Reviewer #1 (Public Review):

Summary:

This manuscript reports the effects of a heterozygous mutation in the KCNT1 potassium channels on the properties of ion currents and firing behavior of excitatory and inhibitory neurons in the cortex of mice expressing KCNT1-Y777H. In humans, this mutation as well as multiple other heterozygotic mutations produce very severe early-onset seizures and produce a major disruption of all intellectual function. In contrast, in mice, this heterozygous mutation appears to have no behavioral phenotype or any increased propensity to seizures. A relevant phenotype is, however, evident in mice with the homozygous mutation, and the authors have previously published the results of similar experiments with the homozygotes. As perhaps expected, the neuronal effects of the heterozygous mutation presented in this manuscript are generally similar but markedly smaller than the previously published findings on homozygotes. There are, however, some interesting differences, particularly on PV+ interneurons, which appear to be more excitable than wild type in the heterozygotes but more excitable in the heterozygotes. This raises the interesting question, which has been explicitly discussed by the authors in the revised manuscript, as to whether the reported changes represent homeostatic events that suppress the seizure phenotype in the mouse heterozygotes or simply changes in excitability that do not reach the threshold for behavioral outcomes.

Strengths and Weaknesses:

(1) The authors find that the heterozygous mutation in PV+ interneurons increases their excitability, a result that is opposite from their previous observation in neurons with the corresponding homozygous mutation. They propose that this results from the selective upregulation of a persistent sodium current INaP in the PV+ interneurons. These observations are very interesting ones, and they raised some issues in the original submission:

A) The protocol for measuring the INaP current could potentially lead to results that could be (mis)interpreted in different ways in different cells. First, neither K currents nor Ca currents are blocked in these experiments. Instead, TTX is applied to the cells relatively rapidly (within 1 second) and the ramp protocol is applied immediately thereafter. It is stated that, at this time, Na currents and INaP are fully blocked but that any effects on Na-activated K currents are minimal. In theory this would allow the pre- to post- difference current to represent a relatively uncontaminated INaP. This would, however, only work if activation of KNa currents following Na entry is very slow, taking many seconds. A good deal of literature has suggested that the kinetics of activation of KNa currents by Na influx vary substantially between cell types, such that single action potentials and single excitatory synaptic events rapidly evoke KNa currents in some cell types. This is, of course, much faster than the time of TTX application. Most importantly, the kinetics of KNa activation may be different in different neuronal types, which would lead to errors that could produce different estimates of INaP in PV+ interneurons vs other cell types.

In their revised manuscript, the authors have provided good data demonstrating that, at least for the PV and SST neurons, loss of KNa currents after TTX application is slow relative to the time course of loss of INaP, justifying the use of this protocol for these neuronal types.

B) As the authors recognize, INaP current provides a major source of cytoplasmic sodium ions for the activation. An expected outcome of increased INaP is, therefore, further activation of KNa currents, rather than a compensatory increase in an inward current that counteracts the increase in KNa currents, as is suggested in the discussion.

The authors comment in the rebuttal that, despite the fact that sodium entry through INaP is known to activate KNa channels, an increase in INaP does not necessarily imply increased KNa current. This issue should be addressed directly somewhere in the text, perhaps most appropriately in the discussion.

C) The numerical simulations, in general, provide a very useful way to evaluate the significance of experimental findings. Nevertheless, while the in-silico modeling suggests that increases in INaP can increase firing rate in models of PV+ neurons, there is as yet insufficient information on the relative locations of the INaP channels and the kinetics of sodium transfer to KNa channels to evaluate the validity of this specific model.

The authors have now put in all of the appropriate caveats on this very nicely in the revised manuscript.

(2) The effects of the KCNT1 channel blocker VU170 on potassium currents are somewhat larger and different from those of TTX, suggesting that additional sources of sodium may contribute to activating KCNT1, as suggested by the authors. Because VU170 is, however, a novel pharmacological agent, it may be appropriate to make more careful statements on this. While the original published description of this compound reported no effect on a variety of other channels, there are many that were not tested, including Na and cation channels that are known to activate KCNT1, raising the possibility of off-target effects.

In the revised version, the authors have added more to the manuscript on this issue and have added a very clear discussion of this to the text (in the discussion section).

This is a very clear and thorough piece of work, and the authors are to be congratulated on this. My one remaining suggestion would be to make an explicit statement about whether increased sodium influx through INaP channels, which is thought to activate KNa channels, would be likely to increase KNa current in these neurons (see comment 1B).

Reviewer #1 (Public Review):

Summary:

Using chromaffin cells as a powerful model system for studying secretion, the authors study the regulatory role of complexin in secretion. Complexin is still enigmatic in its regulatory role, as it both provides inhibitory and facilitatory functions in release. The authors perform an extensive structure-function analysis of both the C- and N-terminal regions of complexin. There are several interesting findings that significantly advances our understanding of cpx/SNARe interactions in regulating release. C-terminal amphipathic helix interferes with SNARE complex assembly and thus clamps fusion. There are acidic residues in the C-term that may be seen as putative interaction partners for Synaptotagmin. The N-terminus of Complexin promoting role may be associated with an interaction with Syt1. In particular the putative interaction with Syt1 is of high interest and supported by quite strong functional and biochemical evidence. The experimental approaches are state of the art, and the results are of the highest quality and convincing throughout. They are adequate and intelligently discussed in the rich context of the standing literature. Whilst there are some concerns about whether the facilitatory actions of complexion have to be tightly linked to Syt1 interactions, the proposed model will significantly advance the field by providing new directions in future research.

Reviewer #1 (Public Review):

Summary:

In this research, Soni and Frank investigate the network mechanisms underlying capacity limitations in working memory from a new perspective, with a focus on visual working memory (VWM). The authors have advanced beyond the classical neural network model, which incorporates the prefrontal cortex and basal ganglia (PBWM), by introducing an adaptive chunking variant. This model is trained using a biologically plausible, dopaminergic reinforcement learning framework. The adaptive chunking mechanism is particularly well-suited to the VWM tasks involving continuous stimuli and elegantly integrates the 'slot' and 'resource' theories of working memory constraints. The chunk-augmented PBWM operates as a slot-like system with resource-like limitations.

Through numerical simulations under various conditions, Soni and Frank demonstrate the performance of the chunk-augmented PBWM model surpasses the no-chunk control model. The improvements are evident in enhanced effective capacity, optimized resource management, and reduced error rates. The retention of these benefits, even with increased capacity allocation, suggests that working memory limitations are due to a combination of factors, including the efficient credit assignments that are learned flexibly through reinforcement learning. In essence, this work addresses fundamental questions related to a computational working memory limitation using a biologically-inspired neural network, and thus has implications for clinical conditions in which working memory is affected, such as Parkinson's disease, ADHD, and schizophrenia.

Strengths:

The integration of mechanistic flexibility, reconciling two theories for WM capacity into a single unified model, results in a neural network that is both more adaptive and human-like. Building on the PBWM framework ensures the robustness of the findings. The addition of the chunking mechanism tailors the original model for continuous visual stimuli. Chunk-stripe mechanisms contribute to the 'resource' aspect, while input-stripes contribute to the 'slot' aspect. This combined network architecture enables flexible and diverse computational functions, enhancing performance beyond that of the classical model.

Moreover, unlike previous studies that design networks for specific task demands, the proposed network model can dynamically adapt to varying task demands by optimizing the chunking gating policy through RL.

The implementation of a dopaminergic reinforcement learning protocol, as opposed to a hard-wired design, leads to the emergence of strategic gating mechanisms that enhance the network's computational flexibility and adaptability. These gating strategies are vital for VWM tasks and are developed in a manner consistent with ecological and evolutionary learning held by humans. Further examination of how reward prediction error signals, both positive and negative, collaborate to refine gating strategies reveals the crucial role of reward feedback in fine-tuning the working memory computations and the model's behavior, aligning with the current neuroscientific understanding that reward matters.

Furthermore, assessing the impact of a healthy balance of dopaminergic reward prediction error signals on information manipulation holds implications for patients with altered striatal dopaminergic signaling.

Weaknesses:

While I appreciate the novelty of the idea presented in this paper, which aligns with common interests in cognitive neuroscience, I believe there are several areas that require further clarification.

First, the method section appears somewhat challenging to follow. To enhance clarity, it might be beneficial to include a figure illustrating the overall model architecture. This visual aid could provide readers with a clearer understanding of the overall network model.

Additionally, the structure depicted in Figure 2 could be potentially confusing. Notably, the absence of an arrow pointing from the thalamus to the PFC and the apparent presence of two separate pathways, one from sensory input to the PFC and another from sensory input to the BG and then to the thalamus, may lead to confusion. While I recognize that Figure 2 aims to explain network gating, there is room for improvement in presenting the content accurately.

Still, for the method part, it would enhance clarity to explicitly differentiate between predesigned (fixed) components and trainable components. Specifically, does the supplementary material state that synaptic connection weights in striatal units (Go&NoGo) are trained using XCAL, while other components, such as those in the PFC and lateral inhibition, are not trained (I found some sentences in 'Limitations and Future Directions')?

I'm not sure about the training process shown in Figure 8. It appears that the training may not have been completed, given that the blue line representing the chunk stripe is still ascending at the endpoint. The weights depicted in panel d) seem to correspond with those shown in panels b) and c), no? Then, how is the optimization process determined to be finished? Alternatively, could it be stated that these weight differences approach a certain value asymptotically? It would be better to clarify the convergence criteria of the optimization process.

Reviewer #1 (Public Review):

Summary:

This paper represents a huge amount of work on a condition whose patients' health and well-being have not always been prioritized, and only relatively recently has the immune dysregulation seen in patients with Down Syndrome (DS) been garnering major research interest.

This paper provides an unparalleled examination of immune disorders in patients with DS. The authors also report the results from a clinical trial with the JAK inhibitor tofacitinib in DS patients.

Strengths:

This manuscript reports a herculean effort and provides an unparalleled examination of immune disorders in a large number of patients with DS.

Weaknesses:

Not a major weakness but, apart from finding an elevation of CD4 T central memory cells and more differentiated plasmablast, several of the alterations reported in this manuscript had already been suggested by a few case reports and a very small series. On the other hand, the number of patients (and controls) utilized for this study is remarkable and allows for drawing much firmer conclusions.

Joint Public Review

Cav1.4 calcium channels control voltage-dependent calcium influx at photoreceptor synapses, and congenital loss of Cav1.4 function causes stationary night blindness CSNB2. Based on a broad portfolio of methodological approaches - genetic mouse models, immunolabeling and microscopic imaging, serial block-face-SEM, ERGs, and electrophysiology - the authors show that cone photoreceptor synapse development is strongly perturbed in the absence of Cav1.4 protein, and that expression of a nonconducting Cav1.4 channel mitigates these perturbations. Further data indicate that Cav3 channels are present, which, according to the authors, may compensate for the loss of Cav1.4 calcium currents and thus maintain cone synaptic transmission. These data, which are in agreement with a similar study by the same authors on rod photoreceptor synapses, help to explain what functional defects exactly cause CSNB2 and why it is accompanied by only mild visual impairment.

The strengths of the present study are its conceptual and experimental soundness, the broad spectrum of cutting-edge methodological approaches pursued, and the convincing differential analysis of mutant phenotypes. Weaknesses mainly concern the mechanism by which Cav3 channels might partially compensate for the loss of Cav1.4 calcium currents.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript the authors use the model organism Drosophila to explore sex and age impacts of a TBI method. They find age and sex differences: older age is susceptible to mild TBI and females are also more susceptible. In particular, they pursue a finding that virgin vs mated females show different responses: virgins are protected but mated females succumb to TBI with climbing deficits. In fact, virgin females compared to mated females are largely protected. They discover this is associated with exposure of the females to Sex Peptide in the reproductive neurons of the female reproductive tract. When they extend to RNAseq of brains, they show that there are very few genes in common between males, mated females, virgins and females mated with males lacking sex peptide. But what the few chronic genes associated with mated females seem associated with the immune system. These findings suggest that mated females have a compromised immune system, which might make them more vulnerable. In a bigger context, these findings point to the idea that the life status of the animal/individual may have an important impact on the outcome of a TBI - here illustrated by the differential state of virgin vs mated females in Drosophila.

Strengths:<br /> This is an interesting paper that allows a detailed comparison of sex and age in TBI which is largely only possible in such a simple model, where large numbers and many variations can be addressed. Overall the findings are interesting.

Weaknesses:<br /> Although the findings beyond Sex peptide are observational, the work sets the stage for more detailed studies to pursue the role of the genes they find by RNAseq and whether for example, boosting the innate immune system would protect the mated females, among other experiments.

Reviewer #1 (Public Review):

The manuscript by Hong-Qian Chen and collaborators describes the development of a mouse model that co-expresses a fluorescent protein ZsGreen marker in gene fusion with the Fshr gene.

The authors are correct in that there is a lack of reliable antibodies against many of the GPCR family members. The approach is novel and interesting, with a potential to help understand the expression pattern of gonadotropin receptors. There has been a very long debate about the expression of gonadotropin receptors in other tissues other than gonads. While their expression of the Fshr in some of those tissues has been detected by a variety of methods, their physiological, or pathophysiological, function(s) remain elusive.

The authors in this manuscript assume that the expression of ZsGren and the Fshr are equal. While this is correct genetically (transcription->translation) it does not go hand in hand to other posttranslational processes.

One of the shocking observations in this manuscript is the expression of Fshr in Leydig cells. Other observations are in the osteoblasts and endothelial cells as well as epithelial cells in different organs. The expression of ZsGreen in these tissues seems high and one shall start questioning if there are other mechanisms at play here.

First, the turnover of fluorescent proteins is long, longer than 48h, which means that they accumulate at a different speed than the endogenous Fshr. This means that ZsGreen will accumulate in time while the Fshr receptor might be degraded almost immediately. This correlated with mRNA expression (by the authors) but does not with the results of other studies in single-cell sequencing (see below).

Then, the expression of ZsGreen in Leydig cells seems much higher than in Sertoli cells, this is "disturbing" to put it mildly. This is visible in both, the ZsGreen expression and the FISH assay (Fig 2 B-D).

The expression in WAT and BAT is also questionable as the expression of ZsGreen is high everywhere. What makes it difficult to actually believe that the images are truly informative? For example, the stainings of the aorta show the ZsGreen expression where elastin and collagen fibres are - these are not "cells" and therefore are not expressing ZsGreen.

FISH expression (for Fshr) in WT mice is missing.

Also, the tissue sections were stained with the IgG only (neg control) but in practice both the KI and the WT tissues should be stained with the primary and secondary antibodies.

The only control that I could think of to truly get a sense of this would be a tagged receptor (N-terminal) that could then be analysed by immunohistochemistry.

The authors also claim:<br /> To functionally prove the presence of Fshr in osteoblasts/osteocytes, we also deleted Fshr in osteocytes using an inducible model. The conditional knockout of Fshr triggered a much more profound increase in bone mass and decrease in fat mass than blockade by Fsh antibodies (unpublished data)

This would be a good control for all their images. I think it is necessary to make the large claim of extragonadal expression, as well as intragonadal such as Leydig cells.

Claiming that the under-developed Leydig cells in FSHR KO animals is due to a direct effect of the FSHR, and not via a cross-talk between Sertoli and Leydig cells, is too much of a claim. It might be speculated to some degree but as written at the moment is suggests this is "proven".

We also do not know if this Fshr expressed is a spliced form that would also result in the expression of ZsGreen but in a non-functional Fshr, or whether the Fshr is immediately degraded after expression. The insertion of the ZsGreen might have disturbed the epigenetics, transcription or biosynthesis of the mRNA regulation.

The authors should go through single-cell data of WT mice to show the existence of the Fshr transcript(s). For example here:<br /> https://www.nature.com/articles/sdata2018192

Comments after revision:

The response by the authors does not seem sufficient or adequate, by any length, for what one would expect for a work having such a large claim as the expression of the Fshr in multiple cell types and organs. It is not the fact that Fshr might be expressed extragonadally or even by other cells in the gonads, but the surprising images where virtually every cell in the provided tissues, and not only cells but structures, glows green.

It is not possible to know, as a reviewer, whether the excitation intensity and exposure for all images is equal. We believe that they cannot be, as control organs such as fat, testes, ovaries, and vasculature have a natural fluorescence background.

Leydig cells cannot simply express more Fshr than Sertoli cells, that would go against what we have known for >50 years in physiology. While it is scientific to question 'old' data, to make extraordinary claims there is a need for "extraordinary evidence". There is very low expression in Sertoli cells (Fig 2) while Leydig cells and spermatozoa glow vividly.

Moreover, even the tails of spermatozoa glow! This is not cytoplasm and cannot contain a soluble fluorescent protein.

The controls should be shown side-by-side to the experimental images. It would be a lot more credible if the WT and the KI tissues were placed on the same slide, with images taken from them side by side not only for ZsGreen but antibody immunofluorescence staining.

Moreover, I noticed that the entire manuscript is based on a single founder mouse, which is not acceptable as an error - either multiple integrations other than in the correct locus or genetic instability created by the KI integration would result in promiscuous expression. The founder mouse is not well enough characterised as it is only performed by Southern blots and PCR, while additional integrations cannot be detected by such. Other methods should be used such as FISH or even whole genome sequencing. Yet, several lines should be used to ensure no other effects exist.

In Fig 5, the section of aorta shows low staining in the elastin/collagen fibres, while there is clearly in Suppl Data 2. In the same figure, the 2nd lung images show green fluorescence in the mucosa (centre) which should not be as there is no cells there.

The additional single-cell data does not truly support their claims, in the sense that while some of the data might go in line e.g. Leydig cells showing as high expression as "tubules", there are many other cell types that show no expression such as hepatocytes and skeletal muscle, where the authors claim to have high expression of Fshr. Moreover, in the datasets presented organs like "ovary" have almost no Fshr expression, which should question the validity of such.

The authors use an Fshr antibody without enough validation. The Fshr KO animals should be used for this. In fact, one of the very first statements in the manuscript is that antibodies against GPCRs in general, and gonadotropin receptors more specifically, are unreliable. The fact that controls show the same pattern as transgenic animals questions the validity, as no single acceptable antibody against FSHR recognises Leydig cells.

The detection of Fshr in e.g. adipocytes of B6 mice is as questionable as many other claims of gonadotropin receptors in extragonadal tissues, which has been questioned a number of times by many researchers.

One question we should ask is, is there any tissue on these mice that does not 'express' (Fshr)-ZsGreen? Because from what I see every single tissue analysed has 'Fshr". Which might be the problem why it is so difficult to find.

Some images seem to be duplicated such as in Fig 2C where the first row and the 3rd row seem to be the same image.

Reviewer #1 (Public Review):

Summary:

Ctnnb1 encodes β-catenin, an essential component of the canonical Wnt signaling pathway. In this study, the authors identify an upstream enhancer of Ctnnb1 responsible for the specific expression level of β-catenin in the gastrointestinal tract. Deletion of this promoter in mice and analyses of its association with human colorectal tumors support that it controls the dosage of Wnt signaling critical to the homeostasis in intestinal epithelia and colorectal cancers.

Strengths:

This study has provided convincing evidence to demonstrate the functions of a gastrointestinal enhancer of Ctnnb1 using combined approaches of bioinformatics, genomics, in vitro cell culture models, mouse genetics, and human genetics. The results support the idea that the dosage of Wnt/β-catenin signaling plays an important role in the pathophysiological functions of intestinal epithelia. The experimental designs are solid and the data presented are of high quality. This study significantly contributes to the research fields of Wnt signaling, tissue-specific enhancers, and intestinal homeostasis.

Weaknesses:

One weakness of this manuscript is an insufficient discussion on the Ctnnb1 enhancers for different tissues. For example, do specific DNA motifs and transcriptional factors contribute to the tissue-specificity of the neocortical and gastrointestinal enhancers? It is also worth discussing the potential molecular mechanisms controlling the gastrointestinal expression of Ctnnb1 in different species since the identified human and mouse enhancers don't seem to share significant similarities in primary sequences.

The paper describes a program developed to identify PPI-hot spots using the free protein structure and compares it to FTMap and SPOTONE, two webservers that they consider as competitive approaches to the problem. We appreciate the effort in providing a new webserver that can be tested by the community but we continue to have major concerns:

(1) The comparison to the FTMap program is problematic. The authors misinterpret the article they refer to, i.e., Zerbe et al. "Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces" J. Chem. Inf. Model. 52, 2236-2244, (2012). FTMap identifies hot spots that bind small molecular ligands. The Zerbe et al. article shows that such hot spots tend to interact with hot spot residues on the partner protein in a protein-protein complex (emphasis on "partner"). Thus, the hot spots identified by FTMap are not the hot spots defined by the authors. In fact, because the Zerbe paper considers the partner protein in a complex, the results cannot be compared to the results of Chen et al. This difference is missed by the authors, and hence the comparison of the FTMap is invalid.

(2) Chen et al. use a number of usual features in a variety of simple machine-learning methods to identify hot spot residues. This approach has been used in the literature for more than a decade. Although the authors say that they were able to find only FTMap and SPOTONE as servers, there are dozens of papers that describe such a methodology. Some examples are given here: (Higa and Tozzi, 2009; Keskin, et al., 2005; Lise, et al., 2011; Tuncbag, et al., 2009; Xia, et al., 2010). There are certainly more papers. Thus, while the web server is a potentially useful contribution, the paper does not provide a fundamentally novel approach.

Reviewer #1 (Public Review):

Summary:

This study addresses the question of how task-relevant sensory information affects activity in the motor cortex. The authors use various approaches to address this question, looking at single units and population activity. They find that there are three subtypes of modulation by sensory information at the single unit level. Population analyses reveal that sensory information affects the neural activity orthogonally to motor output. The authors then compare both single unit and population activity to computational models to investigate how encoding of sensory information at the single unit level is coordinated in a network. They find that an RNN that displays similar orbital dynamics and sensory modulation to the motor cortex also contains nodes that are modulated similarly to the three subtypes identified by the single unit analysis.

Strengths:

The strengths of this study lie in the population analyses and the approach of comparing single-unit encoding to population dynamics. In particular, the analysis in Figure 3 is very elegant and informative about the effect of sensory information on motor cortical activity. The task is also well designed to suit the questions being asked and well controlled.

It is commendable that the authors compare single units to population modulation. The addition of the RNN model and perturbations strengthen the conclusion that the subtypes of individual units all contribute to the population dynamics. However, the subtypes (PD shift, gain, and addition) are not sufficiently justified. The authors also do not address that single units exhibit mixed modulation, but RNN units are not treated as such.

Weaknesses:

The main weaknesses of the study lie in the categorization of the single units into PD shift, gain, and addition types. The single units exhibit clear mixed selectivity, as the authors highlight. Therefore, the subsequent analyses looking only at the individual classes in the RNN are a little limited. Another weakness of the paper is that the choice of windows for analyses is not properly justified and the dependence of the results on the time windows chosen for single-unit analyses is not assessed. This is particularly pertinent because tuning curves are known to rotate during movements (Sergio et al. 2005 Journal of Neurophysiology).

This paper shows sensory information can affect motor cortical activity whilst not affecting motor output. However, it is not the first to do so and fails to cite other papers that have investigated sensory modulation of the motor cortex (Stavinksy et al. 2017 Neuron, Pruszynski et al. 2011 Nature, Omrani et al. 2016 eLife). These studies should be mentioned in the Introduction to capture better the context around the present study. It would also be beneficial to add a discussion of how the results compare to the findings from these other works.

This study also uses insights from single-unit analysis to inform mechanistic models of these population dynamics, which is a powerful approach, but is dependent on the validity of the single-cell analysis, which I have expanded on below.

I have clarified some of the areas that would benefit from further analysis below:

(1) Task:<br /> The task is well designed, although it would have benefited from perhaps one more target speed (for each direction). One monkey appears to have experienced one more target speed than the others (seen in Figure 3C). It would have been nice to have this data for all monkeys.

(2) Single unit analyses:<br /> In some analyses, the effects of target speed look more driven by target movement direction (e.g. Figures 1D and E). To confirm target speed is the main modulator, it would be good to compare how much more variance is explained by models including speed rather than just direction. More target speeds may have been helpful here too.

The choice of the three categories (PD shift, gain addition) is not completely justified in a satisfactory way. It would be nice to see whether these three main categories are confirmed by unsupervised methods.

The decoder analyses in Figure 2 provide evidence that target speed modulation may change over the trial. Therefore, it is important to see how the window considered for the firing rate in Figure 1 (currently 100ms pre - 100ms post movement onset) affects the results.

(3) Decoder:<br /> One feature of the task is that the reach endpoints tile the entire perimeter of the target circle (Figure 1B). However, this feature is not exploited for much of the single-unit analyses. This is most notable in Figure 2, where the use of a SVM limits the decoding to discrete values (the endpoints are divided into 8 categories). Using continuous decoding of hand kinematics would be more appropriate for this task.

(4) RNN:<br /> Mixed selectivity is not analysed in the RNN, which would help to compare the model to the real data where mixed selectivity is common. Furthermore, it would be informative to compare the neural data to the RNN activity using canonical correlation or Procrustes analyses. These would help validate the claim of similarity between RNN and neural dynamics, rather than allowing comparisons to be dominated by geometric similarities that may be features of the task. There is also an absence of alternate models to compare the perturbation model results to.

On responding to the first round of reviews, the authors have nicely adjusted their wording and fairly describe the results of their study. Certain markers were identified for further investigation. Yet, an overall non-obvious relationship between immune markers and HIV reservoirs has been shown previously, and despite the attempt to leverage powerful ML algorithms, they are not magical and cannot reveal strong relationships that fundamentally do not exist. In addition, categorical classification is for now hard to interpret and the more powerful ML algorithms do not seem to outperform more classic regression methods. Therefore, it remains relatively hard to evaluate the utility of this kind of study.

Initial summary:

Semenova et al. have studied a large cross-sectional cohort of people living with HIV on suppressive ART, N=115, and performed high dimensional flow-cytometry to then search for associations between immunological and clinical parameters and intact/total HIV DNA levels.

A number of interesting data science/ML approaches were explored on the data and the project seems a serious undertaking. However, like many other studies that have looked for these kinds of associations, there was not a very strong signal. Of course the goal of unsupervised learning is to find new hypotheses that aren't obvious to human eyes, but I felt in that context, there were (1) results slightly oversold, (2) some questions about methodology in terms mostly of reservoir levels, and (3) results were not sufficiently translated back into meaning in terms of clinical outcomes.

Strengths:

The study is evidently a large and impressive undertaking and combines many cutting edge statistical techniques with a comprehensive experimental cohort of people living with HIV, notably inclusive of populations underrepresented in HIV science. A number of intriguing hypotheses are put forward that could be explored further. Data will be shared and could be a useful repository for more specific analyses.

Weaknesses:

Despite the detailed experiments and methods, there was not a very strong signal for variable(s) predicting HIV reservoir size. The spearman coefficients are ~0.3, (somewhat weak, and acknowledged as such) and predictive models reach 70-80% prediction levels, though of sometimes categorical variables that are challenging to interpret.

There are some questions about methodology, as well as some conclusions that are not completely supported by results, or at minimum not sufficiently contextualized in terms of clinical significance. Edit, authors have substantially revised the text.

On associations: the false discovery rate correction was set at 5%, but data appear underdetermined with fewer observations than variables (144vars > 115ppts), and it isn't always clear if/when variables are related (e.g inverses of one another, for instance %CD4 and %CD8).

The modeling of reservoir size was unusual, typically intact and defective HIV DNA are analyzed on a log10 scale (both for decays and predicting rebound). Also sometimes in this analysis levels are normalized (presumably to max/min?, e.g. S5), and given the large within-host variation of level we see in other works, it is not trivial to predict any downstream impact of normalization across population vs within person. Edit, fixed.

Also, the qualitative characterization of low/high reservoir is not standard, and naturally will split by early/later ART if done as above/below median. Given the continuous nature of these data it seems throughout that predicting above/below median is a little hard to translate into clinical meaning.

Lastly, work is comprehensive and appears solid, but the code was not shared to see how calculations were performed. Edit, fixed.

Reviewer #1 (Public Review):

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

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

Comments on revised version:

The revised manuscript addresses all my concerns.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors report that activation of excitatory DREADDs in the mid-cervical spinal cord can increase inspiratory activity in mice and rats. This is an important first step toward an ultimate goal of using this, or similar, technology to drive breathing in disorders associated with decreased respiratory motor output, such as spinal injury or neurodegenerative disease.

Strengths:

Strengths of this study include a comparison of non-specific DREADD expression in the mid-cervical spinal cord versus specific targeting to ChAT-positive neurons, and the measurement of multiple respiratory-related outcomes, including phrenic inspiratory output, diaphragm EMG activity, and ventilation. The data show convincingly that DREADDs can be used to drive phrenic inspiratory activity, which in turn increases diaphragm EMG activity and ventilation.

Weaknesses:

The main limitation is that the ligand, J60, was not given to control animals without spinal DREADD expression. Since J60 may have off-target effects (PMID: 37530882), a discussion of this limitation is warranted, particularly in light of the one rat that was reported to not have detectible mCherry expression in the mid-cervical spinal cord, yet had robust increases in diaphragm output after J60 administration.

In experiments in ChAT-Cre animals, several neuronal types will express DREADDs, including non-phrenic motor neurons and some interneurons. As such, these experiments do not specifically "target" phrenic motor neurons any more so than experiments in WT animals. Experiments in ChAT-Cre animals also do not avoid inducing "non-specific expression in the vicinity of the phrenic motor nucleus". This is not a study design flaw per se, but an overinterpretation of findings.

Reviewer #1 (Public Review):

Summary:

In the manuscript entitled "Rtf1 HMD domain facilitates global histone H2B monoubiquitination and regulates morphogenesis and virulence in the meningitis-causing pathogen Cryptococcus neoformans" by Jiang et al., the authors employ a combination of molecular genetics and biochemical approaches, along with phenotypic evaluations and animal models, to identify the conserved subunit of the Paf1 complex (Paf1C), Rtf1, and functionally characterize its critical roles in mediating H2B monoubiquitination (H2Bub1) and the consequent regulation of gene expression, fungal development, and virulence traits in C. deneoformans or C. neoformans. Specially, the authors found that the histone modification domain (HMD) of Rtf1 is sufficient to promote H2B monoubiquitination (H2Bub1) and the expression of genes related to fungal mating and filamentation, and restores the fungal morphogenesis and pathogenicity defects caused by RTF1 deletion.

Strengths:

The manuscript is well-written and presents the findings in a clear manner. The findings are interesting and contribute to a better understanding of Rtf1-mediated epigenetic regulation of fungal morphogenesis and pathogenicity in a major human fungal pathogen, and potentially in other fungal species, as well.

Weaknesses:

A major limitation of this study is the absence of genome-wide information on Rtf1-mediated H2B monoubiquitination (H2Bub1), as well as a lack of detail regarding the function of the Plus3 domain. Although overexpression of HMD in the rtf1Δ mutant restored global H2Bub1 levels, it did not rescue certain critical biological functions, such as growth at 39{degree sign}C and melanin production (Figure 4C-D). This suggests that the precise positioning of H2Bub1 is essential for Rtf1's function. A comprehensive epigenetic landscape of H2Bub1 in the presence of HMD or full-length Rtf1 would elucidate potential mechanisms and shed light on the function of the Plus3 domain.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Jiao D et al reported the induction of synthetic lethal by combined inhibition of anti-apoptotic BCL-2 family proteins and WSB2, a substrate receptor in CRL5 ubiquitin ligase complex. Mechanistically, WSB2 interacts with NOXA to promote its ubiquitylation and degradation. Cancer cells deficient in WSB2, as well as heart and liver tissues from Wsb2-/- mice exhibit high susceptibility to apoptosis induced by inhibitors of BCL-2 family proteins. The anti-apoptotic activity of WSB2 is partially dependent on NOXA.

Overall, the finding, that WSB2 disruption triggers synthetic lethality to BCL-2 family protein inhibitors by destabilizing NOXA, is rather novel. The manuscript is largely hypothesis-driven, with experiments that are adequately designed and executed. However, there are quite a few issues for the authors to address, including those listed below.

Specific comments:

(1) At the beginning of the Results section, a clear statement is needed as to why the authors are interested in WSB2 and what brought them to analyze "the genetic co-dependency between WSB2 and other proteins".

(2) In general, the biochemical evidence supporting the role of WSB2 as a SOCS box-containing substrate-binding receptor of CRL5 E3 in promoting NOXA ubiquitylation and degradation is relatively weak. First, since NOXA2 binds to WSB2 on its SOCS box, which consists of a BC box for Elongin B/C binding and a CUL5 box for CUL5 binding, it is crucial to determine whether the binding of NOXA on the SOCS box affects the formation of CRL5WSB2 complex. The authors should demonstrate the endogenous binding between NOXA and the CRL5WSB2 complex. Additionally, the authors may also consider manipulating CUL5, SAG, or ElonginB/C to assess if it would affect NOXA protein turnover in two independent cell lines. Second, in all the experiments designed to detect NOXA ubiquitylation in cells, the authors utilized immunoprecipitation (IP) with FLAG-NOXA/NOXA, followed by immunoblotting (IB) with HA-Ub. However, it is possible that the observed poly-Ub bands could be partly attributed to the ubiquitylation of other NOXA binding proteins. Therefore, the authors need to consider performing IP with HA-Ub and subsequently IB with NOXA. Alternatively, they could use Ni-beads to pull down all His-Ub-tagged proteins under denaturing conditions, followed by the detection of FLAG-tagged NOXA using anti-FLAG Ab. The authors are encouraged to perform one of these suggested experiments to exclude the possibility of this concern. Furthermore, an in vitro ubiquitylation assay is crucial to conclusively demonstrate that the polyubiquitylation of NOXA is indeed mediated by the CRL5WSB2 complex.

(3) In their attempt to map the binding regions between NOXA and WSB2, the authors utilized exogenous proteins of both WSB2 and NOXA. To strengthen their findings, it would be more convincing to perform IP with exogenous wt/mutant WSB2 or NOXA and subsequently perform IB to detect endogenous NOXA or WSB2, respectively. Additionally, an in vitro binding assay using purified proteins would provide further evidence of a direct binding between NOXA and WSB2.

Reviewer #1 (Public Review):

Summary:

Building upon their famous tool for the deconvolution of human transcriptomics data (EPIC), Gabriel et al. implemented a new methodology for the quantification of the cellular composition of samples profiled with Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). To build a signature for ATAC-seq deconvolution, they first created a compendium of ATAC-seq data and derived chromatin accessibility marker peaks and reference profiles for 12 cell types, encompassing immune cells, endothelial cells, and fibroblasts. Then, they coupled this novel signature with the EPIC deconvolution framework based on constrained least-square regression to derive a dedicated tool called EPIC-ATAC. The method was then assessed using real and pseudo-bulk ATAC-seq data from human peripheral blood mononuclear cells (PBMC) and, finally, applied to ATAC-seq data from breast cancer tumors to show it accurately quantifies their immune contexture.

Strengths:

Overall, the work is of very high quality. The proposed tool is timely; its implementation, characterization, and validation are based on rigorous methodologies and results in robust estimates. The newly-generated, validation data and the code are publicly available and well-documented. Therefore, I believe this work and the associated resources will greatly benefit the scientific community.

Weaknesses:

In the benchmarking analysis, EPIC-ATAC was compared also to deconvolution methods that were originally developed for transcriptomics and not for ATAC-seq data. However, the authors described in detail the specific settings used to analyze this different data modality as robustly as possible, and they discussed possible limitations and ideas for future improvement.

Reviewer #1 (Public Review):

Summary:

Heat production mechanisms are flexible, depending on a wide variety of genetic, dietary, and environmental factors. The physiology associated with each mechanism is important to understand since loss of flexibility is associated with metabolic decline and disease.

The phenomenon of compensatory heat production has been described in some detail in publications and reviews, notably by modifying BAT-dependent thermogenesis (for example by deleting UCP1 or impairing lipolysis, cited in this paper).

These authors chose to eliminate exercise as an alternative means of maintaining body temperature. To do this, they cast either one or both mouse hindlimbs.

This paper is set up as an evaluation of a loss of function of muscle on the functionality of BAT.

Strengths:

The study is supported by a variety of modern techniques and procedures.

Weaknesses:

The authors show that cast immobilization (CI) does not work as a (passive) loss of function, instead, this procedure produces a dramatic gain of function, putting the animal under considerable stress, inducing b-adrenergic effectors, increased oxygen consumption, and IL6 expression in a variety of tissues, together with commensurate cachectic effects on muscle and fat. The BAT is put under considerable stress, super-induced but relatively poor functioning.

Thus within hours and days of CI, there is massive muscle loss (leading to high circulating BCAAs), and loss of lipid reserves in adipose and liver. The lipid cycle that maintains BAT thermogenesis is depleted and the mouse is unable to maintain body temperature.

I cannot agree with these statements in the Discussion:

"We have here shown that cast immobilization suppressed skeletal muscle thermogenesis, resulting in failure to maintain core body temperature in a cold environment."<br /> • This result could also be attributed to high stress and decreased calorie reserves. Note also: CI suppresses 50% of locomotor activity, but the actual work done by the mouse carrying bilateral casts is not taken into account.

"Thermoregulatory system in endotherms cannot be explained by thermogenesis based on muscle contraction alone, with nonshivering thermogenesis being required as a component of the ability to tolerate cold temperatures in the long term."<br /> • This statement is correct, and it clearly showcases how difficult it is to interpret results using this CI strategy. The question to the author is- which components of muscle thermogenesis are actually inhibited by CI, and what is the relative heat contribution?

This conclusion is overinterpreted:

"In conclusion, we have shown that cast immobilization induced thermogenesis in BAT that was dependent on the utilization of free amino acids derived from skeletal muscle, and that muscle-derived IL-6 stimulated BCAA metabolism in skeletal muscle. Our findings may provide new insights into the significance of skeletal muscle as a large reservoir of amino acids in the regulation of body temperature".

In terms of the production of the article - the data shown in the heat maps has oddly obscure log10 dimensions. The changes are minimal, approx. 1.5x increase/decrease and therefore significance would be key to reporting these data. Fig.3C heatmap is not suitable. What are the 6 lanes to each condition? Overall, this has little/no information.

Rather than cherry-picking for a few genes, the results could be made more rigorous using RNA-seq profiling of BAT and muscle tissues.

Reviewer #1 (Public Review):

Summary:

Previous research from the Margoliash laboratory has demonstrated that the intrinsic electrophysiological properties of one class of projection neurons in the song nucleus HVC, HVCX neurons, are similar within birds and differ between birds in a manner that relates to the bird's song. The current study builds on this research by addressing how intrinsic properties may relate to the temporal structure of the bird's song and by developing a computational model for how this can influence sequence propagation of activity within HVC during singing.

First, the authors identify that the duration of the song motif is correlated with the duration of song syllables and particularly the length of harmonic stacks within the song. They next found positive correlations between some of the intrinsic properties, including firing frequency, sag ratio, and rebound excitation area with the duration of the birds' longest harmonic syllable and some other measure of motif duration. These results were extended by examining measures of firing frequency and sag ratio between two groups of birds that were experimentally raised to learn songs that only differed by the addition of a long terminal harmonic stack in one of the groups. Lastly, the authors present an HH-based model elucidating how the timing and magnitude of rebound excitation of HVCX neurons can function to support previously reported physiological network properties of these neurons during singing.

Strengths:

By trying to describe how intrinsic properties (IPs) may relate to the structure of learned behavior and providing a potentially plausible model (see below for more on this) for how differences in IPs can relate to sequence propagation in this neural network, this research is addressing an important and challenging issue. An understanding of how cell types develop IPs and how those IPs relate to the function and output of a network is a fundamental issue. Tackling this in the zebra finch HVC is an elegant approach because it provides a quantifiable and reliable behavior that is explicitly tied to the neurons that the authors are studying. Nonetheless, this is a difficult problem, and kudos to the authors for trying to unravel this.

Correlations between harmonic stack durations and song durations are well-supported and interesting. This provides a new insight that can and will likely be used by other research groups in correlating neuronal activity patterns to song behavior and motif duration. Additionally, correlations between IPs associated with rebound excitation are also well supported in this study.

The HH-model presented is important because it meaningfully relates how high or low rebound excitation can set the integration time window for HVCX neurons. Further, the synaptic connectivity of this model provides at least one plausible way in how this functions to permit the bursting activity of HVCX neurons during singing (and potentially during song playback experiments in sleeping birds). Thus, this model will be useful to the field for understanding how this network activity intersects with 'learned' IPs in an important class of neurons in this circuit.

Weaknesses:

The main weakness of the study is that there is somewhat of a disconnect between the physiological measurements described and the key components of the circuit model presented at the end of the paper. Thus, better support could be provided to link the magnitude of rebound excitation with song temporal structure. The rebound excitation area is shown to be positively correlated with the longest harmonic stack. Does this correlation hold when the four birds with unusually long stacks (>150ms) are excluded? Is rebound excitation area positively correlated with motif duration? Additionally, rebound excitation was not considered when examining experimentally tutored birds. Further analysis of these correlations can better link this research to the model presented.

The HH model is of general interest, but I am concerned about the plausibility of some of this circuitry, particularly because synaptic connectivity underlying information flow is a central component of the model. At several steps in the model, excitatory drive onto HVCX neurons is coming from another HVCX neuron. Although disynaptic inhibition between HVCX neurons and between HVCRA and HVCX neurons is well established, I am not aware of any data indicating direct synaptic connections between HVCX neurons.

Thus, how does the model change if all excitatory drive onto HVCX neurons are coming from HVCRA neurons? Currently, the model is realized through neurons being active at syllable or gesture transitions. What does the model predict about the distribution of HVCRA neurons activity across songs if they are the exclusive excitatory input to HVCX neurons? A better consideration of these issues can improve the suitability of the model in the context of known connectivity.

If I understand the model and ideas correctly, birds with longer motifs should exhibit longer pauses in the activity of tonically active HVC interneurons during singing and they should exhibit longer post-rebound integration windows. Experimental evidence supporting either of these ideas is not provided and would strengthen this research.

Reviewer #1 (Public Review):

The authors propose a UPEC TA system in which a metabolite, c-di-GMP, acts as the AT with the toxin HipH. The idea is novel, but several key ideas are missing in regard to the relevant literature, and the experimental design is flawed. Moreover, they are absolutely not studying persister cells as Figure 1b clearly shows they are merely studying dying cells since no plateau in killing (or anything close to a plateau) was reached. So in no way has persistence been linked to c-di-GMP. Moreover, I do not think the authors have shown how the c-di-GMP sensor works. Also, there is no evidence that c-di-GMP is an antitoxin as no binding to HipH has been shown. So at best, this is an indirect effect, not a new toxin/antitoxin system as for all 7 TAs, a direct link to the toxin has been demonstrated for antitoxins.

Weaknesses:

(1) L 53: biofilm persisters are no different than any other persisters (there is no credible evidence of any different persister cells) so this reviewer suggests changing 'biofilm persisters' to 'persisters' throughout the text.

(2) L 51: persister cells do not mutate and, once resuscitated, mutate like any other growing cell so this sentence should be deleted as it promotes an unnecessary myth about persistence.

(3) L 69: please include the only metabolic model for persister cell formation and resuscitation here based on single cells (e.g., doi.org/10.1016/j.bbrc.2020.01.102 , https://doi.org/10.1016/j.isci.2019.100792 ); otherwise, you write as if there are no molecular mechanisms for persistence/resuscitation.

(4) The authors should cite in the Intro or Discussion that others have proposed similar novel TAs including a ppGpp metabolic toxin paired with an enzymatic antitoxin SpoT that hydrolyzes the toxin (http://dx.doi.org/10.1016/j.molcel.2013.04.002).

(5) Figure 1b: there are no results in this paper related to persister cells. Figure 1b simply shows dying cells were enumerated. Hence, the population of stressed cells increased, not 'persister cells' (Figure 1f), in the course of these experiments.

(6) Figure S1: I see no evidence that the authors have shown this c-di-GMP detects different c-di-GMP levels since there appears to be no data related to varying c-di-GMP concentrations with a consistent decrease. Instead, there is a maximum. What are the concentration of c-di-GMP on the X-axis for panels C, D, and E? How were c-di-GMP levels varied such that you know the c-di-GMP concentration?

(7) The viable portion of the VBNC population are persister cells so there is no reason to use VBNC as a separate term. Please see the reported errors often made with nucleic acid staining dyes in regard to VBNCs.

Reviewer #1 (Public Review):

The authors introduce DIPx, a deep learning framework for predicting synergistic drug combinations for cancer treatment using the AstraZeneca-Sanger (AZS) DREAM Challenge dataset. While the approach is innovative, I have the following concerns and comments which hopefully will improve the study's rigor and applicability, making it a more powerful tool in the real clinical world.

(1) Test Set 1 comprises combinations already present in the training set, likely leading overfitting issue. The model might show inflated performance metrics on this test set due to prior exposure to these combinations, not accurately reflecting its true predictive power on unknown data, which is crucial for discovering new drug synergies. The testing approach reduces the generalizability of the model's findings to new, untested scenarios.

(2) The model struggles with predicting synergies for drug combinations not included in its training data (showing only a Spearman correlation of 0.26 in Test Set 2). This limits its potential for discovering new therapeutic strategies. Utilizing techniques such as transfer learning or expanding the training dataset to encompass a wider range of drug pairs could help to address this issue.

(3) The use of pan-cancer datasets, while offering broad applicability, may not be optimal for specific cancer subtypes with distinct biological mechanisms. Developing subtype-specific models or adjusting the current model to account for these differences could improve prediction accuracy for individual cancer types.

(4) Line 127, "Since DIPx uses only molecular data, to make a fair comparison, we trained TAJI using only molecular features and referred to it as TAJI-M.". TAJI was designed to use both monotherapy drug-response and molecular data, and likely won't be able to reach maximum potential if removing monotherapy drug-response from the training model. It would be critical to use the same training datasets and then compare the performances. From Figure 6 of TAJI's paper (Li et al., 2018, PMID: 30054332) , i.e., the mean Pearson correlation for breast cancer and lung cancer is around 0.5 - 0.6.

The following 2 concerns have been included in the Discussion section which is great:

(1) Training and validating the model using cell lines may not fully capture the heterogeneity and complexity of in vivo tumors. To increase clinical relevance, it would be beneficial to validate the model using primary tumor samples or patient-derived xenografts.

(2) The Pathway Activation Score (PAS) is derived exclusively from primary target genes, potentially overlooking critical interactions involving non-primary targets. Including these secondary effects could enhance the model's predictive accuracy and comprehensiveness.

Reviewer #1 (Public Review):

Munday, Rosello, and colleagues compared predictions from a group of experts in epidemiology with predictions from two mathematical models on the question of how many Ebola cases would be reported in different geographical zones over the next month. Their study ran from November 2019 to March 2020 during the Ebola virus outbreak in the Democratic Republic of the Congo. Their key result concerned predicted numbers of cases in a defined set of zones. They found that neither the ensemble of models nor the group of experts produced consistently better predictions. Similarly, neither model performed consistently better than the other, and no expert's predictions were consistently better than the others. Experts were also able to specify other zones in which they expected to see cases in the next month. For this part of the analysis, experts consistently outperformed the models. In March, the final month of the analysis, the models' accuracy was lower than in other months and consistently poorer than the experts' predictions.

A strength of the analysis is the use of consistent methodology to elicit predictions from experts during an outbreak that can be compared to observations, and that are comparable to predictions from the models. Results were elicited for a specified group of zones, and experts were also able to suggest other zones that were expected to have diagnosed cases. This likely replicates the type of advice being sought by policymakers during an outbreak.

A potential weakness is that the authors included only two models in their ensemble. Ensembles of greater numbers of models might tend to produce better predictions. The authors do not address whether a greater number of models could outperform the experts.

The elicitation was performed in four months near the end of the outbreak. The authors address some of the implications of this. A potential challenge to the transferability of this result is that the experts' understanding of local idiosyncrasies in transmission may have improved over the course of the outbreak. The model did not have this improvement over time. The comparison of models to experts may therefore not be applicable to the early stages of an outbreak when expert opinions may be less well-tuned.

This research has important implications for both researchers and policy-makers. Mathematical models produce clearly-described predictions that will later be compared to observed outcomes. When model predictions differ greatly from observations, this harms trust in the models, but alternative forms of prediction are seldom so clearly articulated or accurately assessed. If models are discredited without proper assessment of alternatives then we risk losing a valuable source of information that can help guide public health responses. From an academic perspective, this research can help to guide methods for combining expert opinion with model outputs, such as considering how experts can inform models' prior distributions and how model outputs can inform experts' opinions.

Reviewer #1 (Public Review):

Summary:<br /> The authors demonstrate that, while the loss of Ezrin increases lysosomal biogenesis and function, its presence is required for the specific endocytosis of EGFR. Upon further investigation, the authors reveal that Ezrin is a crucial intermediary protein that links EGFR to AKT, leading to the phosphorylation and inhibition of TSC. TSC is a critical negative regulator of the mTORC1 complex, which is dysregulated in various diseases, making their findings a valuable addition to multiple fields of study. Their cell signaling findings are translatable to an in vivo Medaka fish model and suggest that Ezrin may play a crucial role in retinal degeneration.

Strengths:<br /> Giamundo, Intartaglia, et al. utilized unbiased proteomic and transcriptomic screens in Ezrin KO cells to investigate the mechanistic function of Ezrin in lysosome and cell signaling pathways. The authors' findings are consistent with past literature demonstrating Ezrin's role in the EGFR and mTORC1 signaling pathways. They used several cell lines, small molecule inhibitors, and cellular and in vivo knockout models to validate signaling changes through biochemical and microscopy assays. Their use of multiple advanced microscopy techniques is also impressive.

Weaknesses:<br /> While the authors demonstrated activation of TSC1 (lysosomal accumulation) and inactivation of Akt (decreased phosphorylation in TSC1), as well as decreased mTORC1 signaling in Ezrin knockout cells, direct experiments showing the rescue of mTORC1 activity by AKT and TSC1 mutants are required to confirm the linear signaling pathway and establish Ezrin as a mediator of EGFR-AKT-TSC1-mTORC1 signaling. Although the authors presented representative images from advanced microscopy techniques to support their claims, there is insufficient quantification of these experiments. Additionally, several immunoblots in the manuscript lack vital loading controls, such as input lanes for immunoprecipitations and loading controls for western blots.

Reviewer #1 (Public Review):

This manuscript validates and extends upon the sigh-generating circuit between the NMB/GRP+ RTN/parafacial neurons and the NMBR/GRPR+ preBötC neurons established in Li et al., 2016. The authors generate multiple transgenic lines that enable selective targeting of these various sub-populations of cells and demonstrate the sufficiency of each type in generating a sigh breath. Additionally, they show that NMBR and GPRP preBötC neurons are glutamatergic, have overlapping and distinct expressions, and do not express SST. Beyond this validation, the authors show that ectopic stimulation of SST neurons is sufficient to evoke sighs and that they are necessary for NMB/GRP-induced sighing. This data is the first time that preBötC neurons downstream of NMBR/GRPR neurons have been identified.

The five conclusions stated at the end of the introduction are supported by the data, but a strong emphasis throughout the manuscript is the identification of an unsubstantiated slow sigh rhythm that is produced by NMBR/GRPR neurons. To make such a novel (and quite surprising) claim requires many more studies and the conclusion is dependent on how the authors have defined a sigh. Moreover, some data within the paper conflicts with this idea.

In summary, the optogenetic and chemogenetic characterization of the neuropeptide pathway transgenic lines nicely aligns with and provides important validation of the previous study by Li et. al., 2016 and the SST neuron studies provide a new mechanism for the transformation of NMBR/GRPR neuropeptide activation into a sigh. These are important findings and they should be the points emphasized. The proposal of a slow sigh rhythm should be more rigorously established with new experiments and analysis or should be more carefully described and discussed.

Reviewer #1 (Public Review):

Summary:

Fats and lipids serve many important roles in cancers, including serving as important fuels for energy metabolism in cancer cells by being oxidized in the mitochondria. The process of fatty acid oxidation is initiated by the enzyme carnitine palmitoyltransferase 1A (CPT1A), and the function and targetability of CPT1A in cancer metabolism and biology have been heavily investigated. This includes studies that have found important roles for CPT1A in colorectal cancer growth and metastasis.

In this study, Chen and colleagues use analysis of patient samples and functional interrogation in animal models to examine the role CPT1A plays in colorectal cancer (CRC). The authors find that CPT1A expression is decreased in CRC compared to paired healthy tissue and that lower expression correlates with decreased patient survival over time, suggesting that CPT1A may suppress tumor progression. To functionally interrogate this hypothesis, the authors both use CRISPR to knockout CPT1A in a CRC cell line that expresses CPT1A and overexpress CPT1A in a CRC cell line with low expression. In both systems, increased CPT1A expression decreased cell survival and DNA repair in response to radiation in culture. Further, in xenograft models, CPT1A decreased tumor growth basally and radiotherapy could further decrease tumor growth in CPT1A-expressing tumors. As CRC is often treated with radiotherapy, the authors argue this radiosensitization driven by CPT1A could explain why CPT1A expression correlates with increased patient survival.

Lastly, Chen and colleagues sought to understand why CPT1A suppresses CRC tumor growth and sensitizes the tumors to radiotherapy in culture. The antioxidant capacity of cells can increase cell survival, so the authors examine antioxidant gene expression and levels in CPT1A-expressing and non-expressing cells. CPT1A expression suppresses the expression of antioxidant metabolism genes and lowers levels of antioxidants. Antioxidant metabolism genes can be regulated by the FOXM1 transcription factor, and the authors find that CPT1A expression regulates FOXM1 levels and that antioxidant gene expression can be partially rescued in CPT1A-expressing CRC cells. This leads the authors to propose the following model: CPT1A expression downregulates FOXM1 (via some yet undescribed mechanism) which then leads to decreased antioxidant capacity in CRC cells, thus suppressing tumor progression and increasing radiosensitivity. This is an interesting model that could explain the suppression of CPT1A expression in CRC, but key tenets of the model are untested and speculative.

Strengths:

• Analysis of CPT1A in paired CRC tumors and non-tumor tissue using multiple modalities combined with analysis of independent datasets rigorously show that CPT1A is downregulated in CRC tumors at the RNA and protein level.

• The authors use paired cell line model systems where CPT1A is both knocked out and overexpressed in cell lines that endogenously express or repress CPT1A respectively. These complementary model systems increase the rigor of the study.

• The finding that a metabolic enzyme generally thought to support tumor energetics actually is a tumor suppressor in some settings is theoretically quite interesting.

Weaknesses:

• The authors propose that CPT1A expression modulates antioxidant capacity in cells by suppressing FOXM1 and that this pathway alters CRC growth and radiotherapy response. However, key aspects of this model are not tested. The authors do not show that FOXM1 contributes to the regulation of antioxidant levels in CRC cells and tumors or if FOXM1 suppression is key to the inhibition of CRC tumor growth and radiosensitization by CPT1A. Thus, the model the authors propose is speculative and not supported by the existing data.

• The authors propose two mechanisms by which CPT1A expression triggers radiosensitization: decreasing DNA repair capacity (Figure 3) and decreasing antioxidant capacity (Figure 5). However, while CPT1A expression does alter these capacities in CRC cells, neither is functionally tested to determine if altered DNA repair or antioxidant capacity (or both) are the reason why CRC cells are more sensitive to radiotherapy or are delayed in causing tumors in vivo. Thus, this aspect of the proposed model is also speculative.

• The authors find that CPT1A affects radiosensitization in cell culture and assess this in vivo. In vivo, CPT1A expression slows tumor growth even in the absence of radiotherapy, and radiotherapy only proportionally decreases tumor growth to the same extent as it does in CPT1A non-expressing CRC tumors. The authors propose from this data that CPT1A expression also sensitizes tumors to radiotherapy in vivo. However, it is unclear whether CPT1A expression causes radiosensitization in vivo or if CPT1A expression acts as an independent tumor suppressor to which radiotherapy has an additive effect. Additional experiments would be necessary to differentiate between these possibilities.

• The authors propose in Figure 3 that DNA repair capacity is inhibited in CRC cells by CPT1A expression. However, the gH2AX immunoblots performed in Figure 3H-I that measure DNA repair kinetics are not convincing that CPT1A expression impairs DNA repair kinetics. Separate blots are shown for CPT1A expressing and non-expressing cell lines, not allowing for rigorous comparison of gH2AX levels and resolution as CPT1A expression is modulated.

• There are conflicting studies (PMID: 37977042, 29995871) that suggest that CPT1A is overexpressed in CRC and contributes to tumor progression rather than acting as a tumor suppressor as the authors propose. It would be helpful for readers for the authors to discuss these studies and why there is a discrepancy between them.

Reviewer #1 (Public Review):

In this paper, the authors provide data to support the existence of a regulatory pathway starting with SPI1-driven ZFP36L1 expression, that goes on to downregulate HDAC3 expression at the transcript level, leading to PD-L1 upregulation due to implied enhanced acetylation of its promoter region. This is therefore an interesting pathway that adds to our understanding of how PD-L1 expression is controlled in gastric cancer. However, this is likely one of many possible pathways that impact PD-L1 expression, which is likely equally important. Thus, while potentially interesting, this is more additive information to the literature rather than a fundamentally new concept/finding.

Overall, there are many experiments presented, which appear to be of good quality, however, there are a number of issues with this that need attention. Moreover, the text is often difficult to follow, partly due to the standard of English, but mainly due to the sparsity of detail in the results section and figure legends. Thus providing an overall assessment of data conclusiveness is not possible at this time. This is exacerbated by frequently extrapolating conclusions beyond what is actually shown in an individual experiment.

Major issues:

(1) All the figure legends need to expand significantly, so it is clear what is being presented. All experiments showing data quantification need the numbers of independent biological replicates to be added, plus an indication of what the P-values are associated with the asterisks (and the tests used).

(2) Related to point 1, the description of the data in the text needs to expand significantly, so the figure panels are interpretable. Examples are given below but this is not an exhaustive list.

(3) The addition of "super-enhancer-driven" to the title is a distraction. This is the starting point but the finding is portrayed by the last part of the title. Moreover, it is not clear why this is a super enhancer rather than just a typical enhancer as only one seems to be relevant and functional. I suggest avoiding this term after initial characterisations.

(4) The descriptions of Figures 1B, C, and D are very poor. How for example do you go from nearly 2000 SE peaks to a couple of hundred target genes? What are the other 90% doing? What is the definition of a target gene? This whole start section needs a complete overhaul to make it understandable and this is important as is what leads us to ZFP36L1 in the first place.

(5) It is impossible to work out what Figures 1F, H, and I are from the accompanying text. The same applies to supplementary Figure S1D. Figure 1G is not described in the results.

(6) What is Figure 2A? There is no axis label or description.

(7) Why is CD274 discussed in the text from Figure 2E but none of the other genes? The rationale needs expanding.

(8) Figure 2G needs zooming in more over the putative SE region and the two enhancers labelling. This looks very strange at the moment and does not show typical peak shapes for histone acetylation at enhancers.

(9) The use of JQ1 does not prove something is a super enhancer, just that it is BRD4 regulated and might be a typical enhancer.

(10) An explanation of how the motifs were identified in E1 is needed. Enrichment over what? Were they purposefully looking for multiple motifs per enhancer? Otherwise what it all comes down to later in the figure is a single motif, and how can that be "enriched"?

(11) A major missing experiment is to deplete rather than over-express SPI1 for the various assays in Figure 4.

(12) The authors start jumping around cell lines, sometimes with little justification. Why is MGC803 used in Figure 4I rather than MKN45? This might be due to more endogenous SPI1. However, this does not make sense in Figure 5M, where ZFP36L is overexpressed in this line rather than MKN45. If SPI1 is already high in MGC803, then the prediction is that ZFP36L1 should already be high. Is this the case?

(13) In Figure 5, HDAC3 should also be depleted to show opposite effects to over-expression (as the latter could be artefactual). Also, direct involvement should be proven by ChIP.

(14) Figure 5G and H are not discussed in the text.

(15) Figure 6C needs explaining. Why are three patients selected here? Are these supposed to be illustrative of the whole cohort? What sub-type of GC are these?

(16) In Figure 6E onwards, they switch to MFC cell line. They provide a rationale but the key regulatory axis should be sown to also be operational in these cells to use this as a model system.

Reviewer #1 (Public Review):

Summary:

The manuscript asks the question of whether astrocytes contribute to behavioral deficits triggered by early life stress. This question is tested by experiments that monitor the effects of early life stress on anxiety-like behaviors, long-term potentiation in the lateral amygdala, and immunohistochemistry of astrocyte-specific (GFAP, Cx43, GLT-1) and general activity (c-Fos ) markers. Secondarily, astrocyte activity in the lateral amygdala is impaired by viruses that suppress gap-junction coupling or reduce astrocyte Ca2+ followed by behavioral, synaptic plasticity, and c-Fos staining. Early life stress is found to reduce the expression of GFAP and Cx43 and to induce translocation of the glucocorticoid receptor to astrocytic nuclei. Both early life stress and astrocyte manipulations are found to result in the generalization of fear to neutral auditory cues. All of the experiments are done well with appropriate statistics and control groups. The manuscript is very well-written and the data are presented clearly. The authors' conclusion that lateral amygdala astrocytes regulate amygdala-dependent behaviors is strongly supported by the data. However, the extent to which astrocytes contribute to behavioral and neuronal consequences of early life stress remains open to debate.

Strengths:

A strong combination of behavioral, electrophysiology, and immunostaining approaches is utilized and possible sex differences in behavioral data are considered. The experiments clearly demonstrate that disruption of astrocyte networks or reduction of astrocyte Ca2+ provokes generalization of fear and impairs long-term potentiation in the lateral amygdala. The provocative finding that astrocyte dysfunction accounts for a subset of behavioral effects of early life stress (e.g. not elevated plus or distance traveled observations) is also perceived as a strength.

Weaknesses:

The main weakness is the absence of more direct evidence that behavioral and neuronal plasticity after early life stress can be attributed to astrocytes. It remains unknown what would happen if astrocyte activity were disrupted concurrently with early life stress or if the facilitation of astrocyte Ca2+ would attenuate early life stress outcomes. As is, the only evidence that early life stress involves astrocytes is nuclear translocation of GR and downregulation of GFAP and Cx43 in Figure 3 which may or may not provoke astrocyte Ca2+ or astrocyte network activity changes.

Reviewer #1 (Public Review):<br /> The manuscript by Lucie Oriol et al. revisits the understanding of interneurons in the ventral tegmental area (VTA). The study challenges the traditional notion that VTA interneurons exclusively form local synapses within the VTA. Key findings of the study indicate that VTA GABA and glutamate projection neurons also make local synapses within the VTA. This evidence suggests that functions previously attributed to VTA interneurons could be mediated by these projection neurons.

The study tested four genetic markers-Parvalbumin (PV), Somatostatin (SST), Mu-opioid receptor (MOR), and Neurotensin (NTS)-to determine if they selectively label VTA interneurons. The findings indicate that these markers label VTA projection neurons rather than selectively identifying interneurons. Using a combination of anatomical tracing and brain slice physiological recordings, the study demonstrates that VTA projection neurons make functional inhibitory or excitatory synapses locally within the VTA. These data challenge the conventional view that VTA GABA neurons are purely interneurons and suggest that inhibitory projection neurons can serve functions previously attributed to VTA interneurons. Thus, some functions traditionally ascribed to interneurons may be carried out by projection neurons with local synapses. This has significant implications for understanding the neural circuits underlying reward, motivation, and addiction.

Reviewer #1 (Public Review):

In this study, Franke et al. explore and characterize color response properties across primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The authors use awake 2P imaging to define the spectral response properties of visual interneurons in layer 2/3. They find that opponent responses are more pronounced at photopic light levels, and that diversity in color opponent responses exists across the visual field, with green ON/ UV OFF responses more strongly represented in the upper visual field. This is argued to be relevant for the detection of certain features that are more salient when using chromatic space, possibly due to noise reduction. In the revised version, Franke et al. have addressed the potential pitfalls in the discussion, which is an important point for the non-expert reader. Thus, this study provides a solid characterization of the color properties of V1 and is a valuable addition to visual neuroscience research.

Summary:

This paper studies the genetic factors contributing to childhood obesity. Through a comprehensive analysis integrating genome-wide association study (GWAS) data with 3D genomic datasets across 57 human cell types, consisting of Capture-C/Hi-C, ATAC-seq, and RNA-seq, the study identifies significant genetic contributions to obesity using stratified LD score regression, emphasizing the enrichment of genetic signals in pancreatic alpha cells and identification of significant effector genes at obesity-associated loci such as BDNF, ADCY3, TMEM18, and FTO. Additionally, the study implicated ALKAL2, a gene responsive to inflammation in nerve nociceptors, as a novel effector gene at the TMEM18 locus. This suggests a role for inflammatory and neurological pathways in obesity's pathogenesis which was supported through colocalization analysis using eQTL derived from the GTEx dataset. This comprehensive genomic analysis sheds light on the complex genetic architecture of childhood obesity, highlighting the importance of cellular context for future research and the development of more effective strategies.

Strengths:

Overall, the paper has several strengths, including leveraging large-scale, multi-modal datasets, using computational reasonable tools, and having an in-depth discussion of the significant results.

Weaknesses:

(1) The results are somewhat inconclusive or not validated.

The overall results are carefully designed, but most of the results are descriptive. While the authors are able to find additional evidence either from the literature or explain the results with their existing knowledge, none of the results have been biologically validated. Especially, the last three result sections (signaling pathways, eQTLs, and TF binding) further extended their findings, but the authors did not put the major results into any of the figures in the main text.

(2) Some technical details are missing.

While the authors described all of their analysis steps, a lot of the time, they did not mention the motivation. Sometimes, the details were also omitted. For example:

- The manuscript would benefit from a detailed explanation of the methods used to define cREs, particularly the process of intersecting OCRs with chromatin conformation data. The current description does not fully clarify how the cREs are defined.

- How did the authors define a contact region?

- The manuscript would benefit from an explanation regarding the rationale behind the selection of the 57 human cell types analyzed. it is essential to clarify whether these cell types have unique functions or relevance to childhood development and obesity.

- I wonder whether the used epigenome datasets are all from children. Although the authors use literature to support that body weight and obesity remain stable from infancy to adulthood, it remains uncertain whether epigenomic data from other life stages might overlook significant genetic variants that uniquely contribute to childhood obesity.

- Given that the GTEx tissue samples are derived from adult donors, there appears to be a mismatch with the study's focus on childhood obesity. If possible, identifying alternative validation strategies or datasets more closely related to the pediatric population could strengthen the study's findings.

(3) The writing needs to improve.

Reviewer #1 (Public Review):

Based on previous publications suggesting a potential role for miR-26b in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH), the researchers aim to clarify its function in hepatic health and explore the therapeutical potential of lipid nanoparticles (LNPs) to treat this condition. First, they employed both whole-body and myeloid cell-specific miR-26b KO mice and observed elevated hepatic steatosis features in these mice compared to WT controls when subjected to WTD. Moreover, livers from whole-body miR-26b KO mice also displayed increased levels of inflammation and fibrosis markers. Kinase activity profiling analyses revealed distinct alterations, particularly in kinases associated with inflammatory pathways, in these samples. Treatment with LNPs containing miR-26b mimics restored lipid metabolism and kinase activity in these animals. Finally, similar anti-inflammatory effects were observed in the livers of individuals with cirrhosis, whereas elevated miR-26b levels were found in the plasma of these patients in comparison with healthy control. Overall, the authors conclude that miR-26b plays a protective role in MASH and that its delivery via LNPs efficiently mitigates MASH development.

The study has some strengths, most notably, its employ of a combination of animal models, analyses of potential underlying mechanisms, as well as innovative treatment delivery methods with significant promise. However, it also presents numerous weaknesses that leave the research work somewhat incomplete. The precise role of miR-26b in a human context remains elusive, hindering direct translation to clinical practice. Additionally, the evaluation of the kinase activity, although innovative, does not provide a clear molecular mechanisms-based explanation behind the protective role of this miRNA.

Therefore, to fortify the solidity of their conclusions, these concerns require careful attention and resolution. Once these issues are comprehensively addressed, the study stands to make a significant impact on the field.

Reviewer #2 (Public Review):

Summary:<br /> In this work, the authors aim to better understand how C. elegans detects and responds to heat-killed (HK) E. coli, a low-quality food. They find that HK food activates two canonical stress pathways, ER-UPR and innate immunity, in the nervous system to promote food aversion. Through the creative use of E. coli genetics and metabolomics, the authors provide evidence that the altered carbohydrate content of HK food is the trigger for the activation of these stress responses and that supplementation of HK food with sugars (or their biosynthetic product, vitamin C), reduces stress pathway induction and food avoidance. This work makes a valuable addition to the literature on metabolite detection as a mechanism for evaluation of nutritional value; it also provides some new insight into physiologically relevant roles of well-known stress pathways in modulating behavior.

Strengths:<br /> -The work addresses an important question by focusing on understanding how the nervous system evaluates food quality and couples this to behavioral change.<br /> -The work takes full advantage of the tools available in this powerful system and builds on extensive previous studies on feeding behavior and stress responses in C. elegans.<br /> -Creative use of E. coli genetics and metabolite profiling enabled identification of carbohydrate metabolism as a candidate source of food-quality signals.<br /> -For the most part, the studies are rigorous and logically designed, providing good support for the authors' model.

Weaknesses:<br /> -The authors' claim that they can detect induction of hsp-4 and irg-5 expression in neurons (Fig 1-S2A) requires further support. The two tail cells shown are quite a bit larger than would by typically expected for neurons. The rescue they observe by neuronal expression is largely convincing, so it's quite possible that these pathways do indeed function in neurons, but that their level of induction in the nervous system is below reporter detection limits (or is 'swamped out' by much higher levels of expression in the intestine).<br /> -The authors conclude that "the induction of Pirg-5::GFP was abolished in pmk-1 knockdown animals fed with HK-E. coli" (Fig 2D). Because a negative control for induction (e.g., animals fed with control E. coli) is not shown, this conclusion must be regarded as tentative.<br /> -The effect sizes in the food-preference assay shown in Figure 5 are extremely small and do not provide strong support for the strong conclusions about the role of stress response pathways in food preference behavior.

Reviewer #1 (Public Review):

The manuscript by Li et al. investigates the metabolism-independent role of nuclear IDH1 in chromatin state reprogramming during erythropoiesis. The authors describe accumulation and redistribution of histone H3K79me3, and downregulation of SIRT1, as a cause for dyserythropoiesis observed due to IDH1 deficiency. The authors studied the consequences of IDH1 knockdown, and targeted knockout of nuclear IDH1, in normal human erythroid cells derived from hematopoietic stem and progenitor cells and HUDEP2 cells respectively. They further correlate some of the observations such as nuclear localization of IDH1 and aberrant localization of histone modifications in MDS and AML patient samples harboring IDH1 mutations. These observations are intriguing from a mechanistic perspective and they hold therapeutic significance, however there are major concerns that make the inferences presented in the manuscript less convincing.

(1) The authors show the presence of nuclear IDH1 both by cell fractionation and IF, and employ an efficient strategy to knock out nuclear IDH1 (knockout IDH1/ Sg-IDH1 and rescue with the NES tagged IDH1/ Sg-NES-IDH1 that does not enter the nucleus) in HUDEP2 cells. However, some important controls are missing.<br /> A) In Figure 3C, for IDH1 staining, Sg-IDH1 knockout control is missing.<br /> B) Wild-type IDH1 rescue control (ie., IDH1 without NES tag) is missing to gauge the maximum rescue that is possible with this system.

(2) Considering the nuclear knockout of IDH1 (Sg-NES-IDH1 referenced in the previous point) is a key experimental system that the authors have employed to delineate non-metabolic functions of IDH1 in human erythropoiesis, some critical experiments are lacking to make convincing inferences.<br /> A) The authors rely on IF to show the nuclear deletion of Sg-NES-IDH1 HUDEP2 cells. As mentioned earlier since a knockout control is missing in IF experiments, a cellular fractionation experiment (similar to what is shown in Figure 2F) is required to convincingly show the nuclear deletion in these cells.<br /> B) Since the authors attribute nuclear localization to a lack of metabolic/enzymatic functions, it is important to show the status of ROS and alpha-KG in the Sg-NES-IDH1 in comparison to control, wild type rescue, and knockout HUDEP2 cells. The authors observe an increase of ROS and a decrease of alpha-KG upon IDH1 knockdown. If nuclear IDH1 is not involved in metabolic functions, is there only a minimal or no impact of the nuclear knockout of IDH1 on ROS and alpha-KG, in comparison to complete knockout? These studies are lacking.<br /> C) Authors show that later stages of terminal differentiation are impacted in IDH1 knockdown human erythroid cells. They also report abnormal nuclear morphology, an increase in euchromatin, and enucleation defects. However, the authors only report abnormal nuclear morphology in Sg-NES-IDH1 cells, as evaluated by cytospins. It is important to show the status of the other phenotypes (progression through terminal differentiation, euchromatin %, and enucleation) similar to the quantitations in the IDH1 knockdown cells.

(3) The authors report abnormal nuclear phenotype in IDH1 deficient erythroid cells. It is not clear what parameters are used here to define and quantify abnormal nuclei. Based on the cytospins (eg., Figure 1A, 3D) many multinucleated cells are seen in both shIDH1 and Sg-NES-IDH1 erythroid cells, compared to control cells. Importantly, this phenotype and enucleation defects are not rescued by the administration of alpha-KG (Figures 1E, F). The authors study these nuclei with electron microscopy and report increased euchromatin in Figure 4B. However, there is no discussion or quantification of polyploidy/multinucleation in the IDH1 deficient cells, despite their increased presence in the cytospins.

A) PI staining followed by cell cycle FACS will be helpful in gauging the extent of polyploidy in IDH1 deficient cells and could add to the discussions of the defects related to abnormal nuclei.<br /> B) For electron microscopy quantification in Figures 4B and C, how the quantification was done and the labelling of the y-axis (% of euchromatin and heterochromatin) in Figure 4 C is not clear and is confusingly presented. The details on how the quantification was done and a clear label (y-axis in Figure 4C) for the quantification are needed.<br /> C) As mentioned earlier, what parameters were used to define and quantify abnormal nuclei (e.g. Figure 1A) needs to be discussed clearly. The red arrows in Figure 1A all point to bi/multinucleated cells. If this is the case, this needs to be made clear.

(4) The authors mention that their previous study (reference #22) showed that ROS scavengers did not rescue dyseythropoiesis in shIDH1 cells. However, in this referenced study they did report that vitamin C, a ROS scavenger, partially rescued enucleation in IDH1 deficient cells and completely suppressed abnormal nuclei in both control and IDH1 deficient cells, in addition to restoring redox homeostasis by scavenging reactive oxygen species in shIDH1 erythroid cells. In the current study, the authors used ROS scavengers GSH and NAC in shIDH1 erythroid cells and showed that they do not rescue abnormal nuclei phenotype and enucleation defects. The differences between the results in their previous study with vitamin C vs GSH and NAC in the context of IDH1 deficiency need to be discussed.

(5) The authors describe an increase in euchromatin as the consequential abnormal nuclei phenotype in shIDH1 erythroid cells. However, in their RNA-seq, they observe an almost equal number of genes that are up and down-regulated in shIDH1 cells compared to control cells. If possible, an RNA-Seq in nuclear knockout Sg-NES-IDH1 erythroid cells in comparison with knockout and wild-type cells will be helpful to tease out whether a specific absence of IDH1 in the nucleus (ie., lack of metabolic functions of IDH) impacts gene expression differently.

(6) In Figure 8, the authors show data related to SIRT1's role in mediating non-metabolic, chromatin-associated functions of IDH1.<br /> A) The authors show that SIRT1 inhibition leads to a rescue of enucleation and abnormal nuclei. However, whether this rescues the progression through the late stages of terminal differentiation and the euchromatin/heterochromatin ratio is not clear.<br /> B) In addition, since the authors attribute a role of SIRT1 in mediating non-metabolic chromatin-associated functions of IDH1, documenting ROS levels and alpha-KG is important, to compare with what they showed for shIDH1 cells.

(7) In Figure 4 and Supplemental Figure 8, the authors show the accumulation and altered cellular localization of H3K79me3, H3K9me3, and H3K27me2, and the lack of accumulation of other three histone modifications they tested (H3K4me3, H3K35me4, and H3K36me2) in shIDH1 cells. They also show the accumulation and altered localization of the specific histone marks in Sg-NES-IDH1 HUDEP2 cells.<br /> A) To aid better comparison of these histone modifications, it will be helpful to show the cell fractionation data of the three histone modifications that did not accumulate (H3K4me3, H3K35me4, and H3K36me2), similar to what was shown in Figure 4E for H3K79me3, H3K9me3, and H3K27me2).<br /> B) Further, the cell fractionation and staining for histone marks is done in human primary erythroid cells on day15 of terminal differentiation, and these studies revealed that H3K79me3, H3K9me3, and H3K27me2 were retained in the nucleus in shIDH1 cells unlike a cellular localization observed in control cells. The authors cite reference #40 in relation to the cellular localization of histones - in this study, it was shown that the cellular export of histone to cytosol happens during later stages of terminal differentiation. In the current manuscript, the authors observe nuclear IDH1 throughout erythropoiesis and have shown this at both early and late time points of differentiation (between day7 to day15 of differentiation in primary erythroid cells, between day0 to day8 in HUDEP2 cells) in Figure 2. To help correlate the dynamics of localization and to discuss the mechanism for the retention of histone marks in the nucleus in IDH1 deficient cells, it will be helpful to show the cellular location of histone marks using cell fractionations for both early and late time points in terminal erythroid differentiation, similar to what they showed for IDH1 localization studies.<br /> C) Among the three histone marks that are dysregulated in IDH1 deficient cells (H3K79me3, H3K9me3, and H3K27me2), the authors show via ChIP-seq (Fig5) that H3K79me3 is the critical factor. However, the ChIP-seq data shown here lacks many details and this makes it hard to interpret the data. For example, in Figure 5A, they do not mention which samples the data shown correspond to (are these differential peaks in shIDH1 compared to shLuc cells?). There is also no mention of how many replicates were used for the ChIP seq studies.

Reviewer #1 (Public Review):

Summary:

The authors applied a domain adaptation method using the principal of optimal transport (OT) to superimpose read count data onto each other. While the title suggests that the presented method is independent from and performs better than other methods of bias correction, the presented work uses a self-implemented version of GC bias correction apart of the OT domain adaptation. Performance comparisons were done both on normalized read counts as well as on copy number profiles which is already the complete set of presented use cases. Results involving copy number profiles from iChorCNA were also subjected to the bias correction measures implemented there. It is not clear at many points which correction method actually causes the observed performance.

Strengths:

The quality of superimposing distributions of normalized read counts (and copy number profiles) was sufficiently shown using uniformly distributed p-values in the interval of 0 to 1 for healthy controls D7 and D8 which differed in the choice of library preparation kit.

The ability to select a sample from the source domain for samples in the target domain was demonstrated.

Weaknesses:

Experiment Design:

The chosen bias correction methods are not explicitly designed for nor aimed at domain adaptation. The benchmark against GC bias correction while doing GC bias correction during the OT procedure is probably the most striking flaw of the entire work. GC bias correction has the purpose of correction GC biases, wherever present, NOT correcting categorical pre-analytical variables of undefined character. A more thorough examination of the presented results should address why plain iChor CNA is the best performing "domain adaptation" in some cases. Also, the extent to which the implemented GC bias correction is contributing to the performance increase independent of the OT procedure should be assessed separately in each case.<br /> Moreover, the center-and-scale standardization is probably not the most relevant contestant in domain adaptation that is out there.

Comparison of cohorts (domains) - especially healthy from D7 and D8 - it is not described which type of ChIP analysis was done for the healthy controls of the D7 domain. The utilized library preparation kit implies that D7 represents a subset of available cfDNA in a plasma sample by precipitating only certain cfDNA fragments to which undisclosed type of protein was bound. Even if the type of protein turns out to be histones, the extracted subset of cfDNA should not be regarded as coming from the same distribution of cfNDAs. For example, fragments with sub-mononucleosomal length would be depleted in the ChIP-seq data set while these could be extracted in an untargeted cfDNA sequencing data set. It needs to be clarified why the authors deem D7 and D8 healthy controls to be identical with regards to SCNA analysis. Best start with the protein targets of D7 ChIP-seq samples.

From the Illumina TruSeq ChIP product description page:<br /> "TruSeq ChIP Libary Preparation Kits provide a simple, cost-effective solution for generating chromatin immunoprecipitation sequencing (ChIP-Seq) libraries from ChIP-derived DNA. ChIP-seq leverages next-generation sequencing (NGS) to quickly and efficiently determine the distribution and abundance of DNA-bound protein targets of interest across the genome."

Redundancy:

Some parts throughout the results and discussion part reappear in the methods. The description of the methodology should be concentrated in the method section and only reiterated in a summarizing fashion where absolutely necessary.<br /> Unnecessary repetition inflate the presented work which is not appealing to the reader. Rather include more details of the utilized materials and methods in the corresponding section.

Transparency:

At the time point of review, the code was not available under the provided link.<br /> A part of the healthy controls from D8 is not contained under the provided accession (367 healthy samples are available in the data base vs. sum of D7 and D8 healthy controls is 499)

Neither in the paper nor in reference 4 is an explanation of what was targeted with the ChIP-seq approach.

Consistency:

It is not evident why a ChIP-seq library prep kit was used (sample cohorts designated as D7). The DNA isolation procedure was not presented as having an immunoprecipitation step. Furthermore, it is not clear which DNA bound proteins were targeted during ChIP seq, if such an immunoprecipitation was actually carried out.The authors self-implemented a GC bias correction procedure although they already mentioned other procedures earlier like LIQUORICE. Also, there already exist tools that can be used to correct GC bias, like deepTools (github.com/deeptools/deepTools). Other GC bias correction algorithms designed specifically for cfDNA would be Griffin (github.com/adoebley/Griffin) and GCparagon (github.com/BGSpiegl/GCparagon). When benchmarking against state-of-the-art cfDNA GC bias correction, these algorithms should appear in a relevant scientific work, somewhere other than the introduction, preferably in the results section. It should be shown that the chosen GC bias correction method is performing best under the given circumstances.

Accuracy:

Use clear labels for each group of samples. The domain number is not sufficient to effectively distinguish sample groups. Already the source name plus a simple enumeration would improve the clarity at some points.

The healthy controls of D7 and D8 are described but the numbers do not add up (257 healthy controls in line 227 vs. 260 healthy controls in line 389). Please double check this and use representative sample cohort labels in the materials description for improved clarity!

Avoid statements like "the rest" when talking about a mixed set of samples. It is not clear how many samples from which domain are addressed.

For optimal transport, knowledge about the destination is required ("where do I want to transport to?") and, thus, the proposed method can never be unsupervised. It is always necessary to know the label of both the source and target domains. In practice, this is not often the case and users might fall prey to the error of superimposing data that is actually separated by valid differences in some experimental variables.

Seemingly arbitrary cutoff values are mentioned. For example, it is not clear if choosing "the cutoff that produced the highest MCCs" is meant across methods or for each method separately (are the results for each method reported that also resulted in the highest MCC for that method?).

The Euclidean metric for assessing the similarity of (normalized) read counts is questionable for a high dimensional space: read counts are assessed for 1 Mb genomic intervals which yields around 3000 intervals (dimensions), depending on the number of excluded intervals (which was not described in more detail). There might be more appropriate measures in this high dimensional space.

It is sometimes not clear what data actually is presented. An example would be the caption of Figure 2, (C): it is suggested that all (320) ovarian cancer cases are shown in one copy number profile.

Furthermore, the authors do not make a distinction between male and female samples. A clarification is needed why the authors think SCNAs of ovarian cancer samples should be called against a reference set that contains male controls.<br /> The procedure would likely benefit from a strict separation of male and female cases which would also allow for chrX (and chrY) being included in downstream analysis.

The GC bias and mappability correction implicitly done by iChorCNA for the SCNA profile comparison is presented as "no correction" which is highly misleading. (for clarification, this is also deemed inappropriate, not just inaccurate))

The majority of interpretations presented procedure does not give any significant improvement regarding the similarity of copy number profiles are off and in many instances favor the OT procedure in an unscientific and highly inappropriate manner.

Apart of duplicate marking (which is not specified any further - provide the command(s)!), there is no information on which read (pairs) were used (primary, secondary, supplementary, mapped in a proper pair, fragment length restrictions, clipping restrictions, etc.). The authors should explain why base quality score re-calibration was done as this might be an unnecessary step if the base quality values are not used later on.

The adaptation method presented as "center-and-scale standardization" is inappropriate for unbalanced cancer profiles since it assumes the presence of identical SCNAs in all samples belonging to the same cancer entity.<br /> Please explain why normalizing 1 Mb genomic intervals to the average copy number across different cancer samples should be valid or use another domain adaptation method for performance comparison.

Statements like in line 83 (unsupervised DA) are plain wrong because transport from one domain to another requires the selection of a target domain based on a label, e.g., based on health status, cancer entity, or similar.

Relevance and Appropriateness:

Many of the presented results are not relevant or details of the procedure were incomprehensible or incomplete: the results presented in table 2 - sample assignment. The Euclidean metric seems to be inappropriate for high dimensional data. Also the selection of the cutoff based on Euclidean distance seems to enable the optimization in favor of the OT procedure. It is hypothesized that there might exist other cutoff values for which the selection of samples form the source domain would also work for other correction methods but this is not further described. It could simply be the case that OT can assign a relationship between domains

The statement that there are no continuous pre-analytical variables is wrong (304). The effect of target depth-of-coverage (DoC) was not analyzed although this represents one of the most common (continuous) and difficult to control variables in NGS data analysis. The inclusion of multiple samples from a single patient in a cohort likely represents introduction of a confounding factor ["contamination"] to the model training procedure: the temporal difference that lies between the taken samples of that patient represents leakage of information. As far as can be told from the presented data, this potential bias has not been ruled out (e.g., exclusion of all samples beyond the first from each patient or alternatively: picking all samples of a patient either for the training set or the test set).

Conscientiousness:

Statements like "good"/"best" on their own should be avoided. A clear description of why a certain procedure/methodology/algorithm performs better should be preferred in scientific writing (e.g., "highest MCC values" instead of "best MCC values").<br /> Otherwise, such statements represent mere opinions of the author rather than an unbiased evaluation of the results.<br /> The domain D8 of healthy controls seems to contain samples from multiple sources (some published other in-house). Contrary to the data availability statement (533), not all healthy control samples of the HEMA data set are available from ArrayExpress

Other Major Concerns:

Potential Irrelevance:

The manuscript represents a mere performance assessment of the proposed sWGS per-bin-read-count fitting procedure and, thus, a verification in its character, not a validation (although the model training itself was "validated" - but this is to be viewed separately from the validity of the achieved correction in a biological context). A proper (biological) validation is missing.

It is of utmost importance that parameters of the adapted (transported) samples -that lie outside of what has been optimized to be highly similar- are checked to actually validate the procedure. Especially biological signals and genome-wide parameters (GC content distribution before/after transport) need to be addressed also in hindsight of the rampant criticism towards GC bias correction by the authors. At no point in the manuscript was GC bias addressed properly, i.e., how much of an improvement is expected from GC bias correction if there is no significant GC bias?

The (potential - not clear so far) ability of making ChIP-seq data look like cfDNA data (even if only the copy number profiles SCNAs appear highly similar) raises the concern of potential future users of the tool to superimpose domains that should not be superimposed form a biological point of view because the true domain the superimposed cohorts belong to are different. The ability to superimpose anything onto anything s troubling. There is no control mechanism that allows for failure in cases where the superposition is invalid.

Chromosome X was excluded which could be avoided if data sets were split according to biological sex.

The difference between the distributions was never attributed to GC bias, hence, the benchmark against GC bias correction tools might not be relevant in the first place.

Stability of OT data transformation:

The authors state that the straight forward choice of lambda resulted in many occasions where disruptions (of unspecified nature and amplitude) are introduced in the copy number profiles of transformed data. It is not evident from the proposed work to which extent this behavior was removed from the procedure and if it can occur and how the user could resolve such a problem on their own.

In summary, the presented work needs considerable adaptation and additions before it can actually be considered a valuable contribution to the liquid biopsy field.

Reviewer #1 (Public Review):

Summary:

The paper sets out to examine the social recognition abilities of a 'solitary' jumping spider species. It demonstrates that based on vision alone spiders can habituate and dishabituate to the presence of conspecifics. The data support the interpretation that these spiders can distinguish between conspecifics on the basis of their appearance.

Strengths:

The study presents two experiments. The second set of data recapitulates the findings of the first experiment with an independent set of spiders, highlighting the strength of the results. The study also uses a highly quantitative approach to measuring relative interest between pairs of spiders based on their distance.

Weaknesses:

The study design is overly complicated, missing key controls, and the data presented in the figures are not clearly connected to the study. The discussion is challenging to understand and appears to make unsupported conclusions.

(1) Study design: The study design is rather complicated and as a result, it is difficult to interpret the results. The spiders are presented with the same individual twice in a row, called a habituation trial. Then a new individual is presented twice in a row. The first of these is a dishabituation trial and the second is another habituation trial (but now habituating to a second individual). This is done with three pairings and then this entire structure is repeated over three sessions. The data appear to show the strong effects of differences between habituation and dishabituation trials in the first session. The decrease in differential behavior between the so-called habituation and dishabituation trials in sessions 2 and 3 is explained as a consequence of the spiders beginning to habituate in general to all of the individuals. The claim that the spiders remember specific individuals is somewhat undercut because all of the 'dishabituation' trials in session 2 are toward spiders they already met for 14 minutes previously but seemingly do not remember in session 2. In session 3 it is ambiguous what is happening because the spiders no longer differentiate between the trial types. This could be due to fatigue or familiarity. A second experiment is done to show that introducing a totally novel individual, recovers a large dishabituation response, suggesting that the lack of differences between 'habituation' and 'dishabituation' trials in session 3 is the result of general habituation to all of the spiders in the session rather than fatigue. As mentioned before, these data do support the claim that spiders differentiate among individuals.

The data from session 1 are easy to interpret. The data from sessions 2 and 3 are harder to understand, but these are the trials in which they meet an individual again after a substantial period of separation. Other studies looking at recognition in ants and wasps (cited by the authors) have done a 4 trial design in which focal animal A meets B in the first trial, then meets C in the second trial, meets B again in the third trial, and then meets D in the last trial. In that scenario trials 1, 2, and 4 are between unfamiliar individuals and trial 3 is between potentially familiar individuals. In both the ants and wasps, high aggression is seen in species with and without recognition on trial 1, with low aggression specifically for trials with familiar individuals in species with recognition. Across different tests, species or populations that lack recognition have shown a general reduction in aggression towards all individuals that become progressively less aggressive over time (reminiscent of the session 2 and 3 data) while others have maintained modest levels of aggression across all individuals. The 4 session design used in those other studies provides an unambiguous interpretation of the data while controlling for 'fatigue'. That all trials in sessions 2 and 3 are always with familiar individuals makes it challenging to understand how much the spiders are habituating to each other versus having some kind of associative learning of individual identity and behavior.

The data presentation is also very complicated. How is it the case that a negative proportion of time is spent? The methods reveal that this metric is derived by comparing the time individuals spent in each region relative to the previous time they saw that individual. At the very least, data showing the distribution of distances from the wall would be much easier to interpret for the reader.

(2) "Long-term social memory": It is not entirely clear what is meant by the authors when they say 'long-term social memory', though typically long-term memory refers to a form of a memory that requires protein synthesis. While the precise timing of memory formation varies across species and contexts, a general rule is that long-term memory should last for > 24 hours (e.g., Dreier et al 2007 Biol Letters). The longest time that spiders are apart in this trial setup is something like an hour. There is no basis to claim that spiders have long-term social memory as they are never asked to remember anyone after a long time apart. The odd phrasing of the 'long-term dishabutation' trial makes it seem that it is testing a long-term memory, but it is not. The spiders have never met. The fact that they are very habituated to one set of stimuli and then respond to a new stimulus is not evidence of long-term memory. To clearly test memory (which is the part really lacking from the design), the authors would need to show that spiders - upon the first instance of re-encountering a previously encountered individual are already 'habituated' to them but not to some other individuals. The current data suggest this may be the case, but it is just very hard to interpret given the design does not directly test the memory of individuals in a clear and unambiguous manner.

(3) Lack of a functional explanation and the emphasis on 'asociality': It is entirely plausible that recognition is a pleitropic byproduct of the overall visual cognition abilities in the spiders. However, the discussion that discounts territoriality as a potential explanation is not well laid out. First, many species that are 'asocial' nevertheless defend territories. It is perhaps best to say such species are not group living, but they have social lives because they encounter conspecifics and need to interact with them. Indeed, there are many examples of solitary living species that show the dear enemy effect, a form of individual recognition, towards familiar territorial neighbors. The authors in this case note that territorial competition is mediated by the size or color of the chelicerae (seemingly a trait that could be used to distinguish among individuals). Apparently, because previous work has suggested that territorial disputes can be mediated by a trait in the absence of familiarity has led them to discount the possibility that keeping track of the local neighbors in a potentially cannibalistic species could be a sufficient functional reason. In any event, the current evidence presented certainly does not warrant discounting that hypothesis.

Reviewer #1 (Public Review):

Summary:

The authors aimed to develop a mean-field model that captures the key aspects of activity in the striatal microcircuit of the basal ganglia. They start from a spiking network of individual neuron models tuned to fit striatal data. They show that an existing mean-field framework matches the output firing rates generated by the spiking network both in static conditions and when the network is subject to perfectly periodic drive. They introduce a very simplified representation of dopaminergic cortico-striatal plasticity and show that simulated dopamine exposure makes model firing rates go up or down, in a way that matches the design of the model. Finally, they aim to test the performance of the model in a reinforcement learning scenario, with two very simplified channels corresponding to the selection between two actions. Overall, I do not find that this work will be useful for the field or provide novel insights.

Strengths:

The mean-field model dynamics match well with the spiking network dynamics in all scenarios shown. The authors also introduce a dopamine-dependent synaptic plasticity rule in the context of their reinforcement learning task, which can nicely capture the appropriate potentiation or depression of corticostriatal synapses when dopamine levels change.

Weaknesses:

From the title onwards, the authors refer to a "multiscale" model. They do not, in fact, work with a multiscale model; rather, they fit a spiking model to baseline data and then fit a mean-field model to the spiking model. The idea is then to use the mean-field model for subsequent simulations.

The mean-field modeling framework that is used was already introduced previously by the authors, so that is not a novel aspect of this work in itself. The model includes an adaptation variable for each population in the network. Mean-field models with adaptation already exist, and there is no discussion of why this new framework would be preferable to those. Moreover, as presented, the mean-field model is not a closed system. It includes a variable w (in equation 7) that is never defined.

Overall, the paper shows that a mean-field model behaves similarly to a spiking model in several scenarios. A much stronger result would be to show that the mean-field model captures the activity of neurons recorded experimentally. The spiking model is supposedly fit to data from recordings in some sort of baseline conditions initially, but the quality of this fit is not adequately demonstrated; the authors just show a cursory comparison of data from a single dSPN neuron with the activity of a single model dSPN, for one set of parameters.

The authors purport to test their model via its response to "the main brain rhythms observed experimentally". In reality, this test consists of driving the model with periodic input signals. This is far too simplistic to achieve the authors' goals in this part of the work.

The work also presents model responses to simple simulations of dopamine currents, treated as negative or positive inputs to different model striatal populations. These are implemented as changes in glutamate conductance and possibly in an additional depolarizing/hyperpolarizing current, so the results that are shown are guaranteed to occur by the direct design of the simulation experiment; nothing new is learned from this. The consideration of dopamine also points out that the model is apparently designed and fit in a way that does not explicitly include dopamine, even though the fitting is done to control (i.e., with-dopamine) data, so it's not clear how this modeling framework should be adapted for dopamine-depleted scenarios.

For the reinforcement learning scenario, the model network considered is extremely simplified. Moreover, the behavior generated is unrealistic, with action two selected several times in succession independent of reward outcomes and then an instant change to a pattern of perfectly alternating selection of action 1 and action 2.

Finally, various aspects of the paper are sloppily written. The Discussion section is especially disappointing, because it is almost entirely a summary of the results of the paper, without an actual discussion of their deeper implications, connections to the existing literature, predictions that emerge, caveats or limitations of the current work, and natural directions for future study, as one would expect from a usual discussion section.

Reviewer #1 (Public Review):

Summary:

Starting from an unbiased search for somatic mutations (from COSMIC) likely disrupting binding of clinically approved antibodies the authors focus on mutations known to disrupt binding between two ERBB2 mutations and Pertuzamab. They use a combined computational and experimental strategy to nominate position which when mutated could result in restoring the therapeutic activity of the antibody. Using in vitro assays the authors confirm that the engineered antibody binds to the mutant ERBB2 and prevents ERBB3 phosphorylation

Strengths:

(1) In my assessment, the data sufficiently demonstrates that a modified version of Pertuzamab can bind both the wild-type and S310 mutant forms of ERBB2.

(2) The engineering strategy employed is rational and effectively combines computational and experimental techniques.

(3) Given the clinical activity of HER2-targeting ADCs, antibodies unaffected by ERBB2 mutations would be desired

Weaknesses:

(1) There is no data showing that the engineered antibody is equally specific as Pertuzamab i.e. that it does not bind to other (non-ERBB2) proteins.

(2) There is no data showing that the engineered antibody has the desired pharmacokinetics/pharmacodynamics properties or efficacy in vivo.

(3) Computational approaches are only used to design a phage-screen library, but not used to prioritize mutations that are likely to improve binding (e.g. based on predicted impact on the stability of the interaction). A demonstration how computational pre-screening or lead optimization can improve the time-intensive process would be a welcome advance.

Comments on revised version:

I have nothing to add beyond my first review, because no substantial changes, additional experiments and/or data, have been made to the manuscript.

Reviewer #1 (Public Review):

Summary:

Mutational analysis of diffuse midline glioma (DMG) found that ACVR1 mutations, which up-regulate BMP signaling pathway are found in most H3.1K27M, but not H3.3K27M DMG cases. In this manuscript, Huchede et al attempted to determine whether the BMP signaling pathway has any role in H3.3K27M DMG tumors. They found that the BMP signaling is activated to a similar level in H3.3K27M DMG cells with wild type ACVR1 compared to ACVR1 DMG cells, likely due to the expression of BMP7 or BMP2. They went on to test whether cells treated with BMP7 or BMP2 treatments affected the gene expression and cell fitness of tumor cells with H3.3K27M mutation. They concluded that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. The major issue for this conclusion is that the authors did not use the right models/controls to obtain results to support this conclusion as detailed below. Therefore, in order to strengthen the conclusion, the authors need to address the major concerns below.

Strength:<br /> Address an important question in DMG field.

Major concerns/weakness:<br /> (1) All the results in Fig. 2 utilized two glioma lines SF188 and Res259. The authors should repeat all these experiments in a couple of H3.3K27M DMG lines by deleting H3.3K27M mutation first.<br /> (2) Fig. 3. The experiments of BMP2 treatment should be repeated in another H3.3K27M DMG line using H3.1K27M ACVR1 mutant tumor lines as controls.

Minor concerns<br /> Fig.2A. BMP2 expression increased in H3.3K27M SF188 cells. Therefore, the statement "whereas BMP2 and BMP4 expressions are not significantly modified (Figure 2A and Figure 2-figure supplement A-B)"is not accurate

Comments on revised version:

I had three issues listed above on the initial version. The authors did not address my major concerns of #1 and #2, which are re-listed above.

Reviewer #1 (Public Review):

The authors developed an extension to the pairwise sequentially Markov coalescent model that allows to simultaneously analyze multiple types of polymorphism data. In this paper, they focus on SNPs and DNA methylation data. Since methylation markers mutate at a much faster rate than SNPs, this potentially gives the method better power to infer size history in the recent past. Additionally, they explored a model where there are both local and regional epimutational processes.

Integrating additional types of heritable markers into SMC is a nice idea which I like in principle. However, a major caveat to this approach seems to be a strong dependence on knowing the epimutation rate. In Fig. 6 it is seen that, when the epimutation rate is known, inferences do indeed look better; but this is not necessarily true when the rate is not known. (See also major comment #1 below about the interpretation of these plots.) A roughly similar pattern emerges in Supp. Figs. 4-7; in general, results when the rates have to be estimated don't seem that much better than when focusing on SNPs alone. This carries over to the real data analysis too: the interpretation in Fig. 7 appears to hinge on whether the rates are known or estimated, and the estimated rates differ by a large amount from earlier published ones.

Overall, this is an interesting research direction, and I think the method may hold more promise as we get more and better epigenetic data, and in particular better knowledge of the epigenetic mutational process.

Reviewer #1 (Public Review):

Summary:

In this study, Komarova et al. investigate the clinical prognostic ability of cell-level metabolic heterogeneity quantified via the fluorescence lifetime characteristics of NAD(P)H. Fluorescence lifetime imaging microscopy (FLIM) has been studied as a minimally invasive approach to measure cellular metabolism in live cell cultures, organoids, and animal models. Its clinical translation is spearheaded though macroscopic implementation approaches that are capable of large sampling areas and enable access to otherwise constrained spaces but lack cellular resolution for a one-to-one transition with traditional microscopy approaches, making the interpretation of the results a complicated task. The merit of this study primarily lies in its design by analyzing with the same instrumentation and approach colorectal samples in different research scenarios, namely in vitro cells, in vivo animal xenografts, and ex vivo tumor tissue from human patients. These conform to a valuable dataset to explore the translational interpretation hurdles with samples of increasing levels of complexity. For human samples, which exhibited the highest degree of heterogeneity from the experiments presented, the study specifically investigates the prediction ability of NAD(P)H fluorescence metrics for the binary classification of tumors of low and advanced stage, with and without metastasis, and low and high grade. They find that NAD(P)H fluorescence properties have a strong potential to distinguish between high- and low-grade tumors and a moderate ability to distinguish advanced stage tumors from low stage tumors. This study provides valuable results contributing to the deployment of minimally invasive optical imaging techniques to quantify tumor properties and potentially migrating into tools for human tumor characterization and clinical diagnosis.

Strengths:

The investigation of colorectal samples under multiple imaging scenarios with the same instrument and approach conforms to a valuable dataset that can facilitate interpretation of results across the spectrum of sample complexity.

The manuscript provides a strong discussion reviewing studies that investigated cellular metabolism with FLIM and the metabolic heterogeneity of colorectal cancer in general.

The authors do a thorough acknowledgement of the experimental limitations of investigating human samples ex vivo, and the analytical limitation of manual segmentation, for which they provide a path forward for higher throughput analysis.

Weaknesses:

NAD(P)H fluorescence provides a partial picture of the cell/tissue metabolic characteristics. Including fluorescence from flavins would comprise a more compelling dataset. These additional data should enable the quantification of redox metrics, which could positively contribute to the prognosis potential of metabolic heterogeneity. The authors did attempt to incorporate flavin fluorescence, unfortunately they could not find strong enough signal to proceed with the analysis.

Reviewer #1 (Public Review):

Clostridium thermocellum serves as a model for consolidated bioprocessing (CBP) in lignocellulosic ethanol production. The primary ethanol production pathway involves the enzyme aldehyde-alcohol dehydrogenase (AdhE), which exhibits complex regulation, forming long oligomeric structures known as spirosomes.

The present study describes the cryo-EM structure of C. thermocellum AdhE, resolved at 3.28 Å resolution. By integrating cryo-EM data with molecular dynamics simulations, this study showed that the aldehyde intermediate resides longer in the channel of the extended form, supporting the mechanistic model in which the extended spirosome conformation represents the active form of AdhE.

These findings advance the understanding of the function and regulation of AdhE, a key enzyme involved in the ethanol biosynthesis pathway in Clostridium thermocellum, a model organism for ethanol production in consolidated bioprocessing.

Reviewer #1 (Public Review):

The development of effective computational methods for protein-ligand binding remains an outstanding challenge to the field of drug design. This impressive computational study combines a variety of structure prediction (AlphaFold2) and sampling (RAVE) tools to generate holo-like protein structures of three kinases (DDR1, Abl1, and Src kinases) for binding to type I and type II inhibitors. Of central importance to the work is the conformational state of the Asp-Phy-Gly "DFG motif" where the Asp points inward (DFG-in) in the active state and outward (DFG-out) in the inactive state. The kinases bind to type I or type II inhibitors when in the DFG-in or DFG-out states, respectively.

It is noted that while AlphaFold2 can be effective in generating ligand-free apo protein structures, it is ineffective at generating holo structures appropriate for ligand binding. Starting from the native apo structure, structural fluctuations are necessary to access holo-like structures appropriate for ligand-binding. A variety of methods, including reduced multiple sequence alignment (rMSA), AF2-cluster, and AlphaFlow may be used to create decoy structures. However, those methods can be limited in the diversity of structures generated and lack a physics-based analysis of Boltzmann weight critical to their relative evaluation.

To address this need, the authors combine AlphaFold2 with the Reweighted Autoencoded Variational Bayes for Enhanced Sampling (RAVE) method, to explore metastable states and create a Boltzmann ranking. With that variety of structures in hand, grid-based docking methods Glide and Induced-Fit Docking (IFD) were used to generate protein-ligand (kinase-inhibitor) complexes.

The authors demonstrate that using AlphaFold2 alone, there is a failure to generate DFG-out structures needed for binding to type II inhibitors. By applying the AlphaFold2 with rMSA followed by RAVE (using short MD trajectories, SPIB-based collective variable analysis, and enhanced sampling using umbrella sampling), metastable DFG-out structures with Boltzmann weighting are generated enabling protein-ligand binding. Moreover, the authors found that the successful sampling of DFG-out states for one kinase (DDR1) could be used to model similar states for other proteins (Abl1 and Src kinase). The AF2RAVE approach is shown to result in a set of holo-like protein structures with a 50% rate of docking type II inhibitors.

Overall, this is excellent work and a valuable contribution to the field that demonstrates the strengths and weaknesses of state-of-the-art computational methods for protein-ligand binding. The authors also suggest promising directions for future study, noting that potential enhancements in the workflow may result from the use of binding site prediction models and free energy perturbation calculations.

Reviewer #1 (Public Review):

The study shows a new mechanism of NFkB-p65 regulation mediated by Vangl2-dependent autophagic targeting. Autophagic regulation of p65 has been reported earlier; this study brings an additional set of molecular players involved in this important regulatory event, which may have implications for chronic and acute inflammatory conditions.

Reviewer #1 (Public Review):

The manuscript presents a framework for studying biomechanical principles and their links to morphology and provides interesting insights into a particular question regarding terrestrial locomotion and speed. The goal of the paper is to derive general principals of directed terrestrial locomotion, speed, and symmetry.

Major strengths:

The manuscript is a unique and creative work that explores performance spaces of a complicated question through computational modeling. Overall, the paper is well written and well crafted and was a pleasure to read.

The methods presented here (variable agents used to represent ultra-simplified body configurations that are not inherently constrained) are interesting and there's significant potential in them for a properly constrained question. For the data that is present here their hypotheses (while they can be anticipated from first principles) are very well validated and serve as a robust validation of these expectations and can help.

Of particular interest was the discussion of the transferability of morphologies designed under one system and moving to another. From a deep-time perspective, of particular interest is the transition from subaqueous to terrestrial locomotion which we know was a major earth life transition. The results of this study show that the best suited morphologies for subaqueous movement are ill-suited (from a locomotor speed standpoint at least) to fully terrestrial locomotion which begs the questions on if there are a suite of forms that have balanced performance in both and how that would differ from aquatic morphologies.

Major weaknesses:

(1) There is a major disagreement between target and parameters.

From a biomechanics perspective the target of this study, Directed Locomotion, is a fairly broad behavioral mode. However, what the authors are ultimately evaluating their model organisms on is a single performance parameter (speed, or distance traveled after 30s). Statements such as "bilateral symmetry showed to be a law-like pattern in animal evolution for efficient directed locomotion purposes" (p 12 line 365-366) are problematic for this reason.

Attaining the highest possible speed is a relevant but limited subset of ways one might interpret performance for directed locomotion. Efficiency, power generation, and limb loading/strain are equally relevant components.

The focus on speed coupled with selection for only the highest performing morphologies, rather than setting a minimum performance threshold fundamentally restricts the dynamics of the system in a way that is not representative of their specified target and pulls the simulations toward a specific, anticipatable, result.

Locomotor efficiency is alluded to later in the manuscript as one of the observed outcomes, but speed is not equivalent to locomotor efficiency (in much the same way that it is not the sole metric for describing performance with respect to directed locomotion). Energy/work/power have not been accounted for in the manuscript so this is not a parameter this study weighs in on.

The data and analyses the others present do show an interesting validation of these methods in assessing first order questions relating the shape of a single performance surface to a theoretical morphology, which has significant potential value.

(2) There is significant population and/or sample size and biasing.

Thirty simulations of a population of 101 morphologies seems small for a study of this kind, particularly looking to investigate such a broad question at an abstract level. Particularly when the top 50% of morphologies are chosen to mutate. It would be very easy for artificial biases to rapidly propagate through this system depending on the parameters bounding the formation of the initial generation.

This strong selection choosing the best 50 morphologies and mutating them enforces an aggressive effect that simulates and even more potent phylogenetic inertia than one might anticipate for an actual evolutionary history (it's no surprise then that all of the simulations were able to successfully retrieve a suite of morphotypes that recovered the performance peak for this system within 1500 generations)

Similarly, why is it that a 4^3 voxel limit was chosen? One can imagine that an increase in this voxel limit would allow for the development of more extreme geometries, which might be successful. It is likely that there might be computational resource constraints involved in this, it would be useful for the authors to add additional context here.

Review of resubmission:

I appreciate the clarification of points dealing with the details of computational modeling and methods and clarifications throughout the text.

However, the authors have failed to address the major weaknesses that were previously identified, specifically regarding the broader conclusions of the work, that either 1) the authors need to use an additional metric besides average speed, or 2) the conclusions need to be significantly reigned in to reflect the very narrow nature of the work.

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.<br /> However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts.

Reviewer #1 (Public Review):

Summary:<br /> The study seeks to establish accurate computational models to explore the role of hydrodynamic interactions on energy savings and spatial patterns in fish schools. Specifically, the authors consider a system of (one degree-of-freedom) flapping airfoils that passively position themselves with respect to the streamwise direction, while oscillating at the same frequency and amplitude, with a given phase lag and at a constant cross-stream distance. By parametrically varying the phase lag and the cross-stream distance, they systematically explore the stability and energy costs of emergent configurations. Computational findings are leveraged to distill insights into universal relationships and clarify the role of the wake of the leading foil.

Strengths:<br /> (1) The use of multiple computational models (computational fluid dynamics, CFD, for full Navier-Stokes equations and computationally-efficient inviscid vortex sheet, VS, model) offers an extra degree of reliability of the observed findings and backing to the use of simplified models for future research in more complex settings.

(2) The systematic assessment of the stability and energy savings in multiple configurations of pairs and larger ensembles of flapping foils is an important addition to the literature.

(3) The discovery of a linear phase-distance relationship in the formation attained by pairs of flapping foils is a significant contribution, which helps compare different experimental observations in the literature.

(4) The observation of a critical size effect for in-line formations, above which cohesion and energetic benefits are lost at once, is a new discovery to the field.

Weaknesses:<br /> (1) The extent to which observations on one-degree-of-freedom flapping foils could translate to real fish schools is presently unclear, so that some of the conclusions on live fish schools are likely to be overstated and would benefit from some more biological framing.

(2) The analysis of non-reciprocal coupling is not as novel as the rest of the study and potentially not as convincing due to the chosen linear metric of interaction (that is, the flow agreement).

Overall, this is a rigorous effort on a critical topic: findings of the research can offer important insight into the hydrodynamics of fish schooling, stimulating interdisciplinary research at the interface of computational fluid mechanics and biology.

Reviewer #1 (Public Review):

In the presence of predators, animals display attenuated foraging responses and increased defensive behaviors that serve to protect them from potential predatory attacks. Previous studies have shown that the basolateral nucleus of the amygdala (BLA) and the periaqueductal gray matter (PAG) are necessary for the acquisition and expression of conditioned fear responses. However, it remains unclear how BLA and PAG neurons respond to predatory threats when animals are foraging for food. To address this question, Kim and colleagues conducted in vivo electrophysiological recordings from BLA and PAG neurons and assessed approach-avoidance responses while rats searched for food in the presence of a robotic predator.

The authors observed that rats exhibited a significant increase in the latency to obtain the food pellets and a reduction in the pellet success rate when the predator robot was activated. A subpopulation of PAG neurons showing an increased firing rate in response to the robot activation didn't change their activity in response to food pellet retrieval during the pre- or post-robot sessions. Optogenetic stimulation of PAG neurons increased the latency to procure the food pellet in a frequency- and intensity-dependent manner, similar to what was observed during the robot test. Combining optogenetics with single-unit recordings, the authors demonstrated that photoactivation of PAG neurons increased the firing rate of 10% of BLA cells. A subsequent behavioral test in 3 of these same rats demonstrated that BLA neurons responsive to PAG stimulation displayed higher firing rates to the robot than BLA neurons nonresponsive to PAG stimulation. Next, because the PAG does not project monosynaptically to the BLA, the authors used a combination of retrograde and anterograde neural tracing to identify possible regions that could convey robot-related information from PAG to the BLA. They observed that neurons in specific areas of the paraventricular nucleus of the thalamus (PVT) that are innervated by PAG fibers contained neurons that were retrogradely labeled by the injection of CTB in the BLA. In addition, PVT neurons showed increased expression of the neural activity marker cFos after the robot test, suggesting that PVT may be a mediator of PAG signals to the BLA.

Overall, the idea that the PAG interacts with the BLA via the midline thalamus during a predator vs. foraging test is new and quite interesting. The authors have used appropriate tools to address their questions. However, there are some major concerns regarding the design of the experiments, the rigor of the histological analyses, the presentation of the results, the interpretation of the findings, and the general discussion that largely reduces the relevance of this study.

The authors have fully addressed all my concerns.

Reviewer #1 (Public Review):

Summary:<br /> The author presents the discovery and characterization of CAPSL as a potential gene linked to Familial Exudative Vitreoretinopathy (FEVR), identifying one nonsense and one missense mutation within CAPSL in two distinct patient families afflicted by FEVR. Cell transfection assays suggest that the missense mutation adversely affects protein levels when overexpressed in cell cultures. Furthermore, conditionally knocking out CAPSL in vascular endothelial cells leads to compromised vascular development. The suppression of CAPSL in human retinal microvascular endothelial cells results in hindered tube formation, a decrease in cell proliferation, and disrupted cell polarity. Additionally, transcriptomic and proteomic profiling of these cells indicates alterations in the MYC pathway.

Strengths:<br /> The study is nicely designed with a combination of in vivo and in vitro approaches, and the experimental results are good quality.

Weaknesses:<br /> My reservations lie with the main assertion that CAPSL is associated with FEVR, as the genetic evidence from human studies appears relatively weak. Further careful examination of human genetics evidence in both patient cohorts and the general population will help to clarify. In light of human genetics, more caution needs to be exercised when interpreting results from mice and cell model and how is it related to the human patient phenotype. Future replication by finding more FEVR patients with a mutation in CAPSL will strengthen the findings.

Reviewer #1 (Public Review):

Summary:<br /> The authors analyzed how biotic and abiotic factors impact antagonistic host-parasitoid interaction systems in a large BEF experiment. They found the linkage between the tree community and host-parasitoid community from the perspective of the multi-dimensionality of biodiversity. Their results revealed that the structure of the tree community (habitat) and canopy cover influence host-parasitoid compositions and their interaction pattern. This interaction pattern is also determined by phylogenetic associations among species. This paper provides a nice framework for detecting the determinants of network topological structures.

Strengths:<br /> This study was conducted using a five-year sampling in a well-designed BEF experiment. The effects of the multi-dimensional diversity of tree communities have been well explained in a forest ecosystem with an antagonistic host-parasitoid interaction.

The network analysis has been well conducted. The combination of phylogenetic analysis and network analysis is uncommon among similar studies, especially for studies of trophic cascades. Still, this study has discussed the effect of phylogenetic features on interacting networks in depth.

Weaknesses:<br /> (1) The authors should examine species and interaction completeness in this study to confirm that their sampling efforts are sufficient.<br /> (2) The authors only used Rao's Q to assess the functional diversity of tree communities. However, multiple metrics of functional diversity exist (e.g., functional evenness, functional dispersion, and functional divergence). It is better to check the results from other metrics and confirm whether these results further support the authors' results.<br /> (3) The authors did not elaborate on which extinction sequence was used in robustness analysis. The authors should consider interaction abundance in calculating robustness. In this case, the author may use another null model for binary networks to get random distributions.<br /> (4) The causal relationship between host and parasitoid communities is unclear. Normally, it is easy to understand that host community composition (low trophic level) could influence parasitoid community composition (high trophic level). I suggest using the 'correlation' between host and parasitoid communities unless there is strong evidence of causation.

Reviewer #1 (Public Review):

Summary:

The work by Chuong et al. provides important new insights into the contribution of different molecular mechanisms in the dynamics of CNV formation. It will be of interest to anyone curious about genome architecture and evolution from yeast biologists to cancer researchers studying genome rearrangements.

Strengths:

Their results are especially striking in that the "simplest" mechanism of GAP1 amplification-non-allelic homologous recombination between the flanking Ty-LTR elements is not the most common route taken by the cells, emphasizing the importance of experimentally testing what might seem on the surface to be obvious answers. One of the important developments of their work is the use of their neural network simulation-based inference (nnSBI) model to derive rates of amplicon formation and their fitness effects.

Weaknesses:

The manuscript reads as though two different people wrote two different sections of the manuscript - an experimental evolutionist and a computational scientist. If the goal is to reach both groups of readers, there needs to be more explanation of both types of work. I found the computational sections to be particularly dense but even the experimental sections need clearer explanations and more specific examples of the rearrangements found. I will point out these areas in the detailed remarks to the authors. While I have no reason to question their conclusions, I couldn't independently verify the results that ODIRA was the majority mechanism since the sequence of amplified clones was not made available during the review. I've encouraged the authors to include specific, detailed sequence information for both ODIRA events as well as the specific clones where GAP1 was amplified but the flanking gene GFP was not.

Reviewer #1 (Public Review):

Summary:

This study explores the therapeutic potential of KMO inhibition in endometriosis, a condition with limited treatment options.

Strengths:

KNS898 is a novel specific KMO inhibitor and is orally bioavailable, providing a convenient and non-hormonal treatment option for endometriosis. The promising efficacy of KNS898 was demonstrated in a relevant preclinical mouse model of endometriosis with pathological and behavioural assessments performed.

Weaknesses:

(1) The expression of KMO in human normal endometrium and endometrial lesions was not quantified. Western blot or quantification of IHC images will provide valuable insight. If KMO is not overexpressed in diseased tissues ie it may have homeostatic roles, and inhibition of KMO may have consequences on general human health and wellbeing. In addition, KMO expression in control mice was not shown or quantified. Images of KMO expression in endometriosis mice with treatments should be shown in Figure 4. The images showing quantification analysis (Figure 4A-F) can be moved to supplementary material.

(2) Figure 1 only showed representative images from a few patients. A description of whether KMO expression varies between patients and whether it correlates with AFS stages/disease severity will be helpful. Images from additional patients can be provided in supplementary material.

(3) For Home Cage Analysis, different measurements were performed as stated in methods including total moving distance, total moving time, moving speed, isolation/separation distance, isolated time, peripheral time, peripheral distance, in centre zones time, in centre zones distance, climbing time, and body temperature. However, only the finding for peripheral distance was reported in the manuscript.

(4) The rationale for choosing the different dose levels of KNS898 - 0.01-25mg/kg was not provided. What is the IC50 of a drug?

(5) Statistical significance:<br /> (a) Were stats performed for Fig 3B-E?<br /> (b) Line 141 - 'P = 0.004 for DEGLS per group'<br /> However, statistics were not shown in the figure.<br /> (c) Line 166 - 'the mechanical allodynia threshold in the hind paw was statistically significantly lower compared to baseline for the group'<br /> However, statistics were not shown in the figure.<br /> (d) Line 170 - 'Two-way ANOVA, Group effect P = 0.003, time effect P < 0.0001' The stats need to be annotated appropriately in Figure 5A as two separate symbols.<br /> (e) Figure 5B - multiple comparisons of two-way ANOVA are needed. G4 does not look different to G3 at D42.<br /> (f) Line 565 - 'non-significant improvement in KNS898 treated groups'. However, ** was annotated in Figure 5A.

(6) Discussion is very light. No reference to previous publications was made in the discussion. Discussion on potential mechanistic pathways of KYR/KMO in the pathogenesis of endometriosis will be helpful, as the expression and function of KMO and/or other metabolites in endometrial-related conditions.

The findings in this study generally support the conclusion although some key data which strengthen the conclusion eg quantification of KMO in normal and diseased tissue is lacking. Before KMO inhibitors can be used for endometriosis, the function of KMO in the context of endometriosis should be explored eg KMO knockout mice should be studied.

Reviewer #1 (Public Review):

The study uses nanoscale secondary ion mass spectrometry to show that maize plants inoculated with a bacteria, Gd, incorporated fixed nitrogen into the chloroplast. The authors then state that since "chloroplasts are the chief engines that drive plant growth," that it is this incorporation that explains the maize's enhanced growth with the bacteria.

But the authors don't present the total special distribution of nitrogen in plants. That is, if the majority of nitrogen is in the chloroplast (which, because of Rubisco, it likely is) then the majority of fixed nitrogen should go into the chloroplast.

Also, what are the actual controls? In the methods, the authors detail that the plants inoculated with Gd are grown without nitrogen. But how did the authors document the "enhanced growth rates of the plants containing this nitrogen fixing bacteria." Were there other plants grown without nitrogen and the Gd? If so, of course, they didn't grow as well. Nitrogen is essential for plant growth. If Gd isn't there to provide it in n-free media, then the plants won't grow. Do we need to go into the mechanism for this, really? And it's not just because nitrogen is needed in the chloroplast, even if that might be where the majority ends up.

Furthermore, it is not novel to say that nitrogen from a nitrogen fixing bacteria makes its way into the chloroplast. For any plant ever successfully grown on N free media with a nitrogen fixing bacteria, this must be the case. We don't need a fancy tool to know this.

The experimental setup does not suit the argument the authors are trying to make (and I'm not sure if the argument the authors are trying to make has any legitimacy). The authors contend that their study provides the basis of a "detailed agronomic analysis of the extent of fixed nitrogen fertilizer needs and growth responses in autonomous nitrogen-fixing maize plants." But what is a "fixed nitrogen fertilizer need"? The phrase makes no sense. A plant has nitrogen needs. This nitrogen can be provided via nitrogen fixing bacteria or fertilizer. But are there fixed nitrogen fertilizer needs? It sounds like the authors are suggesting that a plant can distinguish between nitrogen fixed by bacteria nearby and that provided by fertilizer. If that is the contention, then a new set of experiments is needed - with other controls grown on different levels of fertilizer.

What is interesting, and potentially novel, in this study is figure 1D (and lines 90-99). In that image, is the bacteria actually in the plant cell? Or is it colonizing the region between the cells? Either way, it looks to have made its way into the plant leaf, correct? I believe that would be a novel and fascinating finding. If the authors were to go into more detail into how Gd is entering into the symbiotic relationship with maize (e.g. fixing atmospheric nitrogen in the leaf tissue rather than in root nodules like legumes) I believe that would be very significant. But be sure to add to the field in relation to reference 9, and any new references since then.

Also, it would be helpful to have an idea of how fast these plants, grown in n free media but inoculated with the bacteria, grow compared to plants grown on various levels of fertilizer.

Reviewer #1 (Public Review):

Summary:

Advances in machine vision and computer learning have meant that there are now state-of-the-art and open-source toolboxes that allow for animal pose estimation and action recognition. These technologies have the potential to revolutionize behavioral observations of wild primates but are often held back by labor-intensive model training and the need for some programming knowledge to effectively leverage such tools. The study presented here by Fuchs et al unveils a new framework (ASBAR) that aims to automate behavioral recognition in wild apes from video data. This framework combines robustly trained and well-tested pose estimate and behavioral action recognition models. The framework performs admirably at the task of automatically identifying simple behaviors of wild apes from camera trap videos of variable quality and contexts. These results indicate that skeletal-based action recognition offers a reliable and lightweight methodology for studying ape behavior in the wild and the presented framework and GUI offer an accessible route for other researchers to utilize such tools.

Given that automated behavior recognition in wild primates will likely be a major future direction within many subfields of primatology, open-source frameworks, like the one presented here, will present a significant impact on the field and will provide a strong foundation for others to build future research upon.

Strengths:

- Clearly articulated the argument as to why the framework was needed and what advantages it could convey to the wider field.

- For a very technical paper it was very well written. Every aspect of the framework the authors clearly explained why it was chosen and how it was trained and tested. This information was broken down in a clear and easily digestible way that will be appreciated by technical and non-technical audiences alike.

- The study demonstrates which pose estimation architectures produce the most robust models for both within-context and out-of-context pose estimates. This is invaluable knowledge for those wanting to produce their own robust models.

- The comparison of skeletal-based action recognition with other methodologies for action recognition helps contextualize the results.

Weaknesses

While I note that this is a paper most likely aimed at the more technical reader, it will also be of interest to a wider primatological readership, including those who work extensively in the field. When outlining the need for future work I felt the paper offered almost exclusively very technical directions. This may have been a missed opportunity to engage the wider readership and suggest some practical ways those in the field could collect more ASBAR-friendly video data to further improve accuracy.

Reviewer #1 (Public Review):

Summary:

The anatomical connectivity of the claustrum and the role of its output projections has, thus far, not been studied in detail. The aim of this study was to map the outputs of the endopiriform (EN) region of the claustrum complex, and understand their functional role. Here the authors have combined sophisticated intersectional viral tracing techniques, and ex vivo electrophysiology to map the neural circuitry of EN outputs to vCA1, and shown that optogenetic inhibition of the EN→vCA1 projection impairs both social and object recognition memory. Interestingly the authors find that the EN neurons target inhibitory interneurons providing a mechanism for feedforward inhibition of vCA1.

Strengths:

The strength of this study was the application of a multilevel analysis approach combining a number of state-of-the-art techniques to dissect the contribution of the EN→vCA1 to memory function.

Weaknesses:

Some authors would disagree that the vCA1 represents a 'node for recognition of familiarity' especially for object recognition although that is not to say that it might play some role in discrimination, as shown by the authors. I note however that the references provided in the Introduction, concerning the role of vCA1in memory refer to anxiety, social memory, temporal order memory, and not novel object recognition memory. Given the additional projections to the piriform cortex shown in the results, I wonder to what extent the observations may be explained by odour recognition effects. In addition, I wondered whether the impairments in discrimination following Chemo-genetic inhibition of the EN→vCA1 were due to the subject treating the novel and familiar stimuli as either both novel- which might be observed as an increase in exploration, or both stimuli as familiar, with a decrease in overall exploration.

Reviewer #1 (Public Review):

In their manuscript, the authors propose a learning scheme to enable spiking neurons to learn the appearance probability of inputs to the network. To this end, the neurons rely on error-based plasticity rules for feedforward and recurrent connections. The authors show that this enables the networks to spontaneously sample assembly activations according to the occurrence probability of the input patterns they respond to. They also show that the learning scheme could explain biases in decision-making, as observed in monkey experiments. While the task of neural sampling has been solved before in other models, the novelty here is the proposal that the main drivers of sampling are within-assembly connections, and not between-assembly (Markov chains) connections as in previous models. This could provide a new understanding of how spontaneous activity in the cortex is shaped by synaptic plasticity.

The manuscript is well written and the results are presented in a clear and understandable way. The main results are convincing, concerning the spontaneous firing rate dependence of assemblies on input probability, as well as the replication of biases in the decision-making experiment. Nevertheless, the manuscript and model leave open several important questions. The main problem is the unclarity, both in theory and intuitively, of how the sampling exactly works. This also makes it difficult to assess the claims of novelty the authors make, as it is not clear how their work relates to previous models of neural sampling.

Regarding the unclarity of the sampling mechanism, the authors state that within-assembly excitatory connections are responsible for activating the neurons according to stimulus probability. However, the intuition for this process is not made clear anywhere in the manuscript. How do the recurrent connections lead to the observed effect of sampling? How exactly do assemblies form from feedforward plasticity? This intuitive unclarity is accompanied by a lack of formal justification for the plasticity rules. The authors refer to a previous publication from the same lab, but it is difficult to connect these previous results and derivations to the current manuscript. The manuscript should include a clear derivation of the learning rules, as well as an (ideally formal) intuition of how this leads to the sampling dynamics in the simulation.

Some of the model details should furthermore be cleared up. First, recurrent connections transmit signals instantaneously, which is implausible. Is this required, would the network dynamics change significantly if, e.g., excitation arrives slightly delayed? Second, why is the homeostasis on h required for replay? The authors show that without it the probabilities of sampling are not matched, but it is not clear why, nor how homeostasis prevents this. Third, G and M have the same plasticity rule except for G being confined to positive values, but there is no formal justification given for this quite unusual rule. The authors should clearly justify (ideally formally) the introduction of these inhibitory weights G, which is also where the manuscript deviates from their previous 2020 work. My feeling is that inhibitory weights have to be constrained in the current model because they have a different goal (decorrelation, not prediction) and thus should operate with a completely different plasticity mechanism. The current manuscript doesn't address this, as there is no overall formal justification for the learning algorithm.

Finally, the authors should make the relation to previous models of sampling and error-based plasticity more clear. Since there is no formal derivation of the sampling dynamics, it is difficult to assess how they differ exactly from previous (Markov-based) approaches, which should be made more precise. Especially, it would be important to have concrete (ideally experimentally testable) predictions on how these two ideas differ. As a side note, especially in the introduction (line 90), this unclarity about the sampling made it difficult to understand the contrast to Markovian transition models.

There are also several related models that have not been mentioned and should be discussed. In 663 ff. the authors discuss the contributions of their model which they claim are novel, but in Kappel et al (STDP Installs in Winner-Take-All Circuits an Online Approximation to Hidden Markov Model Learning) similar elements seem to exist as well, and the difference should be clarified. There is also a range of other models with lateral inhibition that make use of error-based plasticity (most recently reviewed in Mikulasch et al, Where is the error? Hierarchical predictive coding through dendritic error computation), and it should be discussed how the proposed model differs from these.

Reviewer #2 (Public Review):

This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress and causes the accumulation of mitochondrial precursor proteins in the cytosol.

The data shown are of high quality and well-controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. Moreover, the authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hex-resistant TOM mutant was not identified. Nevertheless, this is an interesting and inspiring study of high quality. The connection of proteostasis, mitochondrial biogenesis and sphingolipid metabolism is exciting and will certainly lead to many follow-up studies.

Reviewer #1 (Public Review):

Summary:

The manuscript considers a mechanistic extension of MacArthur's consumer-resource model to include chasing down of food and potential encounters between the chasers (consumers) that lead to less efficient feeding in the form of negative feedback. After developing the model, a deterministic solution and two forms of stochastic solutions are presented, in agreement with each other. Finally, the model is applied to explain observed coexistence and rank-abundance data.

Strengths:

- The application of the theory to natural rank-abundance curves is impressive.<br /> - The comparison with the experiments that reject the competitive exclusion principle is promising. It would be fascinating to see if in, e.g. insects, the specific interference dynamics could be observed and quantified and whether they would agree with the model.<br /> - The results are clearly presented; the methods adequately described; the supplement is rich with details.<br /> - There is much scope to build upon this expansion of the theory of consumer-resource models. This work can open up new avenues of research.

Weaknesses:

- Though more and better data could be used to constrain and validate the modeling, given this is a theory-driven manuscript, their results are sufficient.

Reviewer #1 (Public Review):

The authors use neural recordings from three different brain areas to assess whether the type of evidence accumulation dynamics in those regions are (1) similar to one another, and (2) similar to best-fitting evidence accumulation dynamics to behavioral choice alone. This is an important theoretical question because it relates to the 'linking hypothesis' that relates neurophysiological data to psychological phenomena. Although the standard evidence accumulation dynamic in describing choice has been the gradual accumulation of evidence, the authors find that those dynamics are not represented equally in all brain regions. Such results suggest that more nuanced computational models are needed to explain how brain areas interact to produce decisions, and the focus of theoretical development should shift away from explaining behavioral patterns alone and more toward explaining both brain and behavioral interactions. Given that the authors simply test the assumption that the same dynamics that best explain behavior should also explain neural data, they accomplish their objective using a sophisticated methodology and find evidence *against* this assumption: they find that each region was best described by a distinct accumulation model, which all differed from the model that best described the rat's choices.

I thought this was an excellent paper with a clear scientific objective, direct analysis to achieve that objective, and a very strong methodological approach to leave little doubt that the conclusions they drew from their analyses were as reasonable and accurate as possible.

Reviewer #1 (Public Review):

Summary:

In their manuscript, Schmidlin, Apodaca et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to enumerate patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.

Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation.

Weaknesses:

(1) The main objective of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. As the authors remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. The general conclusions of the authors regarding the evolution of trade-offs might thus be more focused on multi-drug resistant phenotypes.

(2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay. Previous studies approximated the fraction of lineages that could be overtaken by secondary mutations (Venkataram and Dunn et al 2016). In their calculations, Venkataram and Dunn et al defined adaptive mutations in their data as having a selection coefficient of 5% and highly adaptive mutations at around 10%. From this and an estimation of the mutation rate, they estimate that the fraction of lineages overtaken by adaptive mutations is negligible (10^4) after 32 generations. However, the effects on fitness observed by the authors here tend to be much stronger than 5-10%, with relative fitness advantages above 1 and often reaching 2. This could result in a much higher chance of lineages being overtaken at 40 generations.

(3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach. Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages.

(4) The authors make the decision to use UMAP and a Gaussian mixed model as well as validation data to identify unique clusters, which is one of their main objectives. The choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. All of the data presented in the validations is presented to fit within the 6 clusters structure but does not include evidence against alternative scenarios for additional relevant clusters as might be suggested by Figure S6.

(5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. Reconstructing some of the specific mutants they identified to validate their phenotypes would also have been a good addition. If the phenotypic clusters identified cannot be reproduced outside of the sequencing assay, then their relevance are they as a model for multi-drug resistance scenarios might be reduced.

Reviewer #1 (Public Review):

Summary:

In their manuscript, Schmidlin, Apodaca, et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to examine patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.

Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation.

Weaknesses:

(1) One of the objectives of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. This corresponds to a very small fraction of the starting set of ~21 000 lineages that were combined after experimental evolution for fitness measurements. As the authors briefly remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. This has important implications regarding the general conclusions of the authors regarding the evolution of trade-offs.

(2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay.

(3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach. Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages. This might paint a misleading picture where clusters appear well separate and well defined but are in fact much fuzzier, which would impact the conclusion that the phenotypic space is constricted.

(4) The authors make the decision to use UMAP and a gaussian mixed model to cluster and represent the different fitness landscapes of their lineages of interest. Their approach has many caveats. First, compared to PCA, the axis does not provide any information about the actual dissimilarities between clusters. Using PCA would have allowed a better understanding of the amount of variance explained by components that separate clusters, as well as more interpretable components. Second, the advantages of dimensional reduction are not clear. In the competition experiment, 11/12 conditions (all but the no drug, no DMSO conditions) can be mapped to only three dimensions: concentration of fluconazole, concentration of radicicol, and relative fitness. Each lineage would have its own fitness landscape as defined by the plane formed by relative fitness values in this space, which can then be examined and compared between lineages. Third, the choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. This directly contradicts the statement in the main text that clusters are robust to noise, as more a stringent inclusion threshold appears to increase and not decrease the optimal number of clusters. Additional criteria to BIC could have been used to help choose the optimal number of clusters or even if mixed Gaussian modeling is appropriate for this dataset.

(5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. Having these types of results would help support the accuracy of the main assay in the manuscript and thus better support the claims of the authors.

Reviewer #1 (Public Review):

Mehrdad Kashefi et al. investigated the availability of planning future reaches while simultaneously controlling the execution of the current reach. Through a series of experiments employing a novel sequential arm reaching paradigm they developed, the authors made several findings: 1) participants demonstrate the capability to plan future reaches in advance, thereby accelerating the execution of the reaching sequence, 2) planning processes for future movements are not independent one another, however, it's not a single chunk neither, 3) Interaction among these planning processes optimizes the current movement for the movement that comes after for it.

The question of this paper is very interesting, and the conclusions of this paper are well supported by data.

Reviewer #1 (Public Review):

Summary:

HIV associated nephropathy (HIVAN) is a rapidly progressing form of kidney disease that manifests secondary to untreated HIV infection and is predominantly seen in individuals of African descent. Tg26 mice carrying an HIV transgene lacking gag and pol exhibit high levels of albuminuria and rapid decline in renal function that recapitulates many features of HIVAN in humans. HIVAN is seen predominantly in individuals carrying two copies of missense variants in the APOL1 gene, and the authors have previously shown that APOL1 risk variant mRNA induces activity of the double strand RNA sensor kinase PKR. Because of the tight association between the APOL1 risk genotype and HIVAN, the authors hypothesized that PKR activation may mediate the renal injury in Tg26 mice, and tested this hypothesis by treating mice with a commonly used PKR inhibitory compound called C16. Treatment with C16 substantially attenuated renal damage in the Tg26 model as measured by urinary albumin/creatinine ratio, urinary NGAL/creatinine ratio and improvement in histology. The authors then performed bulk and single-nucleus RNAseq on kidneys from mice from different treatment groups to identify pathways and patterns of cell injury associated with HIV transgene expression as well as to determine the mechanistic basis for the effect of C16 treatment. They show that proximal tubule nuclei from Tg26 mice appear to have more mitochondrial transcripts which was reversed by C16 treatment and suggest that this may provide evidence of mitochondrial dysfunction in this model. They explore this hypothesis by showing there is a decrease in the expression of nuclear encoded genes and proteins involved in oxidative phosphorylation as well as a decrease in respiratory capacity via functional assessment of respiration in tubule and glomerular preparations from these mouse kidneys. All of these changes were reversed by C16 treatment. The authors propose the existence of a novel injured proximal tubule cell-type characterized by the leak of mitochondrial transcripts into the nucleus (PT-Mito). Analysis of HIV transgene expression showed high level expression in podocytes, consistent with the pronounced albuminuria that characterizes this model and HIVAN, but transcripts were also detected in tubular and endothelial cells. Because of the absence of mitochondrial transcripts in the podocytes, the authors speculate that glomerular mitochondrial dysfunction in this model is driven by damage to glomerular endothelial cells.

Strengths:

The strengths of this study include the comprehensive transcriptional analysis of the Tg26 model, including an evaluation of HIV transgene expression, which has not been previously reported. This data highlights that HIV transcripts are expressed in a subset of podocytes, consistent with the highly proteinuric disease seen in mouse and humans. However, transcripts were also seen in other tubular cells, notably intercalated cells, principal cells and injured proximal tubule cells. Though the podocyte expression makes sense, the relevance of the tubular expression to human disease is still an open question.

The data in support of mitochondrial dysfunction are also robust and rely on combined evidence from downregulation of transcripts involved in oxidative phosphorylation, decreases in complex I and II as determined by immunoblot, and assessments of respiratory capacity in tubular and glomerular preparations. These data are largely consistent with other preclinical renal injury model reported in the literature as well as previous, less thorough assessments in the Tg26 model.

Comments on latest version:

The authors have revised the manuscript to acknowledge the potential limitations of the C16 tool compound used and have performed some additional analyses that suggest the PT-Mito population can be identified in samples from KPMP. The authors added some control images for the in situ hybridizations, which are helpful, though they don't get to the core issue of limited resolution to determine whether mitochondrial RNA is present in the nuclei of injured PT cells. Some additional work has been done to show that C16 treatment results in a decrease in phospho-PKR, a readout of PKR inhibition. These changes strengthen the manuscript by providing some evidence for the translatability of the PT-mito cluster to humans and some evidence for on-target activity for C16. It would be helpful if the authors could quantify the numbers of cells in IHC with nuclear transcripts as well as pointing out some specific examples in the images provided, as comparator data for the snRNAseq studies in which 3-6% of cortex cells had evidence of nuclear mitochondrial transcripts.

Reviewer #1 (Public Review):

This study is convincing because they performed time-resolved X-ray crystallography under different pH conditions using active/inactive metal ions and PpoI mutants, as with the activity measurements in solution in conventional enzymatic studies. Although the reaction mechanism is simple and maybe a little predictable, the strength of this study is that they were able to validate that PpoI catalyzes DNA hydrolysis through "a single divalent cation" because time-resolved X-ray study often observes transient metal ions which are important for catalysis but are not predictable in previous studies with static structures such as enzyme-substrate analog-metal ion complexes. The discussion of this study is well supported by their data. This study visualized the catalytic process and mutational effects on catalysis, providing a new insight into the catalytic mechanism of I-PpoI through a single divalent cation. The authors found that His98, a candidate of proton acceptor in the previous experiments, also affects the Mg2+ binding for catalysis without the direct interaction between His98 and the Mg2+ ion, suggesting that "Without a proper proton acceptor, the metal ion may be prone for dissociation without the reaction proceeding, and thus stable Mg2+ binding was not observed in crystallo without His98". In the future, this interesting feature observed in I-PpoI should be investigated by biochemical, structural and computational analyses using other one metal-ion dependent nucleases.

Reviewer #1 (Public Review):

Zheng et al. study the 'glass' transitions that occurs in proteins at ca. 200K using neutron diffraction and differential isotopic labeling (hydrogen/deuterium) of the protein and solvent. To overcome limitations in previous studies, this work is conducted in parallel with 4 proteins (myoglobin, cytochrome P450, lysozyme and green fluorescent protein) and experiments were performed at a range of instrument time resolutions (1ns - 10ps). The author's data looks compelling, and suggests that transitions in the protein and solvent behavior are not coupled and contrary to some previous reports, the apparent water transition temperature is a 'resolution effect'; i.e. instrument response is limited. This is likely to be important in the field, as a reassessment of solvent 'slaving' and the role of the hydration shell on protein dynamics should be reassessed in light of these findings.

Reviewer #1 (Public Review):<br /> Summary:

Zhu et al., investigate the cellular defects in glia as a result of loss in DEGS1/ifc encoding the dihydroceramide desaturase. Using the strength of Drosophila and its vast genetic toolkit, they find that DEGS1/ifc is mainly expressed in glia and its loss leads to profound neurodegeneration. This supports a role for DEGS1 in the developing larval brain as it safeguards proper CNS development. Loss of DEGS1/ifc leads to dihydroceramide accumulation in the CNS and induces alteration in the morphology of glial subtypes and a reduction in glial number. Cortex and ensheathing glia appeared swollen and accumulated internal membranes. Astrocyte-glia on the other hand displayed small cell bodies, reduced membrane extension and disrupted organization in the dorsal ventral nerve cord. They also found that DEGS1/ifc localizes primarily to the ER. Interestingly, the authors observed that loss of DEGS1/ifc drives ER expansion and reduced TGs and lipid droplet numbers. No effect on PC and PE and a slight increase in PS.

The conclusions of this paper are well supported by the data. The study could be further strengthened by a few additional controls and/or analyses.

Strengths:

This is an interesting study that provides new insight into the role of ceramide metabolism in neurodegeneration.

The strength of the paper is the generation of LOF lines, the insertion of transgenes and the use of the UAS-GAL4/GAL80 system to assess the cell-autonomous effect of DEGS1/ifc loss in neurons and different glial subtypes during CNS development.

The imaging, immunofluorescence staining and EM of the larval brain and the use of the optical lobe and the nerve cord as a readout are very robust and nicely done.

Drosophila is a difficult model to perform core biochemistry and lipidomics but the authors used the whole larvae and CNS to uncover global changes in mRNA levels related to lipogenesis and the unfolded protein responses as well as specific lipid alterations upon DEGS1/ifc loss.

Weaknesses:

The authors performed lipidomics and RTqPCR on whole larvae and larval CNS from which it is impossible to define the cell type-specific effects. Ideally, this could be further supported by performing single cell RNAseq on larval brains to tease apart the cell-type specific effect of DEGS1/ifc loss.

It's clear from the data that the accumulation of dihydroceramide in the ER triggers ER expansion but it remains unclear how or why this happens. Additionally, the authors assume that, because of the reduction in LD numbers, that the source of fatty acids comes from the LDs. But there is no data testing this directly.

The authors performed a beautiful EMS screen identifying several LOF alleles in ifc. However, the authors decided to only use KO/ifcJS3. The paper could be strengthened if the authors could replicate some of the key findings in additional fly lines.

The authors use M{3xP3-RFP.attP}ZH-51D transgene as a general glial marker. However, it would be advised to show the % overlap between the glial marker and the RFP since a lot of cells are green positive but not perse RFP positive and vice versa.

The authors indicate that other 3xP3 RFP and GFP transgenes at other genomic locations also label most glia in the CNE. Do they have a preferential overlap with the different glial subtypes?

Reviewer #1 (Public Review):

Summary:

The authors present a theoretical treatment of what they term the "Wright-Fisher-Haldane" model, a claimed modification of the standard model of genetic drift that accounts for variability in offspring number, and argue that it resolves a number of paradoxes in molecular evolution. Ultimately, I found this manuscript quite strange. The notion of effective population size as inversely related to the variance in offspring number is well known in the literature, and not exclusive to Haldane's branching process treatment. However, I found the authors' point about variance in offspring changing over the course of, e.g. exponential growth fairly interesting, and I'm not sure I'd seen that pointed out before. Nonetheless, I don't think the authors' modeling, simulations, or empirical data analysis are sufficient to justify their claims.

Weaknesses:

I have several outstanding issues. First of all, the authors really do not engage with the literature regarding different notions of an effective population. Most strikingly, the authors don't talk about Cannings models at all, which are a broad class of models with non-Poisson offspring distributions that nonetheless converge to the standard Wright-Fisher diffusion under many circumstances, and to "jumpy" diffusions/coalescents otherwise (see e.g. Mohle 1998, Sagitov (2003), Der et al (2011), etc.). Moreover, there is extensive literature on effective population sizes in populations whose sizes vary with time, such as Sano et al (2004) and Sjodin et al (2005). Of course in many cases here the discussion is under neutrality, but it seems like the authors really need to engage with this literature more.

The most interesting part of the manuscript, I think, is the discussion of the Density Dependent Haldane model (DDH). However, I feel like I did not fully understand some of the derivation presented in this section, which might be my own fault. For instance, I can't tell if Equation 5 is a result or an assumption - when I attempted a naive derivation of Equation 5, I obtained E(K_t) = 1 + r/c*(c-n)*dt. It's unclear where the parameter z comes from, for example. Similarly, is equation 6 a derivation or an assumption? Finally, I'm not 100% sure how to interpret equation 7. I that a variance effective size at time t? Is it possible to obtain something like a coalescent Ne or an expected number of segregating sites or something from this?

Similarly, I don't understand their simulations. I expected that the authors would do individual-based simulations under a stochastic model of logistic growth, and show that you naturally get variance in offspring number that changes over time. But it seems that they simply used their equations 5 and 6 to fix those values. Moreover, I don't understand how they enforce population regulation in their simulations---is N_t random and determined by the (independent) draws from K_t for each individual? In that case, there's no "interaction" between individuals (except abstractly, since logistic growth arises from a model that assumes interactions between individuals). This seems problematic for their model, which is essentially motivated by the fact that early during logistic growth, there are basically no interactions, and later there are, which increases variance in reproduction. But their simulations assume no interactions throughout!

The authors also attempt to show that changing variance in reproductive success occurs naturally during exponential growth using a yeast experiment. However, the authors are not counting the offspring of individual yeast during growth (which I'm sure is quite hard). Instead, they use an equation that estimates the variance in offspring number based on the observed population size, as shown in the section "Estimation of V(K) and E(K) in yeast cells". This is fairly clever, however, I am not sure it is right, because the authors neglect covariance in offspring between individuals. My attempt at this derivation assumes that I_t | I_{t-1} = \sum_{I=1}^{I_{t-1}} K_{i,t-1} where K_{i,t-1} is the number of offspring of individual i at time t-1. Then, for example, E(V(I_t | I_{t-1})) = E(V(\sum_{i=1}^{I_{t-1}} K_{i,t-1})) = E(I_{t-1})V(K_{t-1}) + E(I_{k-1}(I_{k-1}-1))*Cov(K_{i,t-1},K_{j,t-1}). The authors have the first term, but not the second, and I'm not sure the second can be neglected (in fact, I believe it's the second term that's actually important, as early on during growth there is very little covariance because resources aren't constrained, but at carrying capacity, an individual having offspring means that another individuals has to have fewer offspring - this is the whole notion of exchangeability, also neglected in this manuscript). As such, I don't believe that their analysis of the empirical data supports their claim.

Thus, while I think there are some interesting ideas in this manuscript, I believe it has some fundamental issues: first, it fails to engage thoroughly with the literature on a very important topic that has been studied extensively. Second, I do not believe their simulations are appropriate to show what they want to show. And finally, I don't think their empirical analysis shows what they want to show.

References:

Möhle M. Robustness results for the coalescent. Journal of Applied Probability. 1998;35(2):438-447. doi:10.1239/jap/1032192859

Sagitov S. Convergence to the coalescent with simultaneous multiple mergers. Journal of Applied Probability. 2003;40(4):839-854. doi:10.1239/jap/1067436085

Der, Ricky, Charles L. Epstein, and Joshua B. Plotkin. "Generalized population models and the nature of genetic drift." Theoretical population biology 80.2 (2011): 80-99

Sano, Akinori, Akinobu Shimizu, and Masaru Iizuka. "Coalescent process with fluctuating population size and its effective size." Theoretical population biology 65.1 (2004): 39-48

Sjodin, P., et al. "On the meaning and existence of an effective population size." Genetics 169.2 (2005): 1061-1070

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes a series of experiments using human intracranial neural recordings designed to evaluate processing of self-generated speech in the setting of feedback delays. Specifically, the authors aim to address the question about the relationship between speech-induced suppression and feedback sensitivity in the auditory cortex, which, relationship has been conflicting in the literature. They found a correlation between speech suppression and feedback delay sensitivity, suggesting a common process. Additional controls were done for possible forward suppression/adaptation, as well as controlling for other confounds due to amplification, etc.

Strengths:<br /> The primary strength of the manuscript is the use of human intracranial recording, which is a valuable resource and gives better spatial and temporal resolution than many other approaches. The use of delayed auditory feedback is also novel and has seen less attention than other forms of shifted feedback during vocalization. Analyses are robust and include demonstrating a scaling of neural activity with the degree of feedback delay, more robust evidence for error encoding than simply using a single feedback perturbation.

Weaknesses:<br /> Some of the analyses performed differ from those used in past work, which limits the ability to directly compare the results. Notably, past work has compared feedback effects between production and listening, which was not done here. There were also some unusual effects in the data, such as increased activity with no feedback delay when wearing headphones, that the authors attempted to control for with additional experiments, but remain unclear. Confounds by behavioral results of delayed feedback are also unclear.

Overall the work is well done and clearly explained. The manuscript addresses an area of some controversy and does so in a rigorous fashion, namely the correlation between speech-induced suppression and feedback sensitivity (or lack thereof). While the data presented overlap that collected and used for a previous paper, this is expected given the rare commodity these neural recordings represent. Contrasting these results to previous ones using pitch-shifted feedback should spawn additional discussion and research, including verification of the previous finding, looking at how the brain encodes feedback during speech over multiple acoustic dimensions, and how this information can be used in speech motor control.

Reviewer #1 (Public Review):

Summary:

The authors want to elucidate which are the mechanisms that regulate the immune response in physiological conditions in cortical development. To achieve this goal, authors used a wide range of mutant mice to analyse the consequences of immune activation in the formation of cortical ectopia in mice.

Strengths:

The authors demonstrated that Abeta monomers are anti-inflammatory and inhibit microglial activation. This is a novel result that demonstrates the physiological role of APP in cortical development.

Weaknesses:

-On the other hand, cortical ectopia has been already described in mouse models in which the amyloid signalling has been disrupted (Herms et al., 2004; Guenette et al., 2006), making the current study less novel.

One of the molecules analysed is Ric8a, a GTPase activator involved in neuronal development. Authors used the conditional mutant mice Emx1-Ric8a to delete Ric8a from early progenitors and glutamatergic neurons in the pallium. Emx1-Ric8a mutant mice present cortical ectopias and authors attributed this malformation to the increase in inflammatory response due to Ric8a deletion in microglia. Several discordances do not fit this interpretation:

-The role of Ric8a in cortical development and function has been already described in several papers, but none of them has been cited in the current manuscript (Kask et al., 2015, 2018; Ruisu et al., 2013; Tonissoo et al., 2006).

-Ectopia formation in the cortex has been already described in Nestin-Ric8a cKO mice (Kask et al., 2015). In the current manuscript, authors analyzed the same mutant mice (Nestin-Ric8a), but they did not detect any ectopia. Authors should discuss this discordance.

-Authors claim that microglia express Emx1, and therefore, Ric8a is deleted in microglia cells. However, the arguments for this assumption are very weak and the evidence suggests that this is not the case. This is an important point considering that authors want to emphasise the role of Ric8a in microglia activation, and therefore, additional experiments should demonstrate that Ric8a is deleted in microglia in Emx1-Ric8a mutant mice.

Reviewer #1 (Public Review):

Summary:<br /> Meissner et al describe an update on the collection of split-GAL4 lines generated by a consortium led by Janelia Research Campus. This follows the same experimental pipeline described before and presents as a significant increment to the present collection. This will strengthen the usefulness and relevance of "splits" as a standard tool for labs that already use this tool and attract more labs and researchers to use it.

Strengths:<br /> This manuscript presents a solid step to establish Split-GAL4 lines as a relevant tool in the powerful Drosophila toolkit. Not only does the raw number of available lines contribute to the relevance of this tool in the "technical landscape" of genetic tools, but additional features of this effort contribute to the successful adoption. These include:<br /> (1) A description of expression patterns in the adult and larvae, expanding the "audience" for these tools<br /> (2) A classification of line combination according to quality levels, which provides a relevant criterion while deciding to use a particular set of "splits".<br /> (3) Discrimination between male and female expression patterns, providing hints regarding the potential role of these gender-specific circuits.<br /> (4) The search engine seems to be user-friendly, facilitating the retrieval of useful information.<br /> Overall, the authors employed a pipeline that maximizes the potential of the Split-GAL4 collection to the scientific community.

Weaknesses:<br /> The following aspects apply:<br /> The use of split-GAL4 lines has improved tremendously the genetic toolkit of Drosophila and this manuscript is another step forward in establishing this tool in the genetic repertoire that laboratories use. Thus, this would be a perfect opportunity for the authors to review the current status of this tool, addressing its caveats and how to effectively implement it into the experimental pipeline.

(1) While the authors do bring up a series of relevant caveats that the community should be aware of while using split-GAL4 lines, the authors should take the opportunity to address some of the genetic issues that frequently arise while using the described genetic tools. This is particularly important for laboratories that lack the experience using split-GAL4 lines and wish to use them. Some of these issues are covertly brought up, but not entirely clarified.<br /> First, why do the authors (wisely) rescreen the lines using UAS-CsChrimson-mVenus? One reason is that using another transgene (such as UAS-GFP) and/or another genomic locus can drive a different expression pattern or intensities. Although this is discussed, this should be made more explicit and the readers should be aware of this.<br /> Second, it would be important to include a discussion regarding the potential of hemidriver lines to suffer from transvection effects whenever there is a genetic element in the same locus. These are serious issues that prevent a more reliable use of split-GAL4 lines that, once again, should be discussed.

(2) The authors simply mention that the goal of the manuscript is to "summarize the results obtained over the past decade.". A better explanation would be welcomed in order to understand the need of a dedicated manuscript to announce the availability of a new batch of lines when previous publications already described the Split-GAL4 lines. At the extreme, one might question why we need a manuscript for this when a simple footnote on Janelia's website would suffice.

Reviewer #1 (Public Review):

Their findings elucidate the mechanisms underlying 2-AA-mediated reduction of pyruvate transport into mitochondria, which impairs the interaction between ERRα and PGC1α, consequently suppressing MPC1 expression and reducing ATP production in tolerized macrophages.

This paper presents a novel discovery regarding the mechanisms through which PA regulates the bioenergetics of tolerized macrophages. This paper will provide valuable insights for the journal's broad readership of scientists.

Reviewer #1 (Public Review):

Summary:

Sumarac et al investigate differences in globus pallidus internus (GPi) spike activity and short- and long-term plasticity of direct pathway projections in patients with Parkinson's disease (PD) and dystonia. Their main claims are that GPi neurons exhibit distinct characteristics in these two disorders, with PD associated with specific power-frequency oscillations and dystonia showing lower firing rates, increased burstiness, and less regular activity. Additionally, long-term plasticity and synaptic depression appear to differ between the two conditions. The authors suggest that these findings support the concept of hyperfunctional GPi output in PD and hypofunctional output in dystonia, possibly driven by variations in plasticity of striato-pallidal synapses. Overall enthusiasm is relatively high, but I think the discussion omits discussing findings that don't align well with standard models.

Strengths:

- These types of studies are valuable as the data arise from patients who have dystonia or PD. This could provide unique insights into disease pathophysiology that might not be recapitulated in animal systems work.

Comments on latest version:

The authors addressed my concerns in their revision.

Reviewer #1 (Public Review):

Summary:<br /> The authors aim to consider the effects of phonotactics on the effectiveness of memory reactivation during sleep. They have created artificial words that are either typical or atypical and showed that reactivation improves memory for the latter but not the former.

Strengths:<br /> This is an interesting design and a creative way of manipulating memory strength and typicality. In addition, the spectral analysis on both the wakefulness data and the sleep data is well done. The article is clearly written and provides a relevant and comprehensive of the literature and of how the results contribute to it.

Weaknesses:<br /> (1) Unlike most research involving artificial language or language in general, the task engaged in this manuscript did not require (or test) learning of meaning or translation. Instead, the artificial words were arbitrarily categorised and memory was tested for that categorisation. This somewhat limits the interpretation of the results as they pertain to language science, and qualifies comparisons with other language-related sleep studies that the manuscript builds on.

(2) Participants had to determine whether words are linked with reward or omission of punishment (if correctly categorised). Therefore, the task isn't a mere item categorisation task (group A/B), but also involves the complicated effects of reward (e.g., reward/loss asymmetries as predicted by prospect theory). This is not, in itself, a flaw, but there isn't a clear hypothesis as to the effects of reward on categorisation, and therefore no real justification for this design. This aspect of the task may add unneeded complexity (at best) or some reward-related contamination of the results (at worst).

(3) The study starts off with a sample size of N=39 but excludes 17 participants for some crucial analyses. This is a high number, and exclusion criteria were not pre-registered. Having said that, some criteria seem very reasonable (e.g., excluding participants who were not fully exposed to words during sleep).

(4) Relatedly, the final N is low for a between-subjects study (N=11 per group). This is adequately mentioned as a limitation, but since it does qualify the results, it seemed important to mention it here.

(5) The linguistic statistics used for establishing the artificial words are all based on American English, and are therefore in misalignment with the spoken language of the participants (which was German). This is a limitation of the study. The experimenters did not check whether participants were fluent in English. In all fairness, the behavioural effects presented in Figure 2A are convincing, providing a valuable manipulation test.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

The genetic data presented is thorough and solid, and the observation is novel.

Weaknesses:

The authors have addressed the main weakness of the study in the revised manuscript, by providing data showing stimulation of exopher production in a single-copy transgenic line. Whether this process is related to the extrusion of cellular damage by the neurons in relatively young day 2 animals should be addressed in future studies.

Reviewer #1 (Public Review):

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

Reviewer #1 (Public Review):

Summary:

The manuscript by Tuberosa et al outlines the generation of a new set of transgenic mice that express different recombinases specifically in Smim32 positive cells. They show that Smim32 is a useful marker of the mouse claustrum. Therefore, these mice could be useful for functional studies focused on measuring claustrum activity or manipulating the claustrum using optogenetic and pharmacogenetic tools.

Strengths:

The manuscript provides a new genetic approach to target claustrum neurons, using Smim32. The work may help future studies where claustrum excitatory neurons are measured or manipulated.

Weaknesses:

A toolbox is only useful if others can use it. Therefore, these mice should be made available to the community through commercial vendors. Without this added step, this toolbox and method does not provide any utility to the research community.

The data presented and quantified in each figure subpanel are from N = 1 mouse. This is not acceptable or conventional. Replication is an important aspect of any paper, and currently, there are no replicates contained in the manuscript. Additional examples of female mice should also be included and separately quantified. Mice from different litters should be used for replicates.

Given the preliminary nature of these data from the minimum possible number of mice, a better characterization of all data should be undertaken.

The tone of the paper implies that this is the superior way to locate the claustrum. A more balanced discussion of the strengths and weaknesses of these mice should be included. Several sentences highlighting the shortfalls of other approaches are overstated and should be toned down.

Reviewer #1 (Public Review):

Summary

In this study, Takagi and colleagues demonstrate that changes in axonal arborization of the segmental wave motor command neurons are sufficient to change behavioral motor output.

The authors identify the Wnt receptors DFz2 and DFz4 and the ligand Wnt4 as modulators of stereotypic segmental arborization patterns of segmental wave neurons along the anterior-posterior body axis. Based on both embryonic expression pattern analysis and genetic manipulation of the signaling components in wave neurons (receptors) and the neuropil (Wnt4) the authors convincingly demonstrate that Wnt4 acts as a repulsive ligand for DFz2 that restricts posterior axon guidance of both anterior and posterior wave neurons. They also provide the first evidence that Wnt4 potentially acts as an attractive ligand for Df4 to promote the posterior extension of p-wave neurons. Interestingly, artificial optogenetic activation of all wave neurons that normally induces backward locomotion due to the activity of anterior wave neurons, fails to induce backward locomotion in a DFz2 knockdown condition with altered axonal extensions of all wave neurons towards posterior segments. In addition, the authors now observe enhanced fast-forward locomotion, a feature normally induced by posterior wave neurons. Consistent with these findings, they observe that the natural response to an anterior tactile stimulus is similarly altered in DFz2 knockdown animals. The animals respond with less backward movement and increased fast forward motion. These results suggest that alterations in the innervation pattern of wave motor command neurons are sufficient to switch behavioral response programs.

Strengths

The authors convincingly demonstrate the importance of Wnt signaling for anterior-posterior axon guidance of a single class of motor command neurons in the larval CNS. The demonstration that alteration of the expression level of a single axon guidance receptor is sufficient to not only alter the innervation pattern but to significantly modify the behavioral response program of the animal provides a potential entry point to understanding behavioral adaptations during evolution.

Weaknesses

While the authors demonstrate an alteration of the behavioral response to a natural tactile stimulus the observed effects, a reduction of backward motion and increased fast-foward locomotion, currently cannot be directly correlated to the morphological alterations observed in the single-neuron analyses. The authors do not report any loss of innervation in the "normal" target region but only a small additional innervation of more posterior regions. An analysis of synaptic connectivity and/or a more detailed morphological analysis that is supported by a larger number of analyzed neurons both in control and experimental animals would further strengthen the confidence of the study. As the authors suggest an alteration of the command circuitry, a direct observation of the downstream activation pattern in response to selective optogenetic stimulation of anterior wave neurons would further strengthen their claims (analogous to Takagi et al., 2017, Figure 4).

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors present a novel CRISPR/Cas9-based genetic tool for the dopamine receptor dop1R2. Based on the known function of the receptor in learning and memory, they tested the efficacy of the genetic tool by knocking out the receptor specifically in mushroom body neurons. The data suggest that dop1R2 is necessary for longer-lasting memories through its action on ⍺/ß and ⍺'/ß' neurons but is dispensable for short-term memory and thus in ɣ neurons. The experiments impressively demonstrate the value of such a genetic tool and illustrate the specific function of the receptor in subpopulations of KCs for longer-term memories. The data presented in this manuscript are significant.

Reviewer #1 (Public Review):

Summary:

This paper explores how diverse forms of inhibition impact firing rates in models for cortical circuits. In particular, the paper studies how the network operating point affects the balance of direct inhibition from SOM inhibitory neurons to pyramidal cells, and disinhibition from SOM inhibitory input to PV inhibitory neurons. This is an important issue as these two inhibitory pathways have largely been studies in isolation. Support for the main conclusions is generally solid, but could be strengthened by additional analyses.

Strengths:

A major strength of the paper is the systematic exploration of how circuit architecture effects the impact of inhibition. This includes scans across parameter space to determine how firing rates and stability depend on effective connectivity. This is done through linearization of the circuit about an effective operating point, and then the study of how perturbations in input effect this linear approximation.

Weaknesses:

The linearization approach means that the conclusions of the paper are valid only on the linear regime of network behavior. The paper would be substantially strengthened with a test of whether the conclusions from the linearized circuit hold over a large range of network activity. Is it possible to simulate the full network and do some targeted tests of the conclusions from linearization? Those tests could be guided by the linearization to focus on specific parameter ranges of interest.

The results illustrated in the figures are generally well described but there is very little intuition provided for them. Are there simplified examples or explanations that could be given to help the results make sense? Here are some places such intuition would be particularly helpful:<br /> page 6, paragraph starting "In sum ..."<br /> Page 8, last paragraph<br /> Page 10, paragraph starting "In summary ..."<br /> Page 11, sentence starting "In sum ..."

Reviewer #1 (Public Review):

Summary:

Dr. Santamaria's group previously utilized antigen-specific nanomedicines to induce immune tolerance in treating autoimmune diseases. The success of this therapeutic strategy has been linked to expanded regulatory mechanisms, particularly the role of T-regulatory type-1 (TR1) cells. However, the differentiation program of TR1 cells remained largely unclear. Previous work from the authors suggested that TR1 cells originate from T follicular helper (TFH) cells. In the current study, the authors aimed to investigate the epigenetic mechanisms underlying the transdifferentiation of TFH cells into IL-10-producing TR1 cells. Specifically, they sought to determine whether this process involves extensive chromatin remodeling or is driven by pre-existing epigenetic modifications. Their goal was to understand the transcriptional and epigenetic changes facilitating this transition and to explore the potential therapeutic implications of manipulating this pathway.

The authors successfully demonstrated that the TFH-to-TR1 transdifferentiation process is driven by pre-existing epigenetic modifications rather than extensive new chromatin remodeling. The comprehensive transcriptional and epigenetic analyses provide robust evidence supporting their conclusions.

Strengths:

(1) The study employs a broad range of bulk and single-cell transcriptional and epigenetic tools, including RNA-seq, ATAC-seq, ChIP-seq, and DNA methylation analysis. This comprehensive approach provides a detailed examination of the epigenetic landscape during the TFH-to-TR1 transition.

(2) The use of high-throughput sequencing technologies and sophisticated bioinformatics analyses strengthens the foundation for the conclusions drawn.

(3) The data generated can serve as a valuable resource for the scientific community, offering insights into the epigenetic regulation of T-cell plasticity.

(4) The findings have significant implications for developing new therapeutic strategies for autoimmune diseases, making the research highly relevant and impactful.

Weaknesses:

(1) While the scope of this study lies in transcriptional and epigenetic analyses, the conclusions need to be validated by future functional analyses.

(2) This study successfully identified key transcription factors and epigenetic marks. How these factors mechanistically drive chromatin closure and gene expression changes during the TFH-to-TR1 transition requires further investigation.

(3) The study provides a snapshot of the epigenetic landscape. Future dynamic analysis may offer more insights into the progression and stability of the observed changes.

Reviewer #1 (Public Review):

Summary:

The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

Strengths:

(1) 7T high-resolution MRS is used.

(2) This study combines the behavioral tests, MRS, and fMRI.

Weaknesses:

Major:

(1) In Melnick (2013) IQ scores were measured by the full set of WAIS-III, including all subtests. However, this study only used visual spatial domain of gF. I wonder why only the visuo-spatial subtest was used not the full WAIS-III? I am wondering whether other subtests were conducted and, if so, please include the results as well to have comprehensive comparisons with Melnick (2013).

Minor:

(1) Table 1 and Table supplementary 1-3 contain many correlation results. But what are the main points of these values? Which values do the authors want to highlight? Why are only p-values shown with significance symbols in Table supplementary 2??

(2) Line 27, it is unclear to me what is "the canonical theory".

(3) Throughout the paper, the authors use "MT+", I would suggest using "hMT+" to indicate the human MT complex, and to be consistent with the human fMRI literature.

(4) At the beginning of the results section, I suggest including the total number of subjects. It is confusing what "31/36 in MT+, and 28/36 in V1" means.

(5) Line 138, "This finding supports the hypothesis that motion perception is associated with neural activity in MT+ area". This sentence is strange because it is a well established finding in numerous human fMRI papers. I think the authors should be more specific about what this finding implies.

(6) There are no unit labels for all x- and y-axies in Figure 1. I only see the unit for Conc is mmol per kg wet weight.

(7) Although the correlations are not significant in Figure supplement 2&3, please also include the correlation line, 95% confidence interval, and report the r values and p values (i.e., similar format as in Figure 1C).

(8) There is no need to separate different correlation figures into Figure supplementary 1-4. They can be combined into the same figure.

(9) Line 213, as far as I know, the study (Melnick et al., 2013) is a psychophysical study and did not provide evidence that the spatial suppression effect is associated with MT+.

(10) At the beginning of the results, I suggest providing more details about the motion discrimination tasks and the measurement of the BDT.

(11) Please include the absolute duration thresholds of the small and large sizes of all subjects in Figure 1.

(12) Figure 5 is too small. The items in plot a and b can be barely visible.

Reviewer #1 (Public Review):

In this revised manuscript, authors have conducted epigenetic and transcriptomic profiling to understand how environmental chemicals such as BPS can cause epimutations that can propagate to future generations. They used isolated somatic cells from mice (Sertoli, granulosa), pluripotent cells to model preimplantation embryos (iPSCs) and cells to model the germline (PGCLCs). This enabled them to model sequential steps in germline development, and when/how epimutations occur. The major findings were that BPS induced unique epimutations in each cell type, albeit with qualitative and quantitative cell-specific differences; that these epimutations are prevalent in regions associated with estrogen-response elements (EREs); and that epimutations induced in iPSCs are corrected as they differentiate into PGCLCs, concomitant with the emergence of de novo epimutations. This study will be useful in understanding the multigenerational effects of EDCs, and underlying mechanisms.

Strengths include:

(1) Using different cell types representing life stages of epigenetic programming and during which exposures to EDCs have different effects. This progression revealed information both about the correction of epimutations and the emergence of new ones in PGCLCs.

(2) Work conducted by exposing iPSCs to BPS or vehicle, then differentiating to PGCLCs, revealed that novel epimutations emerged.

(3) Relating epimutations to promoter and enhancer regions

During the review process, authors improved the manuscript through better organization, clarifying previous points from reviewers, and providing additional data.

Reviewer #1 (Public Review):

Summary:

Kan et al. report the serendipitous discovery of a Bacillus amyloliquefaciens strain that kills N. gonorrhoeae. They use TnSeq to identify that the anti-gonococcal agent is oxydifficidin and show that it acts at the ribosome and that one of the dedA gene products in N. gonorrhoeae MS11 is important for moving the oxydifficidin across the membrane.

Strengths:

This is an impressive amount of work, moving from a serendipitous observation through TnSeq to characterize the mechanism by which Oxydifficidin works.

Weaknesses:

(1) There are important gaps in the manuscript's methods.

(2) The work should evaluate antibiotics relevant to N. gonorrhoeae.

(3) The genetic diversity of dedA and rplL in N. gonorrhoeae is not clear, neither is it clear whether oxydifficidin is active against more relevant strains and species than tested so far.

Reviewer #1 (Public Review):

Summary:

This paper details a study of endothelial cell vessel formation during zebrafish development. The results focus on the role of aquaporins, which mediate the flow of water across the cell membrane, leading to cell movement. The authors show that actin and water flow together drive endothelial cell migration and vessel formation. If any of these two elements are perturbed, there are observed defects in vessels. Overall, the paper significantly improves our understanding of cell migration during morphogenesis in organisms.

Strengths:

The data are extensive and are of high quality. There is a good amount of quantification with convincing statistical significance. The overall conclusion is justified given the evidence.

Weaknesses:

There are two weaknesses, which if addressed, would improve the paper.

(1) The paper focuses on aquaporins, which while mediates water flow, cannot drive directional water flow. If the osmotic engine model is correct, then ion channels such as NHE1 are the driving force for water flow. Indeed this water is shown in previous studies. Moreover, NHE1 can drive water intake because the export of H+ leads to increased HCO3 due to the reaction between CO2+H2O, which increases the cytoplasmic osmolarity (see Li, Zhou and Sun, Frontiers in Cell Dev. Bio. 2021). If NHE cannot be easily perturbed in zebrafish, it might be of interest to perturb Cl channels such as SWELL1, which was recently shown to work together with NHE (see Zhang, et al, Nat. Comm. 2022).

(2) In some places the discussion seems a little confusing where the text goes from hydrostatic pressure to osmotic gradient. It might improve the paper if some background is given. For example, mention water flow follows osmotic gradients, which will build up hydrostatic pressure. The osmotic gradients across the membrane are generated by active ion exchangers. This point is often confused in literature and somewhere in the intro, this could be made clearer.

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.

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

Weaknesses:

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:

Otero-Coronel et al. address an important question for neuroscience - how does a premotor neuron capable of directly controlling behavior integrate multiple sources of sensory inputs to inform action selection? For this, they focused on the teleost Mauthner cell, long known to be at the core of a fast escape circuit. What is particularly interesting in this work is the naturalistic approach they took. Classically, the M-cell was characterized, both behaviorally and physiologically, using an unimodal sensory space. Here the authors make the effort (substantial!) to study the physiology of the M-cell taking into account both the visual and auditory inputs. They performed well-informed electrophysiological approaches to decipher how the M-cell integrates the information of two sensory modalities depending on the strength and temporal relation between them.

Strengths:

The empirical results are convincing and well-supported. The manuscript is well-written and organized. The experimental approaches and the selection of stimulus parameters are clear and informed by the bibliography. The major finding is that multisensory integration increases the certainty of environmental information in an inherently noisy environment.

Weaknesses:

Even though the manuscript and figures are well organised, I found myself struggling to understand key points of the figures.

For example, in Figure 1 it is not clear what are actually the Tonic and Phasic components. The figure will benefit from more details on this matter. Then, in Figure 4 the label for the traces in panel A is needed since I was not able to pick up that they were coming from different sensory pathways.

In line 338 it should be optic tectum and not "optical tectum".

Reviewer #1 (Public Review):

Summary:

This work identified new NMD inhibitors and tested them for cancer treatment, based on the hypothesis that inhibiting NMD could lead to the production of cancer neoantigens from the stabilized mutant mRNAs, thereby enhancing the immune system's ability to recognize and kill cancer cells. Key points of the study include:

• Development of an RNA-seq based method for NMD analysis using mixed isogenic cells that express WT or mutant transcripts of STAG2 and TP53 with engineered truncation mutations.

• Application of this method for a drug screen and identified several potential NMD inhibitors.

• Demonstration that one of the identified compounds, LY3023414, inhibits NMD by targeting the SMG1 protein kinase in the NMD pathway in cultured cells and mouse xenografts.

• Due to the in vivo toxicity observed for LY3023414, the authors developed 11 new SMG1 inhibitors (KVS0001-KVS0011) based on the structures of the known SMG1 inhibitor SMG1i-11 and the SMG1 protein itself.

• Among these, KVS0001 stood out for its high potency, excellent bioavailability and low toxicity in mice. Treatment with KVS0001 caused NMD inhibition and increased presentation of neoantigens on MHC-I molecules, resulting in the clearance of cancer cells in vitro by co-cultured T cells and cancer xenografts in mice by the immune system.

These findings support the strategy of targeting the NMD pathway for cancer treatment and provide new research tools and potential lead compounds for further exploration.

Strengths:

The RNA-seq based NMD analysis, using isogenic cell lines with specific NMD-inducing mutations, represents a novel approach for the high-throughput identification of potential NMD modulators or genetic regulators. The effectiveness of this method is exemplified by the identification of a new activity of AKT1/mTOR inhibitor LY3023414 in inhibiting NMD.

The properties of KVS0001 described in the manuscript as a novel SMG1 inhibitor suggest its potential as a lead compound for further testing the NMD-targeting strategies in cancer treatment. Additionally, this compound may serve as a useful research tool.

The results of the in vitro cell killing assay and in vivo xenograft experiments in both immuno-proficient and immune-deficient mice indicate that inhibiting NMD could be a viable therapeutic strategy for certain cancers.

Weaknesses:

The authors did not address the potential effects of NMD/SMG1 inhibitors on RNA splicing. Given that the transcripts of many RNA-binding proteins are natural targets of NMD, inhibiting NMD could significantly alter splicing patterns. This, in turn, might influence the outcomes of the RNA-seq-based method for NMD analysis and result interpretation.

While the RNA-seq based approach offers several advantages for analyzing NMD, the effects of NMD/SMG1 inhibitors observed through this method should be confirmed using established NMD reporters. This step is crucial to rule out the possibility that mutations in STAG2 or TP53 affect NMD in cells, as well as to address potential clonal variations between different engineered cell lines.

The results from the SMG1/UPF1 knockdown and SMG1i-11 experiments presented in Figure 3 correlate with the effects seen for LY3023414, but they do not conclusively establish SMG1 as the direct target of LY3023414 in NMD inhibition. An epistatic analysis with LY3023414 and SMG1-knockdown is needed.

Comment on the revised version:

Although KVS0001 exhibits promising properties as an SMG1 inhibitor for cancer treatment, it remains unclear if it is superior to existing SMG1 inhibitors, as no direct comparisons have been made.

Reviewer #1 (Public Review):

Summary:

Trutti and colleagues used 7T fMRI to identify brain regions involved in subprocesses of updating the content of working memory. Contrary to past theoretical and empirical claims that the striatum serves a gating function when new information is to be entered into working memory, the relevant contrast during a reference-back task did not reveal significant subcortical activation. Instead, the experiment provided support for the role of subcortical (and cortical) regions in other subprocesses.

Strengths:

The use of high-field imaging optimized for subcortical regions in conjunction with the theory-driven experimental design mapped well to the focus on a hypothetical striatal gating mechanism.

Consideration of multiple subprocesses and the transparent way of identifying these, summarized in a table, will make it easy for future studies to replicate and extend the present experiment.

Weaknesses:

The reference-back paradigm seems to only require holding a single letter in working memory (X or O; Figure 1). It remains unclear how such low demand on working memory influences associated fMRI updating responses. It is also not clear whether reference-switch trials with 'same' response truly tax working-memory updating (and gate opening), as the working-memory content/representation does not need to be updated in this case. These potential design issues, together with the rather low number of experimental trials, raise concerns about the demonstrated absence of evidence for striatal gate opening.

The authors provide a motivation for their multi-step approach to fMRI analyses. Still, the three subsections of fMRI results (3.2.1; 3.2.2; 3.3.3) for 4 subprocesses each (gate opening, gate closing, substitution, updating mode) made the Results section complex and it was not always easy to understand why some but not other approaches revealed significant effects (as the midbrain in gate opening).

The many references to the role of dopamine are interesting, but the discussion of dopaminergic pathways and signals remains speculative and must be confirmed in future studies (e.g., with PET imaging).

Reviewer #1 (Public Review):

The authors investigate pleiotropy in the genetic loci previously associated to a range of neuropsychiatric disorders: Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Parkinson's disease, and schizophrenia. The local statistical fine-mapping and variant colocalisation approaches they use have the potential to uncover not only shared loci but also shared causal variants between these disorders. There is existing literature describing the pleiotropy between ALS and these other disorders but here the authors apply state-of-the-art, local genetic correlation approaches to further refine any relationships.

Complex disease and GWAS is not my area of expertise but the authors managed to present their methods and results in a clear, easy-to-follow manner. Their results statistically support several correlations between the disorders and, for ALS and AD, a shared variant in the vicinity of the lead SNP from the original ALS GWAS. Such findings could have important implications for our understanding of the mechanisms of such disorders and eventually the possibility of managing and treating them.

The authors have built a useful pipeline that plugs together all the gold-standard, existing software to perform this analysis and made it openly available which is commendable. However, there is little discussion of what software is available to perform global and local correlation analysis and, if there are multiple tools available, why they consider the ones they selected to be the gold-standard.

There is some mention of previous findings of genetic pleiotropy between ALS and these other disorders in the introduction, and discussion of their improved ALS-AD evidence relative to previous work. However, detailed comparisons of their other correlations to what was described before for the same pairs of disorders (if any) is missing. Adding this would strengthen the impact of this paper.

Finally, being new to this approach I found the abstract a little confusing. Initially, the shared causal variant between ALS and AD is mentioned but immediately in the following sentence they describe how their study "suggested that disease- implicated variants in these loci often differ between traits". After reading the whole paper I understood that the ALS-AD shared variant was the exception but it may be best to restructure this part of the abstract. Additionally, in the abstract the authors state that different variants "suggests the role of distinct mechanisms across diseases despite shared loci". Is it not possible that different variants in the same regulatory region or protein-coding parts of a gene could be having the same effect and mechanism? Or does the methodology to establish that different variants are involved automatically mean that the variants are too distant for this to be possible?

These concerns were addressed in the revised version of this manuscript.

Reviewer #1 (Public Review):

Summary:

In this paper, Steinemann et al. characterized the nature of stochastic signals underlying the trial-averaged responses observed in lateral intraparietal cortex (LIP) of non-human primates (NHPs), while these performed the widely used random dot direction discrimination task. Ramp-up dynamics in the trial averaged LIP responses were reported in numerous papers before. But the temporal dynamics of these signals at the single-trial level have been subject to debate. Using large scale neuronal recordings with Neuropixels in NHPs, allows the authors to settle this debate rather compellingly. They show that drift-diffusion like computations account well for the observed dynamics in LIP.

Strengths:

This work uses innovative technical approaches (Neuropixel recordings in behaving macaque monkeys). The authors tackle a vexing question that requires measurements of simultaneous neuronal population activity and hence leverage this advanced recording technique in a convincing way.

They use different population decoding strategies to help interpret the results.

They also compare how decoders relying on the data-driven approach using dimensionality reduction of the full neural population space compares to decoders relying on more traditional ways to categorize neurons that are based on hypotheses about their function. Intriguingly, although the functionally identified neurons are a modest fraction of the population, decoders that only rely on this fraction achieve comparable decoding performance to those relying on the full population. Moreover, decoding weights for the full population did not allow the authors to reliably identify the functionally identified subpopulation.

The revision addressed the minor weaknesses to our satisfaction.

Reviewer #1 (Public Review):

This is an interesting and thorough paper describing the modes of locomotion of the nematode C. elegans in the context of random exploration or response to an aversive stimulus. The authors collect extensive statistics on various locomotor states and compare findings to a minimal mathematical model inspired by the data. Their data reveal biases in two modes of turning- gradual and sharp- which define the path structure of the animal moving on an agar plate. The authors also find that animals tend to overcome inherent anatomical/physiological biases to locomotion when escaping aversive stimuli.

Understanding animal navigation is a window for revealing efficient algorithms for exploration of space, and also allows testing of the extent to which we understand how the nervous system produces specific behaviors. This paper adds important analysis towards these goals. I have a couple of comments that may be worth considering:

(1) The authors place a circular barrier of SDS near the edges of their plates and assume that this aversive stimulus is only sensed when the animal is near the barrier. However, it is possible that the SDS diffuses enough into the interior of the plate to affect the navigation statistics. In this case, the data they have accumulated may in fact be some sort of combination of exploratory locomotion and a general background SDS aversive stimulus. Can the authors control for this? Perhaps test the plates at different distances and times for SDS diffusion? Or replace the barrier with a physical one and not a chemical one?

(2) The authors do not look at mutants or perturb the physiology in defined ways relevant to the locomotion being studied to test their model. Specifically, it would be of interest to identify neural circuits that govern some of the parameters in the model. Although the authors bring this up in their Discussion section, it seems appropriate for this paper, as it would considerably bolster the impact of the work.

Public Review:

The study addresses the role of enkephalins, which are specifically expressed by regulatory T cells (Treg), in sensory perception in mice. The authors used a combination of transcriptomic databases available online to characterize the molecular signature of Treg. The proenkephalin gene Penk is among the most enriched transcripts, suggesting that Treg plays an analgesic role through the release of endogenous opioids. In addition, in silico analysis suggests that Penk is regulated by the TNFR superfamily; this being experimentally confirmed. Using flow cytometry analysis, the authors then show that Penk is mostly expressed in Treg of the skin and colon, compared to other immune cells. Finally, genetic conditional excision of Penk, selectively in Treg, results in heat hypersensitivity, as assessed by behavior analysis.

Editors' note: The authors accepted most if not all the suggestions given by the reviewers and the revised version of the manuscript is substantially improved.

Reviewer #2 (Public Review):

Main comment from 1st review:

Weaknesses:<br /> The modeling is interesting, with the integration of tension through tension triangulation around vertices and thus integrating force inference directly in the vertex model. However, the authors are not using it to test their hypothesis and support their analysis at the tissue level. Thus, although interesting, the analysis at the tissue level stays mainly descriptive.

Comments on the revised version:

My main concern was that the author did not use the analysis of mutant contexts such as Snail and Twist to confirm their predictions. They made a series of modifications, clarifying their conclusions. In particular, they now included an analysis of Snail mutant and show that isogonal deformations in the ventro-lateral regions are absent when the external pulling force of the VF is abolished, supporting the idea that isogonal strain could be used as an indicator of external forces (Fig7 and S6).

They further discuss their results in the context of what was published regarding the mutant backgrounds (fog, torso-like, scab, corkscrew, ksr) where midgut invagination is disrupted, and where germ band buckles, and propose that this supports the importance of internal versus external forces driving GBE.<br /> Overall, these modifications, in addition to clarifications in the text, clearly strengthen the manuscript.

Reviewer #1 (Public Review):

Summary and strength:<br /> The authors undertook to assemble and annotate the genome sequence of the Malabar grouper fish, with the aim to provide molecular resources for fundamental and applied research. Even though this is more mainstream, the task is still daunting and labor intensive. Currently, high quality and fully annotated genome sequences are of strategic importance in modern biology. The authors make use of the resource to address the endocrine control of an ecologically and developmentally relevant life cycle transition, metamorphosis. As opposed to amphibian and flat fish where body plan changes, fish metamorphosis is anatomically more subtle and much less known, although it is clear that thyroid hormone (TH) signaling is a key player. The authors thus provide a repertoire of TH-relevant gene expression changes during development and across post-embryonic transitions and correlate developmental stages with changes of gene expression. Overall, this work represents a significant advance in the field.

Fish 'metamorphosis' is well known because it is not as spectacular as amphibians. This work clearly provides technical and theoretical resources to address in a more systematic manner the molecular changes occurring during development and post-embryonic transitions. Heterochrony is a major source of functional and life cycle diversity in fish, which blurs our anatomy-based understanding of fish biology, and has a direct impact on the protocols and rearing procedures used to produce live stocks. This work illustrates how, by using genomics coupled to simple experimental endocrinology, one directly addresses these challenges.

Reviewer #1 (Public Review):

The authors present data on outer membrane vesicle (OMV) production in different mutants, but they state that this is beyond the scope of the current manuscript, which I disagree with. This data could provide valuable physiological context that is otherwise lacking. The preliminary blots suggest that YafK does not alter OMV biogenesis. I recommend repeating these blots with appropriate controls, such as blotting for proteins in the culture media, an IM protein, periplasmic protein and an OM protein to strengthen the reliability of these findings. Including this data in the manuscript, even if it does not directly support the initial hypothesis, would enhance the physiological relevance of the study. Currently, the manuscript relies completely on the experimental setup (labeling-mass spec) previously developed by the authors, which limits the broader scope and interpretability of this study.

Additionally susceptibility of strains to detergents like SDS can be tested to provide a much needed physisological context to the study.

In summary, the authors should consider revising the manuscript to improve clarity, substantiate their claims with more detailed evidence, and include additional experimental results that provide necessary physiological context to their study.

Reviewer #1 (Public Review):

Koren et al. derive and analyse a spiking network model optimised to represent external signals using the minimum number of spikes. Unlike most prior work using a similar setup, the network includes separate populations of excitatory and inhibitory neurons. The authors show that the optimised connectivity has a like-to-like structure, leading to the experimentally observed phenomenon of feature competition. They also characterise the impact of various (hyper)parameters, such as adaptation timescale, ratio of excitatory to inhibitory cells, regularisation strength, and background current. These results add useful biological realism to a particular model of efficient coding. However, not all claims seem fully supported by the evidence. Specifically, several biological features, such as the ratio of excitatory to inhibitory neurons, which the authors claim to explain through efficient coding, might be contingent on arbitrary modelling choices. In addition, earlier work has already established the importance of structured connectivity for feature competition. A clearer presentation of modelling choices, limitations, and prior work could improve the manuscript.

Major comments:

(1) Much is made of the 4:1 ratio between excitatory and inhibitory neurons, which the authors claim to explain through efficient coding. I see two issues with this conclusion: (i) The 4:1 ratio is specific to rodents; humans have an approximate 2:1 ratio (see Fang & Xia et al., Science 2022 and references therein); (ii) the optimal ratio in the model depends on a seemingly arbitrary choice of hyperparameters, particularly the weighting of encoding error versus metabolic cost. This second concern applies to several other results, including the strength of inhibitory versus excitatory synapses. While the model can, therefore, be made consistent with biological data, this requires auxiliary assumptions.

(2) A growing body of evidence supports the importance of structured E-I and I-E connectivity for feature selectivity and response to perturbations. For example, this is a major conclusion from the Oldenburg paper (reference 62 in the manuscript), which includes extensive modelling work. Similar conclusions can be found in work from Znamenskiy and colleagues (experiments and spiking network model; bioRxiv 2018, Neuron 2023 (ref. 82)), Sadeh & Clopath (rate network; eLife, 2020), and Mackwood et al. (rate network with plasticity; eLife, 2021). The current manuscript adds to this evidence by showing that (a particular implementation of) efficient coding in spiking networks leads to structured connectivity. The fact that this structured connectivity then explains perturbation responses is, in the light of earlier findings, not new.

(3) The model's limitations are hard to discern, being relegated to the manuscript's last and rather equivocal paragraph. For instance, the lack of recurrent excitation, crucial in neural dynamics and computation, likely influences the results: neuronal time constants must be as large as the target readout (Figure 4), presumably because the network cannot integrate the signal without recurrent excitation. However, this and other results are not presented in tandem with relevant caveats.

(4) On repeated occasions, results from the model are referred to as predictions claimed to match the data. A prediction is a statement about what will happen in the future - but most of the "predictions" from the model are actually findings that broadly match earlier experimental results, making them "postdictions". This distinction is important: compared to postdictions, predictions are a much stronger test because they are falsifiable. This is especially relevant given (my impression) that key parameters of the model were tweaked to match the data.

Reviewer #1 (Public Review):

Summary:

The question of whether eyespots mimic eyes has certainly been around for a very long time and led to a good deal of debate and contention. This isn't purely an issue of how eyespots work either, but more widely an example of the potential pitfalls of adopting 'just-so-stories' in biology before conducting the appropriate experiments. Recent years have seen a range of studies testing eye mimicry, often purporting to find evidence for or against it, and not always entirely objectively. Thus, the current study is very welcome, rigorously analysing the findings across a suite of papers based on evidence/effect sizes in a meta-analysis.

Strengths:

The work is very well conducted, robust, objective, and makes a range of valuable contributions and conclusions, with an extensive use of literature for the research. I have no issues with the analysis undertaken, just some minor comments on the manuscript. The results and conclusions are compelling. It's probably fair to say that the topic needs more experiments to really reach firm conclusions but the authors do a good job of acknowledging this and highlighting where that future work would be best placed.

Weaknesses:

There are few weaknesses in this work, just some minor amendments to the text for clarity and information.

Reviewer #1 (Public Review):

Summary:

Weiss and co-authors presented a versatile probabilistic tool. aTrack helps in classifying tracking behaviors and understanding important parameters for different types of single particle motion types: Brwonian, Confined, or Directed motion. The tool can be used further to analyze populations of tracks and the number of motion states. This is a stand-alone software package, making it user-friendly for a broad group of researchers.

Strengths:

This manuscript presents a novel method for trajectory analysis.

Weaknesses:

(1) In the results section, is there any reason to choose the specific range of track length for determining the type of motion? The starting value is fine, and would be short enough, but do the authors have anything to report about how much is too long for the model?

(2) Robustness to model mismatches is a very important section that the authors have uplifted diligently. Understanding where and how the model is limited is important. For example, the authors mentioned the limitation of trajectory length, do the authors have any information on the trajectory length range at which this method works accurately? This would be of interest to readers who would like to apply this method to their own data.

(3) aTrack extracts certain parameters from the trajectories to determine the motion types. However, it is not very clear how certain parameters are calculated. For example, is the diffusion coefficient D calculated from fitting, and how is the confinement factor defined and estimated, with equations? This information will help the readers to understand the principles of this algorithm.

(4) The authors mentioned the scenario where a particle may experience several types of motion simultaneously. How do these motions simulated and what do they mean in terms of motion types? Are they mixed motion (a particle switches motion types in the same trajectory) or do they simply present features of several motion types? It is not intuitive to the readers that a particle can be diffusive (Brownian) and direct at the same time.

Reviewer #1 (Public Review):

Summary:

As our understanding of the immune system increases it becomes clear that murine models of immunity cannot always prove an accurate model system for human immunity. However, mechanistic studies in humans are necessarily limited. To bridge this gap many groups have worked on developing humanised mouse models in which human immune cells are introduced into mice allowing their fine manipulation. However, since human immune cells will attack murine tissues, it has proven complex to establish a human-like immune system in mice. To help address this, Vecchione et al have previously developed several models using human cell transfer into mice with or without human thymic fragments that allow negative selection of autoreactive cells. In this report they focus on the examination of the function of the B-helper CD4 T-cell subsets T-follicular helper (Tfh) and T-peripheral helper (Tph) cells. They demonstrate that these cells are able to drive both autoantibody production and can also induce B-cell independent autoimmunity.

Strengths:

A strength of this paper is that currently there is no well-established model for Tfh or Tph in HIS mice and that currently there is no clear murine Tph equivalent making new models for the study of this cell type of value. Equally, since many HIS mice struggle to maintain effective follicular structures Tfh models in HIS mice are not well established giving additional value to this model.

Weaknesses:

A weakness of the paper is that the models seem to lack a clear ability to generate germinal centres. For Tfh it is unclear how we can interpret their function without the structure where they have the greatest influence. In some cases, the definition of Tph does not seem to differentiate well between Tph and highly activated CD4 T-cells in general.

Reviewer #1 (Public Review):

Summary:

Cording et al. investigated how deletion of CNTNAP2, a gene associated with autism spectrum disorder, alters corticostriatal engagement and behavior. Specifically, the authors present slice electrophysiology data showing that striatal projection neurons (SPNs) are more readily driven to fire action potentials in response to stimulation of corticostriatal afferents, and this is due to increases in SPN intrinsic excitability rather than changes in excitatory or inhibitory synaptic inputs. The authors show that CNTNAP2 mice display repetitive behaviors, enhanced motor learning, and cognitive inflexibility. Overall the authors' conclusions are supported by their data, but a few claims could use some more evidence to be convincing.

Strengths:

The use of multiple behavioral techniques, both traditional and cutting-edge machine learning-based analyses, provides a powerful means of assessing repetitive behaviors and behavioral transitions/rigidity. Characterization of both excitatory and inhibitory synaptic responses in slice electrophysiology experiments offers a broad survey of the synaptic alterations that may lead to increased corticostriatal engagement of SPNs.

Weaknesses:

(1) The authors conclude that increased cortical engagement of SPNs is due to changes in SPN intrinsic excitability rather than synaptic strength (either excitatory or inhibitory). One weakness is that only AMPA receptor-mediated responses were measured. Though the holding potential used for experiments in Figure 1F-I wasn't clear, recordings were presumably performed at a hyperpolarized potential that limits NMDA receptor-mediated responses. Because the input-output experiments used to conclude that corticostriatal engagement of SPNs is elevated (Figure 1B-E) were conducted in the current clamp, it is possible that enhanced NMDA receptor engagement contributed to increased SPN responses to cortical stimulation. Confirming that NMDA receptor-mediated EPSC components are not altered would strengthen the main conclusion.

(2) Data clearly show that SPN intrinsic excitability is increased in knockout mice. Given that CNTNAP2 has been linked to potassium channel regulation, it would be helpful to show and quantify additional related electrophysiology data such as negative IV curve responses and action potential hyperpolarization.

(3) As it stands, the reported changes in dorsolateral striatum SPN excitability are only correlative with reported changes in repetitive behaviors, motor learning, and cognitive flexibility.

Reviewer #1 (Public Review):

Summary:

Boldt et al test several possible relationships between trandiagnostically-defined compulsivity and cognitive offloading in a large online sample. To do so, they develop a new and useful cognitive task to jointly estimate biases in confidence and reminder-setting. In doing so, they find that over-confidence is related to less utilization of reminder-setting, which partially mediates the negative relationship between compulsivity and lower reminder-setting. The paper thus establishes that, contrary to the over-use of checking behaviors in patients with OCD, greater levels of transdiagnostically-defined compulsivity predict less deployment of cognitive offloading. The authors offer speculative reasons as to why (perhaps it's perfectionism in less clinically-severe presentations that lowers the cost of expending memory resources), and set an agenda to understand the divergence in cognition between clinical and nonclinical samples. Because only a partial mediation had robust evidence, multiple effects may be at play, whereby compulsivity impacts cognitive offloading via overconfidence and also by other causal pathways.

Strengths:

The study develops an easy-to-implement task to jointly measure confidence and replicates several major findings on confidence and cognitive-offloading. The study uses a useful measure of cognitive offloading - the tendency to set reminders to augment accuracy in the presence of experimentally manipulated costs. Moreover, the utilizes multiple measures of presumed biases - overall tendency to set reminders, the empirically estimated indifference point at which people engage reminders, and a bias measure that compares optimal indifference points to engage reminders relative to the empirically-observed indifference points. That the study observes convergenence along all these measures strengthens the inferences made relating compulsivity to the under-use of reminder-setting. Lastly, the study does find evidence for one of several a priori hypotheses and sets a compelling agenda to try to explain why such a finding diverges from an ostensible opposing finding in clinical OCD samples and the over-use of cognitive offloading.

Weaknesses:

Although I think this design and study are very helpful for the field, I felt that a feature of the design might reduce the tasks's sensitivity to measuring dispositional tendencies to engage cognitive offloading. In particular, the design introduces prediction errors, that could induce learning and interfere with natural tendencies to deploy reminder-setting behavior. These PEs comprise whether a given selected strategy will be or not be allowed to be engaged. We know individuals with compulsivity can learn even when instructed not to learn (e.g., Sharp, Dolan, and Eldar, 2021, Psychological Medicine), and that more generally, they have trouble with structure knowledge (eg Seow et al; Fradkin et al), and thus might be sensitive to these PEs. Thus, a dispositional tendency to set reminders might be differentially impacted for those with compulsivity after an NPE, where they want to set a reminder, but aren't allowed to. After such an NPE, they may avoid more so the tendency to set reminders. Those with compulsivity likely have superstitious beliefs about how checking behaviors leads to a resolution of catastrophes, which might in part originate from inferring structure in the presence of noise or from purely irrelevant sources of information for a given decision problem.

It would be good to know if such learning effects exist if they're modulated by PE (you can imagine PEs are higher if you are more incentivized - e.g., 9 points as opposed to only 3 points - to use reminders, and you are told you cannot use them), and if this learning effect confounds the relationship between compulsivity and reminder-setting.

A more subtle point, I think this study can be more said to be an exploration than a deductive test of a particular model -> hypothesis -> experiment. Typically, when we test a hypothesis, we contrast it with competing models. Here, the tests were two-sided because multiple models, with mutually exclusive predictions (over-use or under-use of reminders) were tested. Moreover, it's unclear exactly how to make sense of what is called the direct mechanism, which is supported by partial (as opposed to complete) mediation.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigate the impact of rare and extreme events on rodents' decision-making under risk, in gain and loss contexts. They describe the behavior of 20 rats performing a four-armed bandit task, where probabilistic gains (sugar pellets) and losses (time-out punishments) can - in some arms - incorporate extremely large - but rare - outcomes. They report that most rats are sensitive to rare and extreme outcomes despite their infrequent occurrence, and that this sensitivity is primarily driven by extreme loss events which they try to avoid, rather than extreme gains that they seek to obtain.

They finally propose a modification of standard reinforcement-learning, which features a specific sensitivity to rare and extreme outcomes and can account for the observed behavior.

Strengths:

The manuscript really taps into a surprisingly neglected but very relevant aspect of decision-making: the effect of rare and extreme events (REE). The authors have developed an experimental setup that seemingly allows investigation of this aspect, which is not trivial given the idiosyncratic properties of rare and extreme events.

The parameters of the experimental setup seem also to be well thought off: basically, in the absence of REE, some options are objectively better than others (because, in expectation, they overall deliver more food, or minimize time-out punishments), but this ordering reverses if REE are taken into account. This allows for a clean test of the integration of REE in the rodent's decision-making model.

The data is presented and analyzed in a very descriptive but exhaustive and transparent way, down to the description of individual rodent's behavior.

Weaknesses:

While the description and analyses of the behavioral patterns are rigorously done under the economic lens of risky decision-making, the authors' interpretation heavily relies on the assumption that rodents have built the correct model of the task during the training. Extensive details are provided about the training procedure, and the observed behavior at the end of the training, but it remains virtually impossible to disambiguate choices due to imperfect learning to choices made due to intrinsic preferences for risk or REE.

By nature, gains (food pellets) and losses (time-out punishments) are somewhat incommensurable so the interpretation of the asymmetry due to outcome valence is also subject to interpretation. There might be some additional subtleties due e.g. satiety that could come from gaining REE (i.e. the delivery of 80 pellets from the Jackpot).

In its current form, the paper is quite hard to digest. This is naturally the case with interdisciplinary work (here mixing economists and neurobiologists). But I am afraid that with the current frame, the paper is going to miss its target, in terms of audience.

The proposed model seems somewhat disconnected from the behavioral patterns: while the model suggests an effect of REE at the decision stage (i.e. with specific decision weights for those rare events), this formalism seems at odds with the observation that REE (notably in the loss domain) has an impact of subsequent behavior - (Black Swans tend to reinforce Total Sensitivity to REE) which rather suggests an effect at the learning stage.

Discussion:

This study convincingly demonstrates that REEs are processed rather uniquely, which makes sense given their evolutionary relevance. REE has indeed been somewhat neglected in previous research, and this study therefore opens an interesting new front on the fundamental aspects of decision under risk. The authors have devised an original theoretical and empirical framework that will be useful for the community, and the combination of economics analysis and rodent behavior constitutes a thought-provoking ground to think about the nature of risk preferences. The interpretation and mechanistic account of these aspects, as well as their generalizability outside the specific context of this study, remain to be strengthened.

Reviewer #1 (Public Review):

Summary:

Lloyd et al employ an evolutionary comparative approach to study how sleep deprivation affects DNA damage repair in Astyanax mexicanus, using the cave vs surface species evolution as a playground. The work shows, convincingly, that the cavefish population has evolved an impaired DNA damage response both following sleep deprivation or a classical paradigm of DNA damage (UV).

Strengths:

The study employs a thorough multidisciplinary approach. The experiments are well conducted and generally well presented.

Weaknesses:

Having a second experimental mean to induce DNA damage would strengthen and generalise the findings.

Overall, the study represents a very important addition to the field. The model employed underlines once more the importance of using an evolutionary approach to study sleep and provides context and caveats to statements that perhaps were taken a bit too much for granted before. At the same time, the paper manages to have an extremely constructive approach, presenting the platform as a clear useful tool to explore the molecular aspects behind sleep and cellular damage in general. The discussion is fair, highlighting the strengths and weaknesses of the work and its implications.

Reviewer #1 (Public Review):

Summary:

In this article, the authors investigated how the brain anticipates sequences of potential sensory events, using temporal predictability to enhance perception. To do so, they combined a tone detection task, electrophysiological recordings, and recurrent neural network models. The stimuli consisted of continuous white noise embedded with either a single tone presented at one of 3 equidistant (500ms) temporal locations, or no tone. The main analyses were carried out on no-tone trials, in which subjects only anticipated future events. First, a modulation power spectrum analysis revealed 4 frequency clusters, and a coupling analysis allowed the authors to group 3 of them together into cluster 234. The time course of the latter aligned with the temporal locations, reaching a local maximum following each of them. The power of cluster 234 during no-tone trials was positively correlated with behavioral performance (d') during tone trials, but not with false alarm rate. Then, the authors trained several continuous-time recurrent neural networks to model the experimental paradigm. After the networks were tuned to reflect the average d' of human subjects, a neural network analogue of EEG was extracted from the activity of neurons. The latter displayed a peak at 2Hz, its time course aligned with the temporal locations, reaching a local maximum both before and after each of them, and its d' score was higher for tones located at one of the temporal locations. A network trained with randomly occurring tones displayed no 2Hz activity and d' independent from tone location. Finally, the authors perturbed the excitatory/inhibitory ratio of neurons within the network, finding that more inhibition resulted in earlier peaks in the neural network activity.

Strengths:

(1) The experimental paradigm introduced in this study is original and well-built, allowing for the study of the targeted phenomenon. The fact that relevant neural signals were found despite the absence of sensory cues proves the setup is promising, opening the way for future works, playing with different parameters: number of tones, time between tones, sequence of temporal locations complexity, sequence of events...etc.

(2) The statistical analysis was exhaustive, the authors consistently introduced controls for different conditions and alternative hypotheses, thoroughly explaining each step of the analysis as well as the choices behind them. The supplementary figures further helped understand the data and answer interrogation one might have. This comprehensive approach was well-appreciated.

(3) The use of more biologically plausible networks, compared to traditional RNNs, to model the response of subjects is a promising approach, which can give clues as to the mechanism at play, but also make predictions that can then be proven (or disproven) by future experiments.

The authors provided a work of good technical quality and reported their methods and findings transparently, making for good reproducibility and evaluation.

Weaknesses:

(1) The most glaring weakness of the paper lies in its interpretation of the different results. Conclusions are scattered around the paper, mostly unclear, and do not always make much sense with regard to the data. For example, the authors never address the absence of a peak before the first temporal location: why would subjects not "suppress" noise before the first temporal location given its (strong) predictability? Moreover, they immediately assume a functional role for the neural signature they found, as well as a direct link between the mechanisms at play in their RNN and the human brain, thus jumping to hasty and unreliable conclusions. The authors seemed to have a strong bias towards a hypothesis (predictive gain control) and tried to fit their data into it.

- The authors cited very few relevant papers on related fields, notably on omission, and therefore did not build efficiently on previous works (e.g., Yabe, Raij, Schröger, Bekinschtein, Chait, Auksztulewicz...). Moreover, at several points in the paper, they make choices about their analysis or model without proper justification or cited sources, even when explicitly pointing to the existence of research supporting said choices.

- Only a single electrode (out of 64) was used (Cz) to carry out every analysis. Without proper justification, this choice could be misinterpreted. Moreover, adopting instead a multivariate approach (incorporating all channels) would give more strength to the paper.

- Overall, the observed electrophysiological results could be more simply explained by a mechanism akin to a go/no-go (a tone/no-tone) or omission response happening after each temporal location, as subjects have learned when to make that inference. The delay of the response with regards to temporal location would change due to error accumulation in time perception, rather than "the anticipation of the first temporal location facilitating the anticipation of the second", which makes little sense. Moreover, a response in Cz could be expected.

- As for the results of RNN, not only is the analogy with actual neurophysiological activity limited, both in principle (simple E/I dynamics) and in implementation (inference is only done at the end of each trial), but the authors do not address the activity before the first temporal location, which is a major difference with human data. Their assumption that both RNN and cluster 234 are functionally related to gain control is thus further flawed. Moreover, the analysis of the RNN is lacking, for example, the authors did not compare false positive/negative of different delays, or analyzed Wout.

- The phrasing and introduction of the paper are misleading, as confusion can arise between predicting a sequence of events (several events in a row) and predicting a single event appearing at different potential locations. It should be clarified that the paper does not address sequences of events at any point.

It seems the authors already drew their conclusion beforehand and fit the data to match this bias. As such, the interpretation of the data is messy, flawed, and often hasty, drawing erroneous conclusions and parallels.

Overall, the manuscript is of good technical quality and communicated results very transparently, but the authors seem to have a strong confirmation bias towards temporal anticipation and gain control, thus leading to flawed interpretations.

Reviewer #1 (Public Review):

Summary:

In this study, Gu et al. employed novel viral strategies, combined with in vivo two-photon imaging, to map the tone response properties of two groups of cortical neurons in A1. The thalamocortical recipient (TR neurons) and the corticothalamic (CT neurons). They observed a clear tonotopic gradient among TR neurons but not in CT neurons. Moreover, CT neurons exhibited high heterogeneity of their frequency tuning and broader bandwidth, suggesting increased synaptic integration in these neurons. By parsing out different projecting-specific neurons within A1, this study provides insight into how neurons with different connectivity can exhibit different frequency response-related topographic organization.

Strengths:

This study reveals the importance of studying neurons with projection specificity rather than layer specificity since neurons within the same layer have very diverse molecular, morphological, physiological, and connectional features. By utilizing a newly developed rabies virus CSN-N2c GCaMP-expressing vector, the authors can label and image specifically the neurons (CT neurons) in A1 that project to the MGB. To compare, they used an anterograde trans-synaptic tracing strategy to label and image neurons in A1 that receive input from MGB (TR neurons).

Weaknesses:

- Perhaps as cited in the introduction, it is well known that tonotopic gradient is well preserved across all layers within A1, but I feel if the authors want to highlight the specificity of their virus tracing strategy and the populations that they imaged in L2/3 (TR neurons) and L6 (CT neurons), they should perform control groups where they image general excitatory neurons in the two depths and compare to TR and CT neurons, respectively. This will show that it's not their imaging/analysis or behavioral paradigms that are different from other labs.  

- Figures 1D and G, the y-axis is Distance from pia (%). I'm not exactly sure what this means. How does % translate to real cortical thickness? 

- For Figure 2G and H, is each circle a neuron or an animal? Why are they staggered on top of each other on the x-axis? If the x-axis is the distance from caudal to rostral, each neuron should have a different distance? Also, it seems like it's because Figure 2H has more circles, which is why it has more variation, thus not significant (for example, at 600 or 900um, 2G seems to have fewer circles than 2H).  

- Similarly, in Figures 2J and L, why are the circles staggered on the y-axis now? And is each circle now a neuron or a trial? It seems they have many more circles than Figure 2G and 2H. Also, I don't think doing a correlation is the proper stats for this type of plot (this point applies to Figures 3H and 3J).

- What does the inter-quartile range of BF (IQRBF, in octaves) imply? What's the interpretation of this analysis? I am confused as to why TR neurons show high IQR in HF areas compared to LF areas, which means homogeneity among TR neurons (lines 213 - 216). On the same note, how is this different from the BF variability?  Isn't higher IQR equal to higher variability?

- Figure 4A-B, there are no clear criteria on how the authors categorize V, I, and O shapes. The descriptions in the Methods (lines 721 - 725) are also very vague.

Reviewer #1 (Public Review):

Summary:

This study resolves a cryo-EM structure of the GPCR, GPR30, which was recently identified as a bicarbonate receptor by the authors' lab. Understanding the ligand and the mechanism of activation is of fundamental importance to the field of receptor signaling. However, the main claim of the paper, the identification of the bicarbonate binding site, is only partly supported by the structural and functional data, leaving the study incomplete.

Strengths:

The overall structure, and proposed mechanism of G-protein coupling seem solid. The authors perform fairly extensive unbiased mutagenesis to identify a host of positions that are important to G-protein signaling. To my knowledge, bicarbonate is the only physiological ligand that has been identified for GPR30, making this study a particularly important contribution to the field.

Weaknesses:

Without higher resolution structures and/or additional experimental assessment of the binding pocket, the assignment of the bicarbonate remains highly speculative. The local resolution is especially poor in the ECL loop region where the ligand is proposed to bind (4.3 - 4 .8 Å range). Of course, sometimes it is difficult to achieve high structural resolution, but in these cases, the assignment of ligands should be backed up by even more rigorous experimental validation.

The functional assay monitors activation of GPR30, and thus reports on not only bicarbonate binding, but also the integrity of the allosteric network that transduces the binding signal across the membrane. Thus, disruption of bicarbonate signaling by mutagenesis of the putative coordinating residues does not necessarily mean that bicarbonate binding has been disrupted. Moreover, the mutagenesis was apparently done prior to structure determination, meaning that residues proposed to directly surround bicarbonate binding, such as E218, were not experimentally validated. Targeted mutagenesis based on the structure would strengthen the story.

Moreover, the proposed bicarbonate binding site is surprising in a chemical sense, as it is located within an acidic pocket. The authors cite several other structural studies to support the surprising observation of anionic bicarbonate surrounded by glutamate residues in an acidic pocket (references 31-34). However, it should be noted that in general, these other structures also possess a metal ion (sodium or calcium) and/or a basic sidechain (arginine or lysine) in the coordination sphere, forming a tight ion pair. Thus, the assigned bicarbonate binding site in GPR30 remains an anomaly in terms of the chemical properties of the proposed binding site.

Reviewer #1 (Public Review):

Summary:

The study "Impact of Maximal Overexpression of a Non-toxic Protein on Yeast Cell Physiology" by Fujita et al. aims to elucidate the physiological impacts of overexpressing non-toxic proteins in yeast cells. By identifying model proteins with minimal cytotoxicity, the authors claim to provide insights into cellular stress responses and metabolic shifts induced by protein overexpression.

Strengths:

The study introduces a neutrality index to quantify cytotoxicity and investigates the effects of protein burden on yeast cell physiology. The study identifies mox-YG (a non-fluorescent fluorescent protein) and Gpm1-CCmut (an inactive glycolytic enzyme) as proteins with the lowest cytotoxicity, capable of being overexpressed to more than 40% of total cellular protein while maintaining yeast growth. Overexpression of mox-YG leads to a state resembling nitrogen starvation probably due to TORC1 inactivation, increased mitochondrial function, and decreased ribosomal abundance, indicating a metabolic shift towards more energy-efficient respiration and defects in nucleolar formation.

Weaknesses:

While the introduction of the neutrality index seems useful to differentiate between cytotoxicity and protein burden, the biological relevance of the effects of overexpression of the model proteins is unclear.