outside-out electrophysiology.<br /> The authors convincingly show that the insertion profoundly impacts the function of GluK1-1a - the channels that have the insertion are slower to desensitize. The data also shows that the insertion changes the modulatory effects of Neto proteins, resulting in altered rates of desensitization and recovery from desensitization. To determine the mechanism by which the insertion exerts these functional effects, the authors perform pull-down assays of Neto proteins, and extensive mutagenesis on the insert.<br /> The electrophysiological part of the study is very rigorous and meticulous.

The biggest weakness of the manuscript is the structural work. Due to issues with preferred orientation (a common problem in cryo-EM), the 3D reconstructions are at a low resolution (in the 5-8 Å range) and cannot offer much mechanistic insight into the effects of the insertion. Based on the available data, the authors posit that the insertion does not change the arrangement of the subunits in the desensitized state. However, there is no comparison with a structure that does not contain the insertion, so while the statement may well be true, no data is shown to support it.

Overall, the cryo-EM contributes little and distracts from the good parts of the manuscript.

Another part that does not contribute much is the RNAseq data that has been pulled from a database and analyzed for the paper. It is being used to show that the exon 9 insertion variant is predominantly expressed in the cerebellar cortex at early stages of brain development. The methods do not describe in detail how the data has been analyzed (e.g., is the data scaled per sample/gene or globally?) so it is hard to know what we can compare in the heat plots. In Figure 1- supplement 1 there aren't striking differences in expression (at least not obviously visible in the current illustration).

Despite these weaknesses, the study is a valuable contribution to the field because it characterizes a GluK1 variant that has not been studied before and highlights the functional diversity that exists within the kainate receptor family.

Revised manuscript:

The authors have clarified some of the issues raised by the reviewers, but no new data has been added to strengthen the manuscript. The structural part of the manuscript remains its weakest point, and the extent of mechanistic insight remains low.

Reviewer #1 (Public Review):

Summary:

This study examines lipid profiles in cancer patients treated with the neurotoxic chemotherapy paclitaxel. Multiple methods, including machine learning as well as more conventional statistical modelling, were used to classify lipid patterns before and after paclitaxel treatment and in conjunction with neuropathy status. Lipid profiles before and after paclitaxel therapy were analysed from 31 patients. The study aimed to characterize from the lipid profile if plasma samples were collected pre paclitaxel or post paclitaxel and their relevance to neuropathy status. Sphingolipids including sphinganine-1-phosphate (SA1P) differed between patients with and without neuropathy. To examine the potential role of SA1P, it was applied to murine primary sensory neuron cultures, and produced calcium transients in a proportion of neurons. This response was abolished by application of a TRPV1 antagonist. The number of neurons responding to SA1P was partially reduced by the sphingosine 1-phosphate receptor (S1PR1) modulator fingolimod.

Strengths:

The strengths of this study include the use of multiple methods to classify lipid patterns and the attempt to validate findings from the clinical cohort in a preclinical model using primary sensory neurons.

Weaknesses:

These still stand from the original review and are repeated here:

There are a number of weaknesses in the study. The small sample size is a significant limitation of the study. Out of 31 patients, only 17 patients were reported to develop neuropathy, with significant neuropathy (grade 2/3) in only 5 patients. The authors acknowledge this limitation in the results and discussion sections of the manuscript, but it limits the interpretation of the results. Also acknowledged is the limited method used to assess neuropathy.

Potentially due to this small number of patients with neuropathy, the machine learning algorithms could not distinguish between samples with and without neuropathy. Only selected univariate analyses identified differences in lipid profiles potentially related to neuropathy.

Three sphingolipid mediators including SA1P differed between patients with and without neuropathy at the end of treatment. These sphingolipids were elevated at end of treatment in the cohort with neuropathy, relative to those without neuropathy. However, across all samples from pre to pos- paclitaxel treatment, there was a significant reduction in SA1P levels. It is unclear from the data presented what the underlying mechanism for this result would be. If elevated SA1P is associated with neuropathy development, it would be expected to increase in those who develop neuropathy from pre to post-treatment timepoints.

Primary sensory neuron cultures were used to examine the effects of SA1P application. SA1P application produced calcium transients in a small proportion of sensory neurons. It is not clear how this experimental model assists in validating the role of SA1P in neuropathy development as there is no assessment of sensory neuron damage or other hallmarks of peripheral neuropathy. These results demonstrate that some sensory neurons respond to SA1P and that this activity is linked to TRPV1 receptors. However, further studies will be required to determine if this is mechanistically related to neuropathy.

Impact:

Taken in total, the data presented do not provide sufficient evidence to support the contention that SA1P has an important role in paclitaxel induced peripheral neuropathy. Further, the results do not provide evidence to support the use of S1PR1 receptor antagonists as a therapeutic strategy. It is important to be careful with language use in the discussion, as the significance of the present results are overstated.

However, based on the results of previous studies, it is likely that sphingolipid metabolism plays a role in chemotherapy induced peripheral neuropathy. Based on this existing evidence, the S1PR1 receptor antagonist fingolimod has already been examined in experimental models and in clinical trials. Further work is needed to examine the links between lipid mediators and neuropathy development and identify additional strategies for intervention.

Reviewer #2 (Public Review):

Summary:

The authors performed a Multi-Omics Factor Analysis (MOFA) on analysis of two published MDS patient cohorts-1 from bone marrow mononuclear cells (BMMNCs) and CD34 cells (ref 17) and another from CD34+ cells (ref 15) --with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, Retrotransposon (RTE) expression, and cell- type composition, were derived from these modalities to attempt to identify the latent factors with significant impact on MDS prognosis.

SF3B1 was found to be the only mutation among 13 mutations in the BMMNC cohort that indicated a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34+ cohort. The MOFA factor representing inflammation showed a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases showed a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Also, MOFA identified RTE expression as a risk factor for MDS. They proposed that this work showed the efficacy of their integrative approach to assess MDS prognostic risk that 'goes beyond all the scoring systems described thus far for MDS'.

Reviewer #1 (Public Review):<br /> The authors present the cryo-EM structure of of PSI-fucoxanthin chlorophyll a/c-binding proteins (FCPs) supercomplex from the diatom Thalassiosira pseudonana CCMP1335 at a global resolution of 2.3 Å. This exceptional resolution allows the authors to construct a near-atomic model of the entire supercomplex and elucidate the molecular details of FCPs arrangement. The high-resolution structure reveals subunits not previously identified in earlier reconstructions and models, as well as sequence analysis of PSI-FCPIs from other diatoms and red algae. Additionally, the authors use their model in conjunction with a phylogenetic analysis to compare and contrast the structural features of the T. pseudonana supercomplex with those of Chaetoceros gracilis, uncovering key structural features that contribute to the efficiency of light energy conversion in diatoms.

The study employs the advanced technique of single particle cryo-electron microscopy to visualize the complex architecture of the PSI supercomplex at near-atomic resolution and analyze the specific roles of FCPs in enhancing photosynthetic performance in diatoms.

Overall, the approach and data are both compelling and of high quality. The paper is well written and will be of wide interest for comprehending the molecular mechanisms of photosynthesis in diatoms. This work provides valuable insights for applications in bioenergy, environmental conservation, plant physiology, and membrane protein structural biology.

Reviewer #1 (Public Review):

Summary:

The study by Wu et al presents interesting data on bacterial cell organization, a field that is progressing now, mainly due to the advances in microscopy. Based mainly on fluorescence microscopy images, the authors aim to demonstrate that the two structures that account for bacterial motility, the chemotaxis complex and the flagella, colocalize to the same pole in Pseudomonas aeruginosa cells and to expose the regulation underlying their spatial organization and functioning.

Strengths:

The subject is of importance.

Weaknesses:

The conclusions are too strong for the presented data. The lack of statistical analysis makes this paper incomplete. The novelty of the findings is not clear.

Major issues:

(1) The novelty is in question since in the Abstract the authors highlight their main finding, which is that both the chemotaxis complex and the flagella localize to the same pole, as surprising. However, in the Introduction they state that "pathway-related receptors that mediate chemotaxis, as well as the flagellum are localized at the same cell pole17,18". I am not a pseudomonas researcher and from my short glance at these references, I could not tell whether they report colocalization of the two structures to the same pole. However, I trust the authors that they know the literature on the localization of the chemotaxis complex and flagella in their organism. See also major issue number 5 on the novelty regarding the involvement of c-di-GMP.

(2) Statistics for the microscopy images, on which most conclusions in this manuscript are based, are completely missing. Given that most micrographs present one or very few cells, together with the fact that almost all conclusions depend on whether certain macromolecules are at one or two poles and whether different complexes are in the same pole, proper statistics, based on hundreds of cells in several fields, are absolutely required. Without this information, the results are anecdotal and do not support the conclusions. Due to the importance of statistics for this manuscript, strict statistical tests should be used and reported. Moreover, representative large fields with many cells should be added as supportive information.

The problem is more pronounced when the authors make strong statements, as in lines 157-158: "The results revealed that the chemoreceptor arrays no longer grow robustly at the cell pole (Figure 2A)". Looking at the seven cells shown in Figure 2A, five of them show polar localization of the chemoreceptors. The question is then: what is the percentage of cells that show precise polar, near-polar, or mid cell localization (the three patterns shown here) in the mutant and in the wild type? Since I know that these three patterns can also be observed in WT cells, what counts is the difference, and whether it is statistically significant.

Even for the graphs shown in Figures 3C and 3D, where the proportion of cells with obvious chemoreceptor arrays and absolute fluorescence brightness of the chemosensory array are shown, respectively, the questions that arise are: for how many individual cells these values hold and what is the significance of the difference between each two strains?

(3) The authors conclude that "Motor structural integrity is a prerequisite for chemoreceptor self-assembly" based on the reduction in cells with chemoreceptor clusters in mutants deleted for flagellar genes, despite the proper polar localization of the chemotaxis protein CheY. They show that the level of CheY in the WT and the mutant strains is similar, based on Western blot, which in my opinion is over-exposed. "To ascertain whether it is motor integrity rather than functionality that influences the efficiency of chemosensory array assembly", they constructed a mutant deleted for the flagella stator and found that the motor is stalled while CheY behaves like in WT cells. The authors further "quantified the proportion of cells with receptor clusters and the absolute fluorescence intensity of individual clusters (Figures 3C-D)". While Figure 3DC suggests that, indeed, the flagella mutants show fewer cells with a chemotaxis complex, Figure 3D suggests that the differences in fluorescence intensity are not statistically significant.

Since it is obvious that the regulation of both structures' production and localization is codependent, I think that it takes more than a Western blot to make such a decision.

(4) I wonder why the authors chose to label CheY, which is the only component of the chemotaxis complex that shuttles back and forth to the base of the flagella. In any case, I think that they should strengthen their results by repeating some key experiments with labeled CheW or CheA.

(5) The last section of the results is very problematic, regarding the rationale, the conclusions, and the novelty. As far as the rationale is concerned, I do not understand why the authors assume that "a spatial separation between the chemoreceptors and flagellar motors should not significantly impact the temporal comparison in bacterial chemotaxis". Is there any proof for that? More surprising for me was to read that "The signal transduction pathways in E. coli are relatively simple, and the chemotaxis response regulator CheY-P affects only the regulation of motor switching". There are degrees of complexity among signal transduction pathways in E. coli, but the chemotaxis seems to be ranked at the top. CheY is part of the adaptation. Perfect adaptation, as many other issues related to the chemotaxis pathway, which include the wide dynamic range, the robustness, the sensitivity, and the signal amplification (gain), are still largely unexplained. Hence, such assumptions are not justified.

More perplexing is the novelty of the authors' documentation of the effect of the chemotaxis proteins on the c-di-GMP level. In 2013, Kulasekara et al. published a paper in eLife entitled "c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility". In the same year, Kulasekara published a paper entitled "Insight into a Mechanism Generating Cyclic di-GMP Heterogeneity in Pseudomonas aeruginosa". The authors did not cite these works and I wonder why.

(6) Throughout the manuscript, the authors refer to foci of fluorescent CheY as "chemoreceptor arrays". If anything, these foci signify the chemotaxis complex, not the membrane-traversing chemoreceptors.

Conclusions:

The manuscript addresses an interesting subject and contains interesting, but incomplete, data.

Reviewer #1 (Public Review):

In this manuscript, the authors employ a combined proteomic and genetic approach to identify the glycoprotein QC factor malectin as an important protein involved in promoting coronavirus infection. Using proteomic approaches, they show that the non-structural protein NSP2 and malectin interact in the absence of viral infection, but not in the presence of viral infection. However, both NSP2 and malectin engage the OST complex during viral infection, with malectin also showing reduced interactions with other glycoprotein QC proteins. Malectin KD reduce replication of coronaviruses, including SARS-COV2. Collectively, these results identify Malectin as a glycoprotein QC protein involved in regulating coronavirus replication that could potentially be targeted to mitigate coronavirus replication.

Overall, the experiments described appear well performed and the interpretations generally reflect the results. Moreover, this work identifies Malectin as an important pro-viral protein whose activity could potentially be therapeutically targeted for the broad treatment of coronavirus infection. However, there are some weaknesses in the work that, if addressed, would improve the impact of the manuscript.

Notably, the mechanism by which malectin regulates viral replication is not well described. It is clear from the work that malectin is a pro-viral protein in the work presented, but the mechanistic basis of this activity is not pursued. Some potential mechanisms are proposed in the discussion, but the manuscript would be strengthened if additional insight was included. For example, does the UPR activated to higher levels in infected cells depleted of malectin? Do glycosylation patterns of viral (or non-viral) proteins change in malectin-depleted cells? Additional insight into this specific question would significantly improve the manuscript.

Further, the evidence for increased interactions between OST and malectin during viral infection is fairly weak, despite being a major talking point throughout the manuscript. The reduced interactions between malectin and other glycoproteostasis QC factors is evident, but the increased interactions with OST are not well supported. I'd recommend backing off on this point throughout the text, instead, continuing to highlight the reduced interactions.

I was also curious as to why non-structural proteins, nsp2 and nsp4, showed robust interactions with host proteins localized to both the ER and mitochondria? Do these proteins localize to different organelles or do these interactions reflect some other type of dysregulation? It would be useful to provide a bit of speculation on this point.

Again, the overall identification of malectin as a pro-viral protein involved in the replication of multiple different coronaviruses is interesting and important, but additional insights into the mechanism of this activity would strengthen the overall impact of this work.

Reviewer #1 (Public Review):

EnvA-pseudotyped glycoprotein-deleted rabies virus has emerged as an essential tool for tracing monosynaptic inputs to genetically defined neuron populations in the mammalian brain. Recently, in addition to the SAD B19 rabies virus strain first described by Callaway and colleagues in 2007, the CVS N2c rabies virus strain has become popular due to its low toxicity and high trans-synaptic transfer efficiency. However, despite its widespread use in the mammalian brain, particularly in mice, the application of this cell-type-specific monosynaptic rabies tracing system in zebrafish has been limited by low labeling efficiency and high toxicity. In this manuscript, the authors aimed to develop an efficient retrograde monosynaptic rabies-mediated circuit mapping tool for larval zebrafish. Given the translucent nature of larval zebrafish, whole-brain neuronal activities can be monitored, perturbed, and recorded over time. Introducing a robust circuit mapping tool for larval zebrafish would enable researchers to simultaneously investigate the structure and function of neural circuits, which would be of significant interest to the neural circuit research community. Furthermore, the ability to track rabies-labeled cells over time in the transparent brain could enhance our understanding of the trans-synaptic retrograde tracing mechanism of the rabies virus.

To establish an efficient rabies virus tracing system in the larval zebrafish brain, the authors conducted meticulous side-by-side experiments to determine the optimal combination of trans-expressed rabies G proteins, TVA receptors, and recombinant rabies virus strains. Consistent with observations in the mouse brain, the CVS N2c strain trans-complemented with N2cG was found to be superior to the SAD B19 combination, offering lower toxicity and higher efficiency in labeling presynaptic neurons. Additionally, the authors tested various temperatures for the larvae post-virus injection and identified 36{degree sign}C as the optimal temperature for improved virus labeling. They then validated the system in the cerebellar circuits, noting evolutionary conservation in the cerebellar structure between zebrafish and mammals. The monosynaptic inputs to Purkinje cells from granule cells were neatly confirmed through ablation experiments.

However, there are a couple of issues that this study should address. Additionally, conducting some extra experiments could provide valuable information to the broader research field utilizing recombinant rabies viruses as retrograde tracers.

(1) It was observed that many radial glia were labeled, which casts doubt on the specificity of trans-synaptic spread between neurons. The issues of transneuronal labeling of glial cells should be addressed and discussed in more detail. In this manuscript, the authors used a transgenic zebrafish line carrying a neuron-specific Cre-dependent reporter and EnvA-CVS N2c(dG)-Cre virus to avoid the visualization of virally infected glial cells. However, this does not solve the real issue of glial cell labeling and the possibility of a non-synaptic spread mechanism.

In addition, wrong citations in Line 307 were made when referring to previous studies discovering the same issue of RVdG-based transneuronal labeling radial glial cells.

"The RVdG-based transneuronal labeling of radial glial cells was commonly observed in larval zebrafish29,30".

The cited work was conducted using vesicular stomatitis virus (VSV). A more thorough analysis and/or discussion on this topic should be included. Several key questions should be addressed:

Does the number of labeled glial cells increase over time?<br /> Do they increase at the same rate over time as labeled neurons?<br /> Are the labeled glial cells only present around the injection site?<br /> Can the phenomenon of transneuronal labeling of radial glial cells be mitigated if the tracing is done in slightly older larvae?<br /> What is the survival rate of the infected glial cells over time?<br /> If an infected glial cell dies due to infection or gets ablated, does the rabies virus spread from the dead glial cells?<br /> If TVA and rabies G are delivered to glial cells, followed by rabies virus injection, will it lead to the infection of other glial cells or neurons?

Answers to any of these questions could greatly benefit the broader research community.

(2) The optimal virus tracing effect has to be achieved by raising the injected larvae at 36C. Since the routine temperature of zebrafish culture is around 28C, a more thorough characterization of the effect on the health of zebrafish should be conducted.

(3) Given the ability of time-lapse imaging of the infected larval zebrafish brain, the system can be taken advantage of to tackle important issues of rabies virus tracing tools.<br /> a) Toxicity.<br /> The toxicity of rabies viruses is an important issue that limits their application and affects the interpretation of traced circuits. For example, if a significant proportion of starter cells die before analysis, the traced presynaptic networks cannot be reliably assigned to a "defined" population of starter cells. In this manuscript, the authors did an excellent job of characterizing the effects of different rabies strains, G proteins derived from various strains, and levels of G protein expression on starter cell survival. However, an additional parameter that should be tested is the dose of rabies virus injection. The current method section states that all rabies virus preparations were diluted to 2x10^8 infection units per ml, and 2-5 nl of virus suspension was injected near the target cells. It would be interesting to know the impact of the dose/volume of virus injection on retrograde tracing efficiency and toxicity. Would higher titers of the virus lead to more efficient labeling but stronger toxicities? What would be the optimal dose/volume to balance efficiency and toxicity? Addressing these questions would provide valuable insights and help optimize the use of rabies viruses for circuit tracing.

b) Primary starters and secondary starters:<br /> Given that the trans-expression of TVA and G is widespread, there is the possibility of coexistence of starter cells from the initial infection (primary starters) and starter cells generated by rabies virus spreading from the primary starters to presynaptic neurons expressing G. This means that the labeled input cells could be a mixed population connected with either the primary or secondary starter cells.

It would be immensely interesting if time-lapse imaging could be utilized to observe the appearance of such primary and secondary starter cells. Assuming there is a time difference between the initial appearance of these two populations, it may be possible to differentiate the input cells wired to these populations based on a similar temporal difference in their initial appearance. This approach could provide valuable insights into the dynamics of rabies virus spread and the connectivity of neural circuits.

Reviewer #1 (Public Review):

Summary:

Wang, Y. et al. used a silicone wire embolus to definitively and acutely clot the pterygopalatine ophthalmic artery in addition to carotid artery ligation to completely block blood supply to the mouse inner retina, which mimic clinical acute retinal artery occlusion. A detailed characterization of this mouse model determined the time course of inner retina degeneration and associated functional deficits, which closely mimic human patients. Whole retina transcriptome profiling and comparison revealed distinct features associated with ischemia, reperfusion, and different model mechanisms. Interestingly and importantly, this team found a sequential event including reperfusion-induced leukocyte infiltration from blood vessels, residual microglial activation, and neuroinflammation that may lead to neuronal cell death.

Strengths:

Clear demonstration of the surgery procedure with informative illustrations, images, and superb surgical videos.<br /> Two time points of ischemia and reperfusion were studied with convincing histological and in vivo data to demonstrate the time course of various changes in retinal neuronal cell survivals, ERG functions, and inner/outer retina thickness.<br /> The transcriptome comparison among different retinal artery occlusion models provides informative evidence to differentiate these models.<br /> The potential applications of the in vivo retinal ischemia-reperfusion model and relevant readouts demonstrated by this study will certainly inspire further investigation of the dynamic morphological and functional changes of retinal neurons and glial cell responses during disease progression and before and after treatments.

Weaknesses:

It would be beneficial to the manuscript and the readers if the authors could improve the English of this manuscript by correcting obvious grammar errors, eliminating many of the acronyms that are not commonly used by the field, and providing a reason why this complicated but clever surgery procedure was designed and a summary table with time course of all the morphological, functional, cellular, and transcriptome changes associated with this model.

Reviewer #1 (Public Review):

Summary:

Satoshi Yamashita et al., investigate the physical mechanisms driving tissue bending using the cellular Potts Model, starting from a planar cellular monolayer. They argue that apical length-independent tension control alone cannot explain bending phenomena in the cellular Potts Model, contrasting with previous works, particularly Vertex Models. They conclude that an apical elastic term, with zero rest value (due to endocytosis/exocytosis), is necessary to achieve apical constriction, and that tissue bending can be enhanced by adding a supracellular myosin cable. Additionally, a very high apical elastic constant promotes planar tissue configurations, opposing bending.

Strengths:

- The finding of the required mechanisms for tissue bending in the cellular Potts Model provides a natural alternative for studying bending processes in situations with highly curved cells.<br /> - Despite viewing cellular delamination as an undesired outcome in this particular manuscript, the model's capability to naturally allow T1 events might prove useful for studying cell mechanics during out-of-plane extrusion.

Weaknesses:

- The authors claim that the cellular Potts Model (CPM) is unable to achieve the results of the vertex model (VM) simulations due to naturally non-straight cellular junctions in the CPM versus the VM. The lack of a substantial comparison undermines this assertion. None of the references mentioned in the manuscript are from a work using vertex model with straight cellular junctions, simulating apical constriction purely by a enhancing a length-independent apical tension. Sherrard et al and Pérez-González et al. use 2D and 3D Vertex Models, respectively, with a "contractility" force driving apical constriction. However, their models allow cell curvature. Both references suggest that the cell side flexibility of the CPM shouldn't be the main issue of the "contractility model" for apical constriction.<br /> - The myosin cable is assumed to encircle the invaginated cells. Therefore, it is not clear why the force acts over the entire system (even when decreasing towards the center), and not locally in the contour of the group of cells under constriction. The specific form of the associated potential is missing. It is unclear how dependent the results of the manuscript are on these not-well-motivated and model-specific rules for the myosin cable.<br /> - The authors are using different names than the conventional ones for the energy terms. Their current attempt to clarify what is usually done in other works might lead to further confusion.

Reviewer #1 (Public Review):

Summary:

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 contribute to seizures is important for future therapeutic strategies. The work by Jain et al., demonstrates that increasing adult-born neurons (not increasing adult neurogenesis because BrdU birthdating was not performed in this study) before status epilepticus (SE) leads to a suppression in chronic seizures in the pilocarpine model of temporal lobe epilepsy. This work is potentially interesting because previous studies showed suppressing adult-born neurons led to reduced chronic seizures.

To increase adult-born neurons, 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. (although this was not shown in this study). After 6 weeks of tamoxifen injection, the authors subject 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, the authors examine chronic seizure number and frequency, seizure duration, postictal depression, and seizure distribution/cluster seizures for 3 weeks after pilocarpine. Overall, the study concludes that increasing adult-born neurons in the normal adult brain can reduce epilepsy in females specifically.

Strengths:

(1) The study is sex matched and reveals differences in response to increasing adult-born neurons in chronic seizures between male and females.

(2) The EEG recording parameters are stringent, and analysis of chronic seizures is comprehensive. In two separate experiments, the electrodes were implanted to record EEG from cortex as well as hippocampus. The recording is 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:

(1) Increased DCX alone (without birthdating with BrdU) could indicate increased survival of adult-born neurons, not proliferation or birth of newborn neurons per se. While prior work has demonstrated that tamoxifen injection in adult mice showed an increase in dentate gyrus neurogenesis based on studies of BrdU, Ki67, and DCX (Sahay et al., 2011), the dynamics of adult-born neurons (proliferation, differentiation, and/or survival) could be different in epileptic (pilocarpine-treated) animals. Other stages, e.g., proliferation of neural precursors or maturation of adult-born dentate granule cells, was not examined. Analysis of additional stages of adult neurogenesis may reveal additional cellular understanding and add impact of the work on the field.

Reviewer #1 (Public Review):

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

Major strengths:

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

Reviewer #1 (Public Review):

Summary:

This paper reports an intracranial SEEG study of speech coordination, where participants synchronize their speech output with a virtual partner that is designed to vary its synchronization behavior. This allows the authors to identify electrodes throughout the left hemisphere of the brain that have activity (both power and phase) that correlates with the degree of synchronization behavior. They find that high-frequency activity in the secondary auditory cortex (superior temporal gyrus) is correlated to synchronization, in contrast to primary auditory regions. Furthermore, activity in the inferior frontal gyrus shows a significant phase-amplitude coupling relationship that is interpreted as compensation for deviation from synchronized behavior with the virtual partner.

Strengths:

(1) The development of a virtual partner model trained for each individual participant, which can dynamically vary its synchronization to the participant's behavior in real-time, is novel and exciting.

(2) Understanding real-time temporal coordination for behaviors like speech is a critical and understudied area.

(3) The use of SEEG provides the spatial and temporal resolution necessary to address the complex dynamics associated with the behavior.

(4) The paper provides some results that suggest a role for regions like IFG and STG in the dynamic temporal coordination of behavior both within an individual speaker and across speakers performing a coordination task.

Weaknesses:

(1) The main weakness of the paper is that the results are presented in a largely descriptive and vague manner. For instance, while the interpretation of predictive coding and error correction is interesting, it is not clear how the experimental design or analyses specifically support such a model, or how they differentiate that model from the alternatives. It's possible that some greater specificity could be achieved by a more detailed examination of this rich dataset, for example by characterizing the specific phase relationships (e.g., positive vs negative lags) in areas that show correlations with synchronization behavior. However, as written, it is difficult to understand what these results tell us about how coordination behavior arises.

(2) In the results section, there's a general lack of quantification. While some of the statistics reported in the figures are helpful, there are also claims that are stated without any statistical test. For example, in the paragraph starting on line 342, it is claimed that there is an inverse relationship between rho-value and frequency band, "possibly due to the reversed desynchronization/synchronization process in low and high frequency bands". Based on Figure 3, the first part of this statement appears to be true qualitatively, but is not quantified, and is therefore impossible to assess in relation to the second part of the claim. Similarly, the next paragraph on line 348 describes optimal clustering, but statistics of the clustering algorithm and silhouette metric are not provided. More importantly, it's not entirely clear what is being clustered - is the point to identify activity patterns that are similar within/across brain regions? Or to interpret the meaning of the specific patterns? If the latter, this is not explained or explored in the paper.

(3) Given the design of the stimuli, it would be useful to know more about how coordination relates to specific speech units. The authors focus on the syllabic level, which is understandable. But as far as the results relate to speech planning (an explicit point in the paper), the claims could be strengthened by determining whether the coordination signal (whether error correction or otherwise) is specifically timed to e.g., the consonant vs the vowel. If the mechanism is a phase reset, does it tend to occur on one part of the syllable?

(4) In the discussion the results are related to a previously-described speech-induced suppression effect. However, it's not clear what the current results have to do with SIS, since the speaker's own voice is present and predictable from the forward model on every trial. Statements such as "Moreover, when the two speech signals come close enough in time, the patient possibly perceives them as its own voice" are highly speculative and apparently not supported by the data.

(5) There are some seemingly arbitrary decisions made in the design and analysis that, while likely justified, need to be explained. For example, how were the cutoffs for moderate coupling vs phase-shifted coupling (k ~0.09) determined? This is noted as "rather weak" (line 212), but it's not clear where this comes from. Similarly, the ROI-based analyses are only done on regions "recorded in at least 7 patients" - how was this number chosen? How many electrodes total does this correspond to? Is there heterogeneity within each ROI?

Reviewer #1 (Public Review):

Summary:

Johnston and Smith used linear electrode arrays to record from small populations of neurons in the superior colliculus (SC) of monkeys performing a memory-guided saccade (MGS) task. Dimensionality reduction (PCA) was used to reveal low-dimensional subspaces of population activity reflecting the slow drift of neuronal signals during the delay period across a recording session (similar to what they reported for parts of the cortex: Cowley et al., 2020). This SC drift was correlated with a similar slow-drift subspace recorded from the prefrontal cortex, and both slow-drift subspaces tended to be associated with changes in arousal (pupil size). These relationships were driven primarily by neurons in superficial layers of the SC, where saccade sensitivity/selectivity is typically reduced. Accordingly, delay-period modulations of both spiking activity and pupil size were independent of saccade-related activity, which was most prevalent in deeper layers of the SC. The authors suggest that these findings provide evidence of a separation of arousal- and motor-related signals. The analysis techniques expand upon the group's previous work and provide useful insight into the power of large-scale neural recordings paired with dimensionality reduction. This is particularly important with the advent of recording technologies which allow for the measurement of spiking activity across hundreds of neurons simultaneously. Together, these results provide a useful framework for comparing how different populations encode signals related to cognition, arousal, and motor output in potentially different subspaces.

The conclusions drawn by this paper, however, are only partially supported by the data. Additional statistical comparisons and clarifications are needed.

Comments:

(1) The authors make fairly strong claims that "arousal-related fluctuations are isolated from neurons in the deep layers of the SC" (emphasis added). This conclusion is based on comparisons between a "slow drift axis", a low-dimensional representation of neuronal drift, and other measures of arousal (Figures 2C, 3) and motor output sensitivity (Figures 2B, 3B). However, the metrics used to compare the slow-drift axis and motor activity were computed during separate task epochs: the delay period (600-1100 ms) and a peri-saccade epoch (25 ms before and after saccade initiation), respectively. As the authors reference, deep-layer SC neurons are typically active only around the time of a saccade. Therefore, it is not clear if the lack of arousal-related modulations reported for deep-layer SC neurons is because those neurons are truly insensitive to those modulations, or if the modulations were not apparent because they were assessed in an epoch in which the neurons were not active. A potentially more valuable comparison would be to calculate a slow-drift axis aligned to saccade onset.

(2) More generally, arousal-related signals may persist throughout multiple different epochs of the task. It would be worthwhile to determine whether similar "slow-drift" dynamics are observed for baseline, sensory-evoked, and saccade-related activity. Although it may not be possible to examine pupil responses during a saccade, there may be systematic relationships between baseline and evoked responses.

(3) The relationships between changes in SC activity and pupil size are quite small (Figures 2C & 5C). Although the distribution across sessions (Figure 2C) is greater than chance, they are nearly 1/4 of the size compared to the PFC-SC axis comparisons. Likewise, the distribution of r2 values relating pupil size and spiking activity directly (Figure 5) is quite low. We remain skeptical that these drifts are truly due to arousal and cannot be accounted for by other factors. For example, does the relationship persist if accounting for a very simple, monotonic (e.g., linear) drift in pupil size and overall firing rate over the course of an individual session?

(4) It is not clear how the final analysis (Figure 6) contributes to the authors' conclusions. The authors perform PCA on: (i) residual spiking responses during the delay period binned according to pupil size, and (ii) spiking responses in the saccade epoch binned according to target location (i.e., the saccade tuning curve). The corresponding PCs are the spike-pupil axis and the saccade tuning axis, respectively. Unsurprisingly, the spike-pupil axis that captures variance associated with arousal (and removes variance associated with saccade direction) was not correlated with a saccade-tuning axis that captures variance associated with saccade direction and omits arousal. Had these measures been related it would imply a unique association between a neuron's preferred saccade direction and pupil control- which seems unlikely. The separation of these axes thus seems trivial and does not provide evidence of a "mechanism...in the SC to prevent arousal-related signals interfering with the motor output." It remains unknown whether, for example, arousal-related signals may impact trial-by-trial changes in neuronal gain near the time of a saccade, or alter saccade dynamics such as acceleration, precision, and reaction time.

Reviewer #1 (Public Review):

Summary:

In the retina, parallel processing of cone photoreceptor output under bright light conditions dissects critical features of our visual environment and is fundamental to visual function. Cone photoreceptor signals are sampled by several types of bipolar cells and passed onto the ganglion cells. At the output of retinal processing, retinal ganglion cells send about 40 different codes of the visual scene to the brain for further processing. In this study, the authors focus on whether subtype-specific differences in the size of synaptic ribbon-associated vesicle pools of bipolar cells contribute to different retinal ganglion cell (RGC) responses. Specifically, inputs to ON alpha RGCs producing transient versus sustained kinetics (ON-S vs. ON-T, respectively) are compared. The authors first demonstrate that ON-S vs. ON-T RGCs are readily identifiable in a whole mount preparation and respond differently to both static and to a spatially uniform, randomly fluctuating (Gaussian noise) light stimulus. Liner-nonlinear (LN) models were used to estimate the transformation between visual input and excitatory synaptic input for each RGCs; these models suggested the presence of transient versus sustained kinetics already in the excitatory inputs to ON-T and ON-S RGCs. Indeed, the authors show that (glutamatergic) excitatory inputs to ON-S vs. ON-T RGCs are of distinct kinetics. The subtypes of bipolar cells providing input to ON-S are known (i.e., type 6 and 7), but the source of excitatory bipolar inputs to ON-T RGCs needed to be determined. In a tedious process, it is elegantly shown here that ON-T RGCs receive most of their excitatory inputs from type 5 and 6 bipolars. Interestingly, the temporal properties of light-evoked responses of type 5, 6, and 7 bipolars recorded from the somas were indistinguishable and rather sustained, suggesting that the origin of transient kinetics of excitatory inputs to ON-T RGCs suggested by the LN model might be found in the processing of visual signals at the bipolar cell axon terminal. Blocking GABA- or glycinergic inhibitory inputs did not alter the light-evoked excitatory input kinetics to ON-T and ON-S RGCs. Two-photon glutamate sensor imaging revealed significantly faster kinetics of light-evoked glutamate signals at ON-T versus ON-S RGCs. Detailed EM analysis of bipolar cell ribbon synapses onto ON-T and ON-S RGCs revealed fewer ribbon-associated vesicles at ON-T synapses, which is consistent with stronger paired-flash depression of light-evoked excitatory currents in ON-T RGCS versus ON-S RGCs. This study suggests that bipolar subtype-specific differences in the size of synaptic ribbon-associated vesicle pools contribute to transient versus sustained kinetics in RGCs.

Strengths:

The use of multiple, state-of-the-art tools and approaches to address the kinetics of bipolar to ganglion cell synapse in an identified circuit.

Weaknesses:

For the most part, the data in the paper support the conclusions, and the authors were careful to try to address questions in multiple ways. Two-photon glutamate sensor imaging experiment showing that blocking GABA- and glycinergic inhibition does not change the kinetics of light-evoked glutamate signals at ON-T RGCs would strengthen the conclusion that bipolar subtype-specific differences in the size of synaptic ribbon-associated vesicle pools contribute to transient versus sustained kinetics in RGCs.

Reviewer #1 (Public Review):

In the study "Re-focusing visual working memory during expected and unexpected memory tests" by Sisi Wang and Freek van Ede, the authors investigate the dynamics of attentional re-orienting within visual working memory (VWM). Utilizing a robust combination of behavioral measures, electroencephalography (EEG), and eye tracking, the research presents a compelling exploration of how attention is redirected within VWM under varying conditions. The research question addresses a significant gap in our understanding of cognitive processes, particularly how expected and unexpected memory tests influence the focus and re-focus of attention. The experimental design is meticulously crafted, enabling a thorough investigation of these dynamics. The figures presented are clear and effectively illustrate the findings, while the writing is concise and accessible, making the complex concepts understandable. Overall, this study provides valuable insights into the mechanisms of visual working memory and attentional re-orienting, contributing meaningfully to the field of cognitive neuroscience. Despite the strengths of the manuscript, there are several areas where improvements could be made.

Microsaccades or Saccades?

In the manuscript, the terms "microsaccades" and "saccades" are used interchangeably. For instance, "microsaccades" are mentioned in the keywords, whereas "saccades" appear in the results section. It is crucial to differentiate between these two concepts. Saccades are large, often deliberate eye movements used for scanning and shifting attention, while microsaccades are small, involuntary movements that maintain visual perception during fixation. The authors note the connection between microsaccades and attention, but it is not well-recognized that saccades are directly linked to attention. Despite the paradigm involving a fixation point, it remains unclear whether large eye movements (saccades) were removed from the analysis. The authors mention the relationship between microsaccades and attention but do not clarify whether large eye movements (saccades) were excluded from the analysis. If large eye movements were removed during data processing, this should be documented in the manuscript, including clear definitions of "microsaccades" and "saccades." If such trials were not removed, the contribution of large eye movements to the results should be shown, and an explanation provided as to why they should be considered.

Alpha Lateralization in Attentional Re-orienting

In the attentional orienting section of the results (Figure 2), the authors effectively present EEG alpha lateralization results with time-frequency plots and topographic maps. However, in the attentional re-orienting section (Figure 3), these visualizations are absent. It is important to note that the time period in attentional orienting differs from attentional re-orienting, and consequently, the time-frequency plots and topographic maps may also differ. Therefore, it may be invalid to compute alpha lateralization without a clear alpha activity difference. The authors should consider including time-frequency plots and topographic maps for the attentional re-orienting period to validate their findings.

Onset and Offset Latency of Saccade Bias

The use of the 50% peak to determine the onset and offset latency of the saccade bias is problematic. For example, if one condition has a higher peak amplitude than another, the standard for saccade bias onset would be higher, making the observed differences between the onset/offset latencies potentially driven by amplitude rather than the latencies themselves. The authors should consider a more robust method for determining saccade bias onset and offset that accounts for these amplitude differences.

Control Analysis for Trials Not Using the Initial Cue

The control analysis for trials where participants did not use the initial cue raises several questions:

(1) The authors claim that "unlike continuous alpha activity, saccades are events that can be classified on a single-trial level." However, alpha activity can also be analyzed at the single-trial level, as demonstrated by studies like "Alpha Oscillations in the Human Brain Implement Distractor Suppression Independent of Target Selection" by Wöstmann et al. (2019). If single-trial alpha activity can be used, it should be included in additional control analyses.

(2) The authors aimed to test whether the re-orienting signal observed after the test is not driven exclusively by trials where participants did not use the initial cue. They hypothesized that "in such a scenario, we should only observe attention deployment after the test stimulus in trials in which participants did not use the preceding retro cue." However, if the saccade bias is the index for attentional deployment, the authors should conduct a statistical test for significant saccade bias rather than only comparing toward-saccade after-cue trials with no-toward-saccade after-cue trials. The null results between the two conditions do not immediately suggest that there is attention deployment in both conditions.

(3) Even if attention deployment occurs in both conditions, the prolonged re-orienting effect could also be caused by trials where participants did not use the initial cue. Unexpected trials usually involve larger and longer brain activity. The authors should perform the same analysis on the time after the removal of trials without toward-saccade after the cue to address this potential confound.

Reviewer #1 (Public Review):

Summary:

Mosshammer et al. studied the oxygenic photosynthetic productivity of beachrock samples containing cyanobacteria with different pigment compositions. The use of longer wavelength absorbing chlorophylls in some cyanobacteria (chlorophylls d and f) allows their photosystems to use light further in the red than canonical chlorophyll a photosystems. As such, their distribution in visible light-shaded environments, such as the beachrock studied by Mosshammer et al., allows them to perform oxygenic photosynthesis using wavelengths not capable of driving photosynthesis in most cyanobacteria, algae, or plants.

By adapting measuring systems they have previously used to study these types of beachrock samples, the authors attempt to mimic a more natural light penetration through the beachrock in order to measure oxygen production. By doing so with different wavelengths and intensities, the authors are able to show that far-red light-driven oxygen production is potentially capable of driving high levels of gross primary production.

Strengths:

The manuscript builds on previous measurement techniques used by the authors while focussing on illumination from the top of a sample rather than the specific microbial layers themselves. This provides a more environmentally realistic understanding of the beachrock community, as well as far-red light-driven photosynthesis.

The manuscript benefits from using previously defined methods to further characterize complex environmental samples.

Weaknesses:

The manuscript suffers from a lack of discussion and interpretation of the findings, and as such is more of a report.

Using the envionmental beachrock samples has inherent complications, from the variation in rock morphology, to the microbial community composition of different samples as well as within a single sample. It would benefit the authors to discuss these technical difficulties in more detail, as the light penetration through the beachrock is likely greatly limiting measurements of chlorophyll f and/or chlorophyll d-driven photosynthesis in the beachrock.

This can be seen in the different luminescence measurements (Figure 2 and supplements), that the different samples have clear differences in far-red light-driven oxygen production. While the BLACK sample produces oxygen with 740nm LED filtered with a NIR-75N filter, neither of the other two samples produce measureable oxygen under this condition. Conversely, this sample results in the lowest level of gross photosynthesis when measuring dissolved oxygen. A more detailed discussion of the variation between and within samples and measurements would benefit the overall results of the manuscript.

The PINK beachrock sample has the highest level of chlorophyll d per chlorophyll a. As FaRLiP cyanobacteria only incorporate 1 chlorophyll d per photosystem II, and none in photosytem I, is there a (relatively) high composition of Acaryochloris species in the PINK sample? If normalized to the reflectance minima can more distinct populations be identified?

For Figure 1, multiple points should be clarified. The first is that the HPLC methods are estimates of concentrations, as the extinction coefficients are not correct for the solvent solution for which the pigments elute, and are likely to be differently incorrect for each pigment. This results in quantitatively incorrect data, but qualitative comparisons between samples likely remain valid. Secondly, the pigment concentrations can also be misleading. Within the cyanobacterial cells, photosystem I harbors approximately 3 times as many chlorophylls as photosystem II. While the community numbers and photosystem stoichiometry are not necessarily relevant to the current study, the red shift in absorbance between photosystem II and photosystem I is of importance for the measurements performed. How cyanobacterial cells with differing concentrations of photosystems will absorb the red tail of the far-red LEDs, as well as impact the light penetration would be a useful discussion point.

The different samples used are from varying beachrock zonations but have the same chlorophyll f per chlorophyll a concentrations. A discussion of why this might be would be useful.

For the luminescence measurements (Figure 2 and supplements), no oxygen production is seen in the BROWN or PINK beachrock samples when the 740nm LED is filtered with a NIR-75N filter. This is likely due to multiple factors (low initial intensity compounded by penetration depth, community composition, etc.) but should be discussed. While the authors say that Chrooccidiopsis species dominate the samples, variation of absorbance between different chlorophyll f containing cyanobacteria has also been measured (see Tros et al. 2021, Chem), and the extent to which even chlorophyll f species extend into the far-red varies. Discussions about these implications would help with their characterization of the luminescence data. While the authors discuss that based on their respiration measurements the oxygen may be being consumed, resulting in an inability to measure it (lines 147-150), other explanations are clearly viable.

For the luminescence measurements, no oxygen production is discernable in the endolithic region when excited with visible light, which is at a much stronger intensity than the near-infrared light used. However, both Acaryochloris and chlorophyll f cyanobacteria are capable of driving photosynthesis with visible light. As the intensities used are much brighter than for the NIR measurements, presumably generated oxygen would be higher than what could be immediately consumed by respiration. It is important that the authors address this.

A highlighted point by the authors is the >20% of photosynthesis driven by NIR in the beachrock at comparable irradiation. However, this statement is deceiving for multiple reasons.<br /> (1) The irradiation is likely not comparable for what is reaching the cells. This is not a problem per se as illumination from above is the point, but does skew the interpretation.<br /> (2) The >20% value comes from the maximum amount of gross photosynthesis driven by NIR at ~1400 umol photons m-2s-1, whereas at other comparable illuminations the value is much, much lower (<1%). A likely interpretation of such data is that while the chlorophyll f endolithic layer is capable of producing a relatively large amount of oxygen, it is likely far less productive under most illuminations, though not zero.

The authors have the difficult task of weaving in results from laboratory, uniculture or isolated photosystem measurements with their environmental-based results. This is especially clear in lines 172-183. While the authors are correct that measurements of trapping times in chlorophyll f containing photosystems have been measured and are slower in chlorophyll f photosystem II and photosystem I relative to all chlorophyll a photosystems, the quantum yield for trapping remains high in chlorophyll f photosystem I (Tros et al. 2021, Chem). The quantum yield of trapping for chlorophyll f photosystem II is much lower for chlorophyll f than chlorophyll a complex, though improved by the attachment of phycobilisomes. However, these are intrinsic physical properties of the complexes that are not modulated in response to the environments. This could be interpreted that at low photon flux densities as measured in these experiments, the endolithic near infrared-driven oxygen production could be limited by an overall lower quantum efficiency of trapping the captured light and thus minimizing photosynthetic productivity relative to a theoretical level based on the efficiency of the chlorophyll a photosystem II. How the variations in intensity and spectral composition impact the cyanobacterial community likely involves many other factors and has not been addressed (though see Nurnberg et al. 2018, Science and Viola et al. 2022 eLife for further discussions).

Reviewer #1 (Public Review):

Summary:

In this work, the authors study whether the human brain uses long term priors (acquired during our lifetime) regarding the statistics of auditory stimuli to make predictions respecting auditory stimuli. This is an important open question in the field of predictive processing.

To address this question, the authors cleverly profit from the naturally existing differences in two linguistic groups. While speakers of Spanish use phrases in which function-words (short words like, articles and prepositions) are followed by content-words (longer words like nouns, adjectives and verbs), speakers of Basque use phrases with the opposite order. Because of this, speakers of Spanish usually hear phrases in which short words are followed by longer words, and speakers of Basque experience the opposite. This difference in the order of short and longer words is hypothesized to result in a long term duration prior that is used to make predictions regarding the likely durations of incoming sounds, even if they are not linguistic in nature.

To test this, the authors used MEG to measure the mismatch responses (MMN) elicited by the omission of short and long tones that were presented in alternation. The authors report an interaction between the language background of the participants (Spanish, Basque) and the type of omission MMN (short, long), which goes in line with their predictions. They supplement these results with a source level analysis.

Strengths:

This work has many strengths. To test the main question, the authors profit from naturally occurring differences in the everyday auditory experiences of two linguistic groups, which allows to test the effect of putative auditory priors consolidated over the years. This is a direct way of testing the effect of long term priors.

The fact that the priors in question are linguistic and that the experiment was conducted using non-linguistic stimuli (i.e. simple tones), allows to test if these long term priors generalize across auditory domains.

The experimental design is elegant and the analysis pipeline appropriate. This work is very well written. In particular the introduction and discussion sections are clear and engaging. The literature review is complete.

Weaknesses:

The authors report a widespread omission response, which resembles the classical mismatch response (in MEG planar gradiometers) with strong activations in sensors over temporal regions. However the interaction reported is circumscribed to four sensors that do not overlap with the peaks of activation of the omission response.

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, expression of Sex-lethal (Sxl) protein determines sexual identity and drives female development. Functional Sxl protein is absent from males where splicing includes a termination codon-containing "poison" exon. Early during development, in the soma of female individuals, Sxl expression is initiated by an X chromosome counting mechanism that activates the Sxl establishment promoter (SxlPE) to produce an initial amount of Sxl protein. This then suppresses the inclusion of the "poison" exon, directing the constructive splicing of Sxl transcripts emerging from the Sxl maintenance promotor (SxlPM) which is activated at a later stage during development irrespective of sex. This autoregulatory loop maintains Sxl expression and commits to female development.

Sxl also determines the sexual identity of the germline. Here Sxl expression generally follows the same principles as in somatic tissues, but the way expression is initiated differs from the soma. This regulation has so far remained elusive.

In the presented manuscript, Goyal et al. show that activation of Sxl expression in the germline depends on additional regulatory DNA sequences, or sequences different from the ones driving initial Sxl expression in the soma. They further demonstrate that sisterless A (sisA), a transcription factor that is required for activation of Sxl expression in the soma, is also necessary, but not sufficient, to initiate the expression of functional Sxl protein in female germ cells. sisA expression precedes Sxl induction in the germline and its ablation by RNAi results in impaired expression of Sxl, formation of ovarian tumors, and germline loss, phenocopying the loss of Sxl. Intriguingly, this phenotype can be rescued by the forced expression of Sxl, demonstrating that the primary function of sisA in the germline is the induction of Sxl expression.

Strengths:

The clever design of probes (for RNA FISH) and reporters allowed the authors to dissect Sxl expression from different promoters to get novel insight into sex-specific gene regulation in the germline. All experiments are carefully controlled. Since Sxl regulation differs between the soma and the germline, somatic tissues provide elegant internal controls in many experiments, ensuring e.g. functionality of the reporters. Similarly, animals carrying newly generated alleles (e.g. genomic tagging of the Sxl locus) are fertile and viable, demonstrating that the genetic manipulation does not interfere with protein function. The conclusions drawn from the experimental data are sound and advance our understanding of how Sxl expression is induced in the female germline.

Weaknesses:

The assays employed by the authors provide valuable information on when Sxl promoters become active. However, since no information on the stability of the gene products (i.e. RNA and protein) is available, it remains unclear when the SxlPE promoter is switched off in the germline (conceptually it only needs to be active for a short time period to initiate production of functional Sxl protein). As correctly stated by the authors, the persisting signals observed in the germline might therefore not reflect the continuous activity of the SxlPE promoter.

Mapping of regulatory elements and their function: SxlPE with 1.5 kb of flanking upstream sequence is sufficient to recapitulate early Sxl expression in the soma. The authors now provide evidence that beyond that, additional DNA sequences flanking the SxlPE promoter are required for germline expression. However, a more precise mapping was not performed. Also, due to technical limitations, the authors could not precisely map the sisA binding sites. Since this protein is also involved in the somatic induction of Sxl, its binding sites likely reside in the region 1.5kb upstream of the SxlPE promoter, which has been reported to be sufficient for somatic regulation. The regulatory role of the sequences beyond SxlPE-1.5kb therefore remains unaddressed and it remains to be investigated which trans-acting factor(s) exert(s) its/their function(s) via this region.

The central question of how Sxl expression is initiated and controlled in the germline still remains unanswered. Since sisA is zygotically expressed in both the male and the female germline (Figure 4D), it is unlikely the factor that restricts Sxl expression to the female germline.

How does weak expression of Sxl in male tissues or expression above background after knockdown of sisA reconcile with the model that an autoregulatory feedback loop enforces constant and clonally inheritable Sxl expression once Sxl is induced? Is the current model for Sxl expression too simple or are we missing additional factors that modulate Sxl expression (such as e.g. Sister of Sex-lethal)? While I do not expect the authors to answer these questions, I would expect them to appropriately address these intriguing aspects in the discussion.

Reviewer #1 (Public Review):

In this manuscript, Liu et al. used scRNA-seq to characterize cell type-specific responses during allergic contact dermatitis (ACD) in a mouse model, specifically the hapten-induced DNFB model. Using the scRNA-seq data, they deconvolved the cell types responsible for the expression of major inflammatory cytokines such as IFNG (from CD4 and CD8 T cells), IL4/13 (from basophils), IL17A (from gd T cells), and IL1B from neutrophils and macrophages. They found the highest upregulation of a type 1 inflammatory response, centering around IFNG produced by CD4 and CD8 T cells. They further identified a subpopulation of dermal fibroblasts (pre-adipocytes found in the dermal white adipose tissue layer) that upregulate CXCL9/10 during ACD and provide functional genetic evidence in their mouse model that disrupting IFNG signaling in fibroblasts decreases CD8 T cell infiltration and overall inflammation. They identify an increase in IFNG-expressing CD8 T cells in human patient samples of ACD vs. healthy control skin and co-localization of CD8 T cells with PDGFRA+ fibroblasts, which suggests this mechanism is relevant to human ACD. This mechanism is reminiscent of recent work showing that IFNG signaling in dermal fibroblasts upregulates CXCL9/10 to recruit CD8 T cells in a mouse model of vitiligo. Overall, this is a well-presented, clear, and comprehensive manuscript. The conclusions of the study are well supported by the data, with thoughtful discussion on study limitations by the authors. One such limitation was the use of one ACD model (DNFB), which prevents an assessment of how broadly relevant this axis is. The human sample validation is limited by the multiplexing capacity of immunofluorescence markers but shows a predominance of CD8+/IFNG+ cells and PDGFRA+/CXCL10+ cells in ACD (which are virtually absent in healthy control), along with co-localization of CD8+ cells with PDGFRA+ cells. Thus, this mechanism is likely active in human ACD.

Strengths:<br /> Through deep characterization of the in vivo ACD model using scRNA-seq, the authors were able to determine which cell types were expressing the major cytokines involved in ACD inflammation, such as IFNG, IL4/13, IL17A, and IL1B. These analyses are well-presented and thoughtful, showing first that the response is IFNG-dominant, then focusing on deeper characterization of lymphocytes, myeloid cells, and fibroblasts, which are also validated and complemented by FACS experiments using canonical markers of these cell types as well as IF staining. Crosstalk analyses from the scRNA-seq data led the authors to focus on IFNG signaling fibroblasts, and in vitro experiments demonstrate that CXCL9 and CXCL10 are expressed by fibroblasts stimulated by IFNG. In vivo functional genetic evidence demonstrates an important role for IFNG signaling in fibroblasts, as KO of Ifngr1 using Pdgfra-Cre Ifngr1 fl/fl mice, showed a reduction in inflammation and CD8 T cell recruitment. Human ACD sample staining demonstrates the likely activity of the CD8 T cell IFNG-driven fibroblast response in human disease.

Weaknesses:<br /> The use of one model limits an understanding of how broad this fibroblast-T cell axis is during ACD. However, the authors chose the most commonly employed model and compared their data to work in a vitiligo model (another type 1 immune response) to demonstrate similar mechanisms at play. Human patient samples of ACD were co-stained with two markers at a time, demonstrating the presence of CD8+IFNG+ T cells, PDGFRA+CXCL10+ fibroblasts, and co-localization of PDGFRA+ fibroblasts and CD8+ T cells. However, no IF staining demonstrates co-expression of all 4 markers at once; thus, the human validation of co-localization of CD8+IFNG+ T cells and PDGFRA+CXCL10+ fibroblasts is ultimately indirect, although more likely than not to be true.

Reviewer #1 (Public Review):

Review after revision

Of note the main results of this article are very similar to the results present in the previous manuscript (same Figures 1 to 9, addition of Figure 10 with no quantification).<br /> Unfortunately, the main weaknesses of the article have not been addressed:

(1) The main findings have been obtained in clones of Jurkat cells. They have not been confirmed in primary T cells. The only experiment performed in primary cells is shown in Figure S7 (primary human T lymphoblasts) for which only the distribution of FMNL1 is shown without quantification. No results presenting the effect of FMNL1 KO and expression of mutants in primary T cells are shown.

(2) Analysis in- depth of the defect in actin remodeling (quantification of the images, analysis of some key actors of actin remodeling) is still lacking. Only F-actin is shown, no attempt to look more precisely at actors of actin remodeling has been done.

(3) The defect in the secretion of extracellular vesicles is still very preliminary. Examples of STED images given by the authors are nice, yet no quantification is performed.

(4) Results shown in Figure S12 on the colocalization of proteins phosphorylated on Ser/Thr are still not convincing. It seems indeed that "phospho-PKC" is labeling more preferentially the CMAC positive cells (Raji) than the Jurkat T cells. It is thus particularly difficult to conclude on the co-localization and even more on the recruitment of phosphorylated-FMNL1 at the IS. Thus, these experiments are not conclusive and cannot be the basis even for their cautious conclusion: "Although all these data did not allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limit of confocal and STED microscopes, the results are compatible with the idea that both endogenous FMNL1 and YFP-FMNL1bWT are specifically phosphorylated at the cIS".

The study would benefit from a more careful statistical analysis. The dot plots showing polarity are presented for one experiment. Yet, the distribution of the polarity is broad. Results of the 3 independent experiments should be shown and a statistical analysis performed on the independent experiments.

Reviewer #1 (Public Review):

Summary:

This paper presents valuable findings that gustation and feeding state influence the preferred environmental temperature preference in flies. Interestingly, the authors showed that by refeeding starved animals with non-nutritive sugar sucralose, they are able to tune their preference towards a higher temperature in addition to nutrient-dependent warm preference. The authors show that temperature sensing and sweet sensing gustatory neurons (SGNs) are involved in the former but not the latter. In addition, their data indicate that peptidergic signals involved in internal state and clock genes are required for taste-dependent warm preference behavior.

The authors made an analogy of their results to the cephalic phase response (CPR) in mammals where the thought, sight and taste of food prepares the animal for the consumption of food and nutrients. The authors showed that taste triggers CPR-induced temperature preference behaviors in flies. The authors also briefly covered that the combined modalities of smell and taste induced CPR responses, showing that starved orco mutant flies failed to recover temperature preference after refeeding with sucralose.

The findings of this work hold promising future research prospects, for example, whether the sight of food influences temperature preference behavior in hungry flies, or whether taste, smell and sight work together or independently in promoting CPR responses.

Futhermore, these valuable behavioral results can be further investigated in flies with the advantage of being able to dissect the neural circuitry underlying CPR and nutrient homeostasis.

Strengths:

(1) The authors convincingly showed that tasting is sufficient to drive warm temperature preference behavior in starved flies and show that it is independent of nutrient-driven warm preference.<br /> (2) By using the genetic manipulation of key internal sensors and genes controlling internal feeding and sleep state such as DH44 neurons and the per genes for eg the authors linked gustation and temperature preference behavior control to the internal state of the animal.

Weaknesses:

Most of the weaknesses of the paper have been addressed in the revision. The points mentioned below are meant to improve readability of the paper and to promote understanding of the significance of the work.<br /> (1) Supplementary fig 1 could replace Figure 1A. The purpose of Figure 1F is not clear to me as the comparison between the different food substances is not separately addressed anywhere in the text.<br /> (2) The data for the orco receptor mutant could be placed in the main figures to justify the discussion emphasising CPR-like responses.

Reviewer #1 (Public Review):

Summary:

This paper uses a model of binge alcohol consumption in mice to examine how the behaviour and its control by a pathway between the anterior insular cortex (AIC) to the dorsolateral striatum (DLS) may differ between males and females. Photometry is used to measure the activity of AIC terminals in the DLS when animals are drinking and this activity seems to correspond to drink bouts in males but not females. The effects appear to be lateralized with inputs to the left DLS being of particular interest.

Strengths:

Increasing alcohol intake in females is of concern and the consequences for substance use disorder and brain health are not fully understood, so this is an area that needs further study. The attempt to link fine-grained drinking behaviour with neural activity has the potential to enrich our understanding of the neural basis of behaviour, beyond what can be gleaned from coarser measures of volumes consumed etc.

Weaknesses:

The introduction to the drinking in the dark (DID) paradigm is rather narrow in scope (starting line 47). This would be improved if the authors framed this in the context of other common intermittent access paradigms and gave due credit to important studies and authors that were responsible for the innovation in this area (particularly studies by Wise, 1973 and returned to popular use by Simms et al 2010 and related papers; e.g., Wise RA (1973). Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia 29: 203-210; Simms, J., Bito-Onon, J., Chatterjee, S. et al. Long-Evans Rats Acquire Operant Self-Administration of 20% Ethanol Without Sucrose Fading. Neuropsychopharmacol 35, 1453-1463 (2010).) The original drinking in the dark demonstrations should also be referenced (Rhodes et al., 2005). Line 154 Theile & Navarro 2014 is a review and not the original demonstration.

When sex differences in alcohol intake are described, more care should be taken to be clear about whether this is in terms of volume (e.g. ml) or blood alcohol levels (BAC, or at least g/kg as a proxy measure). This distinction was often lost when lick responses were being considered. If licking is similar (assuming a single lick from a male and female brings in a similar volume?), this might mean males and females consume similar volumes, but females due to their smaller size would become more intoxicated so the implications of these details need far closer consideration. What is described as identical in one measure, is not in another.

While the authors have some previous data on the AIC to DLS pathway, there are many brain regions and pathways impacted by alcohol and so the focus on this one in particular was not strongly justified. Since photometry is really an observational method, it's important to note that no causal link between activity in the pathway and drinking has been established here.

It would be helpful if the authors could further explain whether their modified lickometers actually measure individual licks. While in some systems contact with the tongue closes a circuit which is recorded, the interruption of a photobeam was used here. It's not clear to me whether the nose close to the spout would be sufficient to interrupt that beam, or whether a tongue protrusion is required. This detail is important for understanding how the photometry data is linked to behaviour. The temporal resolution of the GCaMP signal is likely not good enough to capture individual links but I think more caution or detail in the discussion of the correspondence of these events is required.

Even if the pattern of drinking differs between males and females, the use of the word "strategy" implies a cognitive process that was never described or measured.

Reviewer #1 (Public Review):

Summary:

This paper introduces an efficient approach to infer properties of receptive-field subunits from the ensemble of spike-triggered stimuli. This is an important general problem in sensory coding. The results introduced in the paper make a solid contribution to both how subunits can be identified and how subunits of different types are coordinated in space.

Strengths:

A primary strength of the paper is the development of approaches that substantially speed non-negative matrix factorization and by doing so create an opportunity for a more systematic exploration of how the procedure depends on various control parameters. The improved procedure is well documented and the direct comparisons with previous procedures are helpful. The improved efficiency enabled several improvements in the procedure - notably tests of good procedures for initializing NNMF and tests of the dependence of the results on the sparsity regularization parameter.

A second strength of the paper is the exploration of the spatial relationship between different subunits. This, to my knowledge, is new and is an interesting direction. There are some concerns about this analysis (see weaknesses below), but if this analysis can be strengthened it will provide new information that will be important both functionally and developmentally.

Weaknesses:

A primary concern is that choices made about parameters for several aspects of the analysis appear to be made subjectively. Much of this centers around how much of the structure in the extracted subunits is imposed by the procedure itself, and how much reflects the underlying neural circuitry. Some specific issues related to this concern are:

- Sparsity: the use of the autocorrelation function to differentiate real vs spurious subunits should be documented and validated. For example, can the authors split data in half and show that the real subunits are stable?

- Choice of regularization: the impact of the regularization parameter on subunit properties is nicely documented. However, the choice of an appropriate regularization parameter seems somewhat arbitrary. Line 253-256 is an example of this problem: this sentence sounds circular - as if the sparsity factor was turned up until the authors obtained what they expected to obtain. Could the choice of this parameter significantly impact the properties of the extracted subunits? How sensitive are the subunit properties to that parameter? Some additional control analyses are needed to validate the parameter choice (see the crossvalidation comment below).

- Crossvalidation was not used to identify the regularization constraint value because the weight matrix from NNMF does not generalize beyond the data it was fit to. Could the authors instead hold the components matrix fixed and recompute the weight matrix, and use that approach for cross-validation (especially since it is really the components matrix that needs validating)?

The paper would benefit from a more complete comparison with known anatomy. For example, can the authors estimate the number of cones within each subunit? This is well-constrained both anatomically (at least in macaque) and, especially for midget ganglion cell subunits, functionally. In macaque, most midget bipolar cells get input from single cones, so the number of extracted subunits should be close to the number of cones. This would be a useful point of comparison for the current work.

Is the analysis of the spatial relationship between different subunit mosaics robust to the incompleteness of those mosaics? The argument on lines 496-503 should be backed up by more analysis. For example, if subunits are removed from regions where the mosaic is pretty complete, do the authors change the spatial dependence? Alternatively, could they use synthetic mosaics with properties like those measured to check the sensitivity to missing cells?

NNMF relies on accounting for each spike-triggered stimulus with a linear combination of components. Would nonlinearities - e.g. those in the bipolar cell outputs - substantially change the results?

Does the approach work for cells that receive input from multiple bipolar types? Some ganglion cells, e.g. in mice, receive input from multiple bipolar types, each accounting for a sizable percentage of the total input. There is similar anatomical work indicating that parasol cells may receive input from multiple diffuse bipolar types. It is not clear whether the current approach works in cases where the subunits of a single ganglion cell overlap. Some discussion of this would be useful.

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 describe how mutations and deletions that disrupt inter-subunit interactions at the cytoplasmic gating ring assembly affect Kv10.1 channel gating and modulation by CaM. In the revised manuscript, additional information is provided to allow readers to identify within the Kv10.1 channel structure the location of E600R, one of the key channel mutants analyzed in this study. However, the mechanistic role of the cytoplasmic domains that this study focuses on, as well as the location of the ΔPASCap deletion and other perturbations investigated in the study remain difficult to visualize without additional graphical information.

The authors focused mainly on two structural perturbations that disrupt interactions within the cytoplasmic domain, the E600R mutant and the ΔPASCap deletion. 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 conductance-voltage (G-V) relations and exhibit activation and deactivation kinetics with multiple voltage-dependent components. Importantly, the mutant-specific component in the G-V relations is observed at negative voltages where WT channels remain closed. The authors argue that the biphasic behavior in the G-V relations is unlikely to result from two different populations of channels in the oocytes, because they found that the relative amplitude between the two components in the G-V relations was highly reproducible across individual oocytes that otherwise tend to show high variability in expression levels. Instead, the G-V relations for all mutant channels could be well described by an equation that considers two open states O1 and O2, and a transition between them; O1 appeared to be unaffected by any of the structural manipulations tested (i.e. E600R, ΔPASCap, and other deletions) whereas the parameters for O2 and the transition between the two open states were different between constructs. The O1 state is not observed in WT channels and is hypothesized to be associated with voltage sensor activation. O2 represents the open state that is normally observed in WT channels and is speculated to be associated with conformational changes within the cytoplasmic gating ring that follow voltage sensor activation, which could explain why the mutations and deletions disrupting cytoplasmic interactions affect primarily O2.

Severing the covalent link between the voltage sensor and pore reduced O1 occupancy in one of the deletion constructs. Although this observation is consistent with the hypothesis that voltage-sensor activation drives entry into O1, this result is not conclusive. 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 covalent linker between the sensor and the pore, and thus the severed construct could still retain coupling through other mechanisms, which is consistent with the prominent voltage dependence that is observed. If both states O1 and O2 require voltage sensor activation, it is unclear why the severed construct would affect state O1 primarily, as suggested in the manuscript, as opposed to decreasing occupancy of both open states. In line with this argument, the presence of Mg2+ in the extracellular solution affected both O1 and O2. This finding suggests that entry into both O1 and O2 requires voltage-sensor activation because Mg2+ ions are known to stabilize the voltage sensor in its most deactivated conformations.

Activation towards and closure from O1 is slow, whereas channels close rapidly from O2. A rapid alternating pulse protocol was 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 towards 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 O1 has a larger macroscopic conductance than O2. In the revised manuscript, the authors performed single-channel recordings to determine why O1 and O2 have different macroscopic conductance. The results show that at voltages where the state O1 predominates, channels exhibited longer open times and overall higher open probability, whereas at more depolarized voltages where occupancy of O2 increases, channels exhibited more flickery gating behavior and decreased open probability. These results are informative but not conclusive since single-channel amplitudes could not be resolved at strong depolarizations, limiting the extent to which the data could be analyzed. In the last revision, the authors have included one representative example showing inhibition of single channel activity by the Kv10-specific inhibitor astemizole. Group data analysis would be needed to conclusively establish that the currents that were recorded indeed correspond to Kv10 channels.

It is shown that conditioning pulses to very negative voltages result in mutant channel 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, O1 occupancy is shown to be favored by increasingly negative conditioning voltages. This is interpreted as indicating that O1 is primarily accessed from deeply closed states in which voltage sensors are in their most deactivated position. 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.

The authors then address the role of the hidden O1 state in channel regulation by calcium-calmodulin (CaM). Stimulating calcium entry into oocytes with ionomycin and thapsigargin, assumed to enhance CaM-dependent modulation, resulted in preferential potentiation of the first component in ΔPASCap and E600R channels. This potentiation was attenuated by including an additional mutation that disfavors deeply closed states. Together, these results are interpreted as an indication that calcium-CaM preferentially stabilizes deeply closed states from which O1 can be readily accessed in mutant channels, thus favoring current activation. In WT channels lacking a conducting O1 state, CaM stabilizes deeply closed states and is therefore inhibitory. It is found that the potentiation of ΔPASCap and E600R by CaM is more strongly attenuated by mutations in the channel that are assumed to disrupt interaction with the C-terminal lobe of CaM than mutations assumed to affect interaction with the N-terminal lobe. These results are intriguing but difficult to interpret in mechanistic terms. The strong effect that calcium-CaM had on the occupancy of the O1 state in the mutants raises the possibility that O1 can be only observed in channels that are constitutively associated with CaM. To address this, a biochemical pull-down assay was carried out to establish that only a small fraction of channels are associated with CaM under baseline conditions. These 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.

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. Although the model used is highly symmetric and appears simple, the mathematical form used for the rate constants in the model adds a layer of complexity to the model that makes mechanistic interpretations difficult. In addition, many transitions that from a mechanistic standpoint should not depend on voltage were assigned a voltage dependence in the model. These limitations diminish the mechanistic insight that can be reliably extracted from the model.

Reviewer #1 (Public Review):

In this manuscript, Yang et al. conduct a comprehensive investigation to demonstrate the role of adipose tissue miR-802 in obesity-associated inflammation and metabolic dysfunction. Using multiple models and techniques, they propose a mechanism where elevated levels of miR-802 in adipose tissue (both in mouse models and humans) trigger fat accumulation and inflammation, leading to increased adiposity and insulin resistance. They suggest that increased miR-802 levels in adipocytes during obesity result in the downregulation of TRAF3, a negative regulator of canonical and non-canonical NF-κB pathways. This downregulation induces inflammation through the production of cytokines/chemokines that attract and polarize macrophages. Concurrently, the NF-κB pathway induces the lipogenic transcriptional factor SREBP1, which promotes fat accumulation and further recruits pro-inflammatory macrophages. While the proposed model is supported by multiple experiments and consistent data, there are areas where the manuscript could be improved. Some improvements can be addressed in the text, while others require additional controls, experiments, or analyses.

(1) The manuscript should provide measurements of lipid droplet/adipocyte size for all models, both in vitro and in vivo. In vivo studies should also include fat weight measurements. This is crucial to determine whether miR-802, TRAF3, and SREBP1 promote adiposity/fat accumulation across all models.<br /> (2) The rationale for co-culture experiments using WAT SVF is unclear, given that miR-802 is upregulated by obesity in adipocytes, not in the stromal-vascular fraction. These experiments would be more relevant if performed using isolated adipocytes or differentiated WAT SVF.<br /> (3) Figures 1G and 1H lack a control group (time 0 or NCD). Without this control, it is impossible to determine if inflammation precedes miR-802 upregulation.<br /> (4) The statement, "The knockout of miR-802 in adipose tissue did not alter food intake, body weight, glucose level, and adiposity (data not shown)," needs more detail regarding the age and sex of the animals. These data are important and should be reported, perhaps in a supplementary figure.<br /> (5) The terms "KO" (knockout) and "KI" (knock-in) are misleading for AAV models, as they do not modify the genome. "KD" (knockdown) and "OE" (overexpression) are more accurate.<br /> (6) The statement, "miR-802 expression was unaffected in other organs (Figure S3O)," should clarify that this is except for BAT.

By addressing these points, the manuscript would present a more robust and clear demonstration of the role of miR-802 in obesity-associated inflammation and metabolic dysfunction.

Reviewer #1 (Public Review):

In this paper, Tompary & Davachi present work looking at how memories become integrated over time in the brain, and relating those mechanisms to responses on a priming task as a behavioral measure of memory linkage. They find that remotely but not recently formed memories are behaviorally linked and that this is associated with a change in the neural representation in mPFC. They also find that the same behavioral outcomes are associated with the increased coupling of the posterior hippocampus with category-sensitive parts of the neocortex (LOC) during a post-learning rest period-again only for remotely learned information. There was also correspondence in rest connectivity (posterior hippocampus-LOC) and representational change (mPFC) such that for remote memories specifically, the initial post-learning connectivity enhancement during rest related to longer-term mPFC representational change.

This work has many strengths. The topic of this paper is very interesting, and the data provide a really nice package in terms of providing a mechanistic account of how memories become integrated over a delay. The paper is also exceptionally well-written and a pleasure to read. There are two studies, including one large behavioral study, and the findings replicate in the smaller fMRI sample. I do however have two fairly substantive concerns about the analytic approach, where more data will be required before we can know whether the interpretations are an appropriate reflection of the findings. These and other concerns are described below.

(1) One major concern relates to the lack of a pre-encoding baseline scan prior to recent learning.

a) First, I think it would be helpful if the authors could clarify why there was no pre-learning rest scan dedicated to the recent condition. Was this simply a feasibility consideration, or were there theoretical reasons why this would be less "clean"? Including this information in the paper would be helpful for context. Apologies if I missed this detail in the paper.

b) Second, I was hoping the authors could speak to what they think is reflected in the post-encoding "recent" scan. Is it possible that these data could also reflect the processing of the remote memories? I think, though am not positive, that the authors may be alluding to this in the penultimate paragraph of the discussion (p. 33) when noting the LOC-mPFC connectivity findings. Could there be the reinstatement of the old memories due to being back in the same experimental context and so forth? I wonder the extent to which the authors think the data from this scan can be reflected as strictly reflecting recent memories, particularly given it is relative to the pre-encoding baseline from before the remote memories, as well (and therefore in theory could reflect both the remote + recent). (I should also acknowledge that, if it is the case that the authors think there might be some remote memory processing during the recent learning session in general, a pre-learning rest scan might not have been "clean" either, in that it could have reflected some processing of the remote memories-i.e., perhaps a clean pre-learning scan for the recent learning session related to point 1a is simply not possible.)

c) Third, I am thinking about how both of the above issues might relate to the authors' findings, and would love to see more added to the paper to address this point. Specifically, I assume there are fluctuations in baseline connectivity profile across days within a person, such that the pre-learning connectivity on day 1 might be different from on day 2. Given that, and the lack of a pre-learning connectivity measure on day 2, it would logically follow that the measure of connectivity change from pre- to post-learning is going to be cleaner for the remote memories. In other words, could the lack of connectivity change observed for the recent scan simply be due to the lack of a within-day baseline? Given that otherwise, the post-learning rest should be the same in that it is an immediate reflection of how connectivity changes as a function of learning (depending on whether the authors think that the "recent" scan is actually reflecting "recent + remote"), it seems odd that they both don't show the same corresponding increase in connectivity-which makes me think it may be a baseline difference. I am not sure if this is what the authors are implying when they talk about how day 1 is most similar to prior investigation on p. 20, but if so it might be helpful to state that directly.

d) Fourth and very related to my point 1c, I wonder if the lack of correlations for the recent scan with behavior is interpretable, or if it might just be that this is a noisy measure due to imperfect baseline correction. Do the authors have any data or logic they might be able to provide that could speak to these points? One thing that comes to mind is seeing whether the raw post-learning connectivity values (separately for both recent and remote) show the same pattern as the different scores. However, the authors may come up with other clever ways to address this point. If not, it might be worth acknowledging this interpretive challenge in the Discussion.

(2) My second major concern is how the authors have operationalized integration and differentiation. The pattern similarity analysis uses an overall correspondence between the neural similarity and a predicted model as the main metric. In the predicted model, C items that are indirectly associated are more similar to one another than they are C items that are entirely unrelated. The authors are then looking at a change in correspondence (correlation) between the neural data and that prediction model from pre- to post-learning. However, a change in the degree of correspondence with the predicted matrix could be driven by either the unrelated items becoming less similar or the related ones becoming more similar (or both!). Since the interpretation in the paper focuses on change to indirectly related C items, it would be important to report those values directly. For instance, as evidence of differentiation, it would be important to show that there is a greater decrease in similarity for indirectly associated C items than it is for unrelated C items (or even a smaller increase) from pre to post, or that C items that are indirectly related are less similar than are unrelated C items post but not pre-learning. Performing this analysis would confirm that the pattern of results matches the authors' interpretation. This would also impact the interpretation of the subsequent analyses that involve the neural integration measures (e.g., correlation analyses like those on p. 16, which may or may not be driven by increased similarity among overlapping C pairs). I should add that given the specificity to the remote learning in mPFC versus recent in LOC and anterior hippocampus, it is clearly the case that something interesting is going on. However, I think we need more data to understand fully what that "something" is.

(3) The priming task occurred before the post-learning exposure phase and could have impacted the representations. More consideration of this in the paper would be useful. Most critically, since the priming task involves seeing the related C items back-to-back, it would be important to consider whether this experience could have conceivably impacted the neural integration indices. I believe it never would have been the case that unrelated C items were presented sequentially during the priming task, i.e., that related C items always appeared together in this task. I think again the specificity of the remote condition is key and perhaps the authors can leverage this to support their interpretation. Can the authors consider this possibility in the Discussion?

(4) For the priming task, based on the Figure 2A caption it seems as though every sequence contributes to both the control and primed conditions, but (I believe) this means that the control transition always happens first (and they are always back-to-back). Is this a concern? If RTs are changing over time (getting faster), it would be helpful to know whether the priming effects hold after controlling for trial numbers. I do not think this is a big issue because if it were, you would not expect to see the specificity of the remotely learned information. However, it would be helpful to know given the order of these conditions has to be fixed in their design.

(5) The authors should be cautious about the general conclusion that memories with overlapping temporal regularities become neurally integrated - given their findings in MPFC are more consistent with overall differentiation (though as noted above, I think we need more data on this to know for sure what is going on).

(6) It would be worth stating a few more details and perhaps providing additional logic or justification in the main text about the pre and post-exposure phases were set up and why. How many times each object was presented pre and post, and how the sequencing was determined (were any constraints put in place e.g., such that C1 and C2 did not appear close in time?). What was the cover task (I think this is important to the interpretation & so belongs in the main paper)? Were there considerations involving the fact that this is a different sequence of the same objects the participants would later be learning - e.g., interference, etc.?

Reviewer #1 (Public Review):

In this study, Girardello et al. use proteomics to reveal the membrane tension sensitive caveolin-1 interactome in migrating cells. The authors use EM and surface rendering to demonstrate that caveolae formed at the rear of migrating cells are complex membrane-linked multilobed structures, and they devise a robust strategy to identify caveolin-1 associated proteins using APEX2-mediated proximity biotinylation. This important dataset is further validated using proximity ligation assays to confirm key interactions, and follows up with an interrogation of a surprising relationship between caveolae and RhoGTPase signalling, where caveolin-1 recruits ROCK1 under high membrane tension conditions, and ROCK1 activity is required to reform caveolae upon reversion to isotonic solution. However, caveolin-1 recruits the RhoA inactivator ARHGAP29 when membrane tension is low and ARHGAP29 overexpression leads to disassembly of caveolae and reduced cell motility. This study builds on previous findings linking caveolae to positive feedback regulation of RhoA signalling, and provides further evidence that caveolae serve to drive rear retraction in migration but also possess an intrinsic brake to limit RhoA activation, leading the authors to suggest that cycles of caveolae assembly and disassembly could thereby be central to establish a stable cell rear for persistent cell migration

A major strength of the manuscript is the robust proteomic dataset. The experimental set up is well defined and mostly well controlled, and there is good internal validation in that the high abundance of core caveolar proteins in low membrane tension (isotonic) conditions, and absence under high membrane tension (brief hypo-osmotic shock) conditions, correlating very well with previous finding. The data could however be better presented to show where statically robust changes occur, and supplementary information should include a table of showing abundance. It's very good to see a link to PRIDE, providing a useful resource for the community.

The authors detail several known interactions and their mechanosensitivty, but also report new interactors of caveolin-1. Several mechanosensitive interactions of caveolin-1 take place at the cell rear, but others are more diffuse across the cell looking at the PLA data (e.g FLN1, CTTN, HSPB1; Figure 4A-F and Figure 4 supplement 1). It is interesting to speculate that those at the cell rear are involved in caveolae, whilst others are linked specifically to caveolin-1 (e.g. dolines). PLA or localisation analysis with Cavin1/PTRF may be able to resolve this and further specify caveolae versus non-caveolae mechanosensitive interactions.

The Cav1/ARHGAP29 influence on YAP signalling is interesting, but appear to be quite isolated from the rest of the manuscript. Does overexpression of ARHGAP29 influence YAP signalling and/or caveolar protein expression/Cav1pY14?<br /> ARHGAP29 and RhoA/ROCK1 related observations are very interesting and potentially really important. However, the link between ARHGAP29 and caveolae is not well established (other than in proteomic data). PLA or FRET could help establish this.<br /> The relationship between ARHGAP29 and RhoA signalling is not well defined. Is GAP activity important in determining the effect on migration and caveolae formation? What is the effect on RhoA activity? Alternatively, the authors could investigate YAP dependent transcriptional regulation downstream of overexpression.

Reviewer #3 (Public Review):

Although the authors findings are interesting, they do little to demonstrate new scientific information or advancements in producing genetically modified livestock with improved production characteristics. While the MSTNDel273 sheep exhibited an increased number of muscle fibers, the data provided did not demonstrate a significant improvement in meat production, quality or quantity in the MSTNDel273 sheep vs WT.

The manuscript is very long, complicated and difficult to read, given the minimum amount of significant information that is provided. It reads more like a graduate student thesis than a scientific manuscript ready for publication. Given the significant findings are so minimal, the amount of text provided, figures and tables are excessive. A large number of different molecular techniques are employed to try and decipher the mechanism(s) that result in the observed phenotype = double muscling. The authors focus on the MEK-ERK-FOSL1 pathway and suggest this is the key pathway/mechanism resulting in the phenotype observed in MSTNDel273sheep. However, they provide very little "significant" evidence to support this. RNA-Seq data demonstrated that hundreds of different genes were either upregulated or down-regulated, but the authors chose to only focus on FOSL1 and associated genes. The findings do not support the idea that FOSL1 is not involved, but neither do they strongly support FOSL1 involvement. The observations made by the authors could be co-incidental and not causative in nature.

The authors indicate that sgRNA design changes in addition to changing the molar ratio of Cas9MRNA:sgRNA improved the ability to generate biallelic homozygous mutant sheep; however, the data provided to not demonstrate any significant difference. Given the small number of sheep that were actually produced and evaluated, it is extremely difficult to demonstrate anything that was analyzed to be significantly (statistically) different between MSTNDel273 sheep and WT, yet the authors seem to ignore this in much of their discussion. There is no explanation as to why the authors started with sheep that were FGF5 knockouts. The reviewer assumes that this was simply a line of sheep available from previous studies and the goal was to produce sheep with both improved hair/wool characteristics in addition to improved muscle development. However, the use of FGF5 knockout sheep complicates the ability to accurately decipher the unique aspects associated with targeting only myostatin for knock-out. At minimum, this is a variable that has to be considered in the statistical analysis. No information is provided on the methods used to produce the MSTNDel273 sheep, which seems fundamentally important. It is assumed they were produced by injecting one-cell zygotes then transferring these into surrogate females, but given the information provided, it is impossible to know. Certainly, the methods employed could have a profound effect on the outcome. There is no information provided on the sex of the animals produced and then analyzed.

Comments on revised version:

The manuscript by Chen et al. is improved and demonstrates successful gene editing in sheep embryos to obtain biallelic mutation of Mstn and FGF5. Despite the improvements in the revised manuscript, the cellular and molecular mechanism remain inadequate to conclude whether Fosl1 indeed acts downstream of myostatin. In addition, there is little that is new direction versus confirmatory for what is already well know regarding Mstn and FGF5

There are also a number of editorial mistakes e.g. the authors refer to tables S1-S4 in the materials and methods and results section, but there is no table S1-S4 provided.

Reviewer #1 (Public Review):

Summary:

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:

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:

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.

Reviewer #1 (Public Review):

Summary:

BMP signaling is, arguably, best known for its role in the dorsoventral patterning, but not in nematodes, where it regulates body size. In their paper, Vora et al. analyze ChIP-Seq and RNA-Seq data to identify direct transcriptional targets of SMA-3 (Smad) and SMA-9 (Schnurri) and understand the respective roles of SMA-3 and SMA-9 in the nematode model Caenorhabditis elegans. The authors use publicly available SMA-3 and SMA-9 ChIP-Seq data, own RNA-Seq data from SMA-3 and SMA-9 mutants, and bioinformatic analyses to identify the genes directly controlled by these two transcription factors (TFs) and find approximately 350 such targets for each. They show that all SMA-3-controlled targets are positively controlled by SMA-3 binding, while SMA-9-controlled targets can be either up or downregulated by SMA-9. 129 direct targets were shared by SMA-3 and SMA-9, and, curiously, the expression of 15 of them was activated by SMA-3 but repressed by SMA-9. Since genes responsible for cuticle collagen production were eminent among the SMA-3 targets, the authors focused on trying to understand the body size defect known to be elicited by the modulation of BMP signaling. Vora et al. provide compelling evidence that this defect is likely to be due to problems with the BMP signaling-dependent collagen secretion necessary for cuticle formation.

Strengths:

Vora et al. provide a valuable analysis of ChIP-Seq and RNA-Seq datasets, which will be very useful for the community. They also shed light on the mechanism of the BMP-dependent body size control by identifying SMA-3 target genes regulating cuticle collagen synthesis and by showing that downregulation of these genes affects body size in C. elegans.

Weaknesses:

(1) Although the analysis of the SMA-3 and SMA-9 ChIP-Seq and RNA-Seq data is extremely useful, the goal "to untangle the roles of Smad and Schnurri transcription factors in the developing C. elegans larva", has not been reached. While the role of SMA-3 as a transcriptional activator appears to be quite straightforward, the function of SMA-9 in the BMP signaling remains obscure. The authors write that in SMA-9 mutants, body size is affected, but they do not show any data on the mechanism of this effect.

(2) The authors clearly show that both TFs can bind independently of each other, however, by using distances between SMA-3 and SMA-9 ChIP peaks, they claim that when the peaks are close these two TFs act as complexes. In the absence of proof that SMA-3 and SMA-9 physically interact (e.g. that they co-immunoprecipitate - as they do in Drosophila), this is an unfounded claim, which should either be experimentally substantiated or toned down.

(3) The second part of the paper (the collagen story) is very loosely connected to the first part. dpy-11 encodes an enzyme important for cuticle development, and it is a differentially expressed direct target of SMA-3. dpy-11 can be bound by SMA-9, but it is not affected by this binding according to RNA-Seq. Thus, technically, this part of the paper does not require any information about SMA-9. However, this can likely be improved by addressing the function of the 15 genes, with the opposing mode of regulation by SMA-3 and SMA-9.

(4) The Discussion does not add much to the paper - it simply repeats the results in a more streamlined fashion.

Reviewer #1 (Public Review):

Summary:

This manuscript by Aybar-Torres et al investigated the effect of common human STING1 variants on STING-mediated T cell phenotypes in mice. The authors previously made knock-in mice expressing human STING1 alleles HAQ or AQ, and here they established a new knock-in line Q293. The authors stimulated cells isolated from these mice with STING agonists and found that all three human mutant alleles resist cell death, leading to the conclusion that R293 residue is essential for STING-mediated cell death (there are several caveats with this conclusion, more below). The authors also bred HAQ and AQ alleles to the mouse Sting1-N153S SAVI mouse and observed varying levels of rescue of disease phenotypes with the AQ allele showing more complete rescue than the HAQ allele. The Q293 allele was not tested in the SAVI model. They conclude that the human common variants such as HAQ and AQ have a dominant negative effect over the gain-of-function SAVI mutants.

Strengths:

The authors and Dr. Jin's group previously made important observations of common human STING1 variants, and these knock-in mouse models are essential for understanding the physiological function of these alleles.

Weaknesses:

However, although some of the observations reported here are interesting, the data collectively does not support a unified model. The authors seem to be drawing two sets of conclusions from in vitro and in vivo experiments, and neither mechanism is clear. Several experiments need better controls, and these knock-in mice need more comprehensive functional characterization.

Summary:

This paper described the dynamics of the nuclear substructure called PML Nucleolar Association (PNA) in response to DNA damage on ribosomal DNA (rDNA) repeats. The authors showed that the PNA with rDNA repeats is induced by the inhibition of topoisomerases and RNA polymerase I and that the PNA formation is modulated by RAD51, thus homologous recombination. Artificially induced DNA double-strand breaks (DSBs) in rDNA repeats stimulate the formation of PNA with DSB markers. This DSB-triggered PNA formation is regulated by DSB repair pathways.

Strengths:

This paper illustrates a unique DNA damage-induced sub-nuclear structure containing the PML body, which is specifically associated with the nucleolus. Moreover, the dynamics of this PML Nucleolar Association (PNA) require topoisomerases and RNA polymerase I and are modulated by RAD51-mediated homologous recombination and non-homologous end-joining. This study provides a unique regulation of DSB repair at rDNA repeats associated with the unique-membrane-less subnuclear structure.

Weaknesses:

Although the PNA formation on rDNA repeat is nicely shown by cytological analysis, the biological significance of PNA in DSB repair is not fully addressed.

Reviewer #1 (Public Review):

Summary:<br /> In this work, the authors examine the activity and function of D1 and D2 MSNs in dorsomedial striatum (DMS) during an interval timing task. In this task, animals must first nose poke into a cued port on the left or right; if not rewarded after 6 seconds, they must switch to the other port. Thus, this task requires animals to estimate if at least 6 seconds have passed after the first nose poke. After verifying that animals estimate the passage of 6 seconds, the authors examine striatal activity during this interval. They report that D1-MSNs tend to decrease activity, while D2-MSNs increase activity, throughout this interval. They suggest that this activity follows a drift-diffusion model, in which activity increases (or decreases) to a threshold after which a decision is made. The authors next report that optogenetically inhibiting D1 or D2 MSNs, or pharmacologically blocking D1 and D2 receptors, increased the average wait time. This suggests that both D1 and D2 neurons contribute to the estimate of time, with a decrease in their activity corresponding to a decrease in the rate of 'drift' in their drift-diffusion model. Lastly, the authors examine MSN activity while pharmacologically inhibiting D1 or D2 receptors. The authors observe most recorded MSNs neurons decrease their activity over the interval, with the rate decreasing with D1/D2 receptor inhibition.

Major strengths:<br /> The study employs a wide range of techniques - including animal behavioral training, electrophysiology, optogenetic manipulation, pharmacological manipulations, and computational modeling. The question posed by the authors - how striatal activity contributes to interval timing - is of importance to the field and has been the focus of many studies and labs. This paper contributes to that line of work by investigating whether D1 and D2 neurons have similar activity patterns during the timed interval, as might be expected based on prior work based on striatal manipulations. However, the authors find that D1 and D2 neurons have distinct activity patterns. They then provide a decision-making model that is consistent with all results. The data within the paper is presented very clearly, and the authors have done a nice job presenting the data in a transparent manner (e.g., showing individual cells and animals). Overall, the manuscript is relatively easy to read and clear, with sufficient detail given in most places regarding the experimental paradigm or analyses used.

Major weaknesses:<br /> One weakness to me is the impact of identifying whether D1 and D2 had similar or different activity patterns. Does observing increasing/decreasing activity in D2 versus D1, or different activity patterns in D1 and D2, support one model of interval timing over another, or does it further support a more specific idea of how DMS contributes to interval timing?

I found the results presented in Figures 2 and 3 to be a little confusing or misleading. In Figure 2, the authors appear to claim that D1 neurons decrease their activity over the time interval while D2 neurons increase activity. The authors use this result to suggest that D1/D2 activity patterns are different. In Figure 3, a different analysis is done, and this time D2 neurons do not significantly increase their activity with time, conflicting with Figure 2. While in both figures, there is a significant difference between the mean slopes across the population, the secondary effect of positive/negative slope for D2/D1 neurons changes. I find this especially confusing as the authors refer back to the positive/negative slope for D2/D1 neurons result throughout the rest of the text.

It is a bit unclear to me how the authors chose the parameters for the model, and how well the model explains behavior is quantified. It seems that the authors didn't perform cross-validation across trials (i.e., they chose parameters that explained behavior across all trials combined, rather than choosing parameters from a subset of trials and determining whether those parameters are robust enough to explain behavior on held-out trials). I think this would increase the robustness of the result.

In addition, it remains a bit unclear to me how the authors changed the specific parameters they did to model the optogenetic manipulation. It seems these parameters were chosen because they fit the manipulation data. This makes me wonder if this model is flexible enough that there is almost always a set of parameters that would explain any experimental result; in other words, I'm not sure this model has high explanatory power.

Lastly, the results are based on a relatively small dataset (tens of cells).

Impact:<br /> The task and data presented by the authors are very intriguing, and there are many groups interested in how striatal activity contributes to the neural perception of time. The authors perform a wide variety of experiments and analysis to examine how DMS activity influences time perception during an interval-timing task, allowing for insight into this process. However, the significance of the key finding -- that D1 and D2 activity is distinct across time -- remains somewhat ambiguous to me.

Reviewer #1 (Public Review):

Summary:

This review evaluates the SCellBOW framework, which applies phenotype algebra to obtain vectors from cancer subclusters or user-defined subclusters.

Strengths:

SCellBOW employs an innovative application of NLP-inspired techniques to analyze scRNA-seq data, facilitating the identification and visualization of phenotypically divergent cell subpopulations.

The framework demonstrates robustness in accurately representing various cell types across multiple datasets, highlighting its versatility and utility in different biological contexts.

By simulating the impact of specific malignant subpopulations on disease prognosis, SCellBOW provides valuable insights into the relative risk and aggressiveness of cancer subpopulations, which is crucial for personalized therapeutic strategies.

The identification of a previously unknown and aggressive AR−/NElow subpopulation in metastatic prostate cancer underscores the potential of SCellBOW in uncovering clinically significant findings.

Weaknesses:

The reliance on bulk RNA-seq data as a reference raises concerns about potentially misleading results due to the presence of RNA expression from immune cells in the TME. It is unclear if SCellBOW adequately addresses this issue, which could affect the accuracy of the cancer subcluster vectors.

The method of extracting vectors in phenotype algebra appears to be a straightforward subtraction operation. This simplicity might limit its efficiency in excluding associations with phenotypes from specific subpopulations, potentially leading to inaccurate interpretations of the data.

The review would benefit from additional validation studies to assess the effectiveness of SCellBOW in distinguishing between cancerous and non-cancerous signals, particularly in heterogeneous tumor environments.

Further clarification on how SCellBOW handles mixed-cell populations within bulk RNA-seq data would strengthen the evaluation of its applicability and reliability in diverse research settings.

Reviewer #1 (Public Review):

This study unveils a novel role for ferritin in Drosophila larval brain development. Furthermore, it pinpoints that the observed defects in larval brain development resulting from ferritin knockdown are attributed to impaired Fe-S cluster activity and ATP production. Overall this is a well-conducted and novel study.

The author have adequately addressed the concerns.