Reviewer #1 (Public Review):

This study shows that activation of α1-adrenergic receptors in hippocampal neurons in culture increases nPo of single L-type calcium channels. This pathway is dissected using a large number of activating agents and blockers to involve PKC, Pyk2 and src. The pathway is further examined using PC12 cells, where it is activated by bradykinin. Finally, a form of LTP which is dependent on L-type calcium channels is augmented in young mice by use of the α1-AR agonist, phenylephrine.

1) My main critique would be that the study, while very well executed and rigorous, is fragmented, consisting of three parts that each feel incomplete: i, hippocampal neuron studies, mainly single channel recordings; ii, biochemical studies mainly in PC12 cells, using a different agonist bradykinin, and iii, the examination of LTP in young mice.

Reviewer #1 (Public Review):

The paper addresses why and how odor discrimination ability achieved after learning occurs in select contexts. The finding is that two related odors trigger near identical Kenyon cell responses when tested in isolation, but trigger different responses to the second odor if these are experienced in sequence within a small temporal window. The authors argue that this template comparison requires some activity downstream of Kenyon cells, that is recruited by MBONs. Overall, the experiments provide very nice physiological evidence for a neural mechanism that underlies a contextual basis for the precision of memory recall.

The experiments were well designed and done. The findings are interesting, but the pitch (e.g. the last paragraph of the discussion and the title of the paper) seems to both ignore the main finding of the paper and overstate the novelty of the idea that memory recall can be flexibly regulated by context. There should be more space dedicated to clearly articulated statements/descriptions of hypotheses and candidate mechanisms to explain the interesting phenomenon described here. For instance, explaining "enhanced template mismatch detection" by potential " real-time and delay line summation" of MBON activity is not super useful for the reader as seems to use one abstraction to explain another. The authors cite Lin et al, 2014 from Miesenbock's lab which shows a key role for GABAergic APL neurons in discrimination. Is there increased activation of APL neurons when similar odourants are being compared and discrimination is required? This seems like a simple physically embodied mechanism that could/ should be examined.

Overall, I think the idea that memories are recalled with high precision (less generalisation) only when increased precision is demanded, is a fact that sure is well appreciated by behavioral biologists even beyond the two papers cited here (Campbell et al., J Neurosci 2013; Xu and Südhof, Science 2013). The new findings fill in a physiological gap in this phenomenology. I think the paper would be greatly improved if the authors highlighted what and focused on the physiological correlate uncovered, and tried to communicate (or test) possible mechanistic origins for this in more physically accessible terms.

Reviewer #1 (Public Review):

This study characterizes the localization of the lone voltage-gated Na channel in Drosophila Para in motor and sensory neurons. Like previous studies, the authors identify an enrichment of Na (and importantly the K+ channel Shal) in axon initial segment-like (AIS-) areas in motor neuron axons, and show that this structure is not apparent in axons of sensory neurons. Upon ablation of wrapping glia in the periphery, the authors find this AIS-like organization of Para is lost. Finally, compelling EM analyses of peripheral nerves suggest an intriguing area devoid of glia around AIS-like structures, and some evidence for myelin-like structures along the distal axon. The author propose several interesting ideas for how these structures might be involved in AP signaling and as evolutionary precursors to conventional myelination and saltatory conductance in vertebrates.

Clearly, the evolution of myelination, and how glia contribute to neuronal firing in systems without classically accepted myelination and saltatory conductance are important questions. Although much of the Para clustering in AIS-like domains and regular densities along motor axons have been described in previous studies, the ultrastructural analyses and dependence on wrapping glia might be important advances in the field. In particular, major strengths of this study are the detailed analysis of AIS-like Para clusters, spanning molecular genetic, confocal and super resolution imaging, and ultrastructural approaches and clear writing. However, these strengths are somewhat tempered by a lack of functional approaches to test the idea of a lacunar structure that promotes ion exchange at putative AIS regions as well as little mechanistic insight into how glia may specifically coordinate the formation of Para clusters in AIS-like regions.

Reviewer #1 (Public Review):

Inhibition of translation has been found as a conserved intervention to extend lifespan across a number of species. In this work, the authors systematically investigate the similarities and differences between pharmacological inhibition of protein synthesis at the initiation or elongation steps on longevity and stress resistance. They find that translation elongation inhibition is beneficial during times when proteostasis collapse is the primary phenotype such as proteasome dysfunction, hsf-1 mutants, and heat shock, but this intervention does not extend the lifespan of wt worms. While translation initiation inhibition extends the lifespan of wt worms and heat shock, but in an HSF-1 dependent manner. This work shows that a simple explanation of just inhibiting total protein synthesis and reduced folding load cannot explain all of the phenotypes seen from protein synthesis inhibition, as initiation and elongation inhibition repress overall translation similarly, but have different effects depending on the experiment tested. Using multiple interventions that target both initiation and elongation lends further support to their findings. These experiments are important for conceptualizing how translation inhibition actually extends lifespan and promotes proteostasis.

Major Comment:

The authors acknowledge that lifespan extension must not necessarily arise just from reducing protein synthesis, as elongation inhibition reduced protein synthesis but did not extend lifespan. Yet for the converse effects from elongation inhibition they seem to suggest that it arises from reducing protein synthesis. For example, regarding how elongation inhibition extends lifespan in an hsf-1 mutant, the authors suggest that "inhibition of elongation lowers the production of newly synthesized proteins and thus reduces the folding load on the proteostasis machinery", even though initiation inhibitors do not extend lifespan in an hsf-1 background (while presumably lowering the production of newly synthesized proteins).

Reviewer #1 (Public Review):

The role of HCO3 (or possibly CO2) in regulating sACs is well established yet its physiological context is less clear. The heart is indeed an excellent choice of organ to study this. Isolated mitochondria offer a tractable model for studying the model, although are not without limitations. The quality of recordings is very high, as judged by the consistency of results (i.e. lack of clustering between biological repeats). My primary concern is about distinguishing the effect of pH and HCO3. A rise in HCO3 will also raise pH unless this had been compensated by CO2. It is unclear, from the legend or results, if the bicarbonate effect is due to HCO3 or pH. Was pH controlled by matching the rise in HCO3 with an appropriate level of CO2? The swings in pH are likely to be very large and, potentially, a confounding factor. Certainly, there will be an effect on the proton motive force. A more informative test would compare the effect of 0 CO2/0HCO3 at a pH set to say 7.2, 2.5% CO2/7.5 mM HCO3, and then 5% CO2/15 mM HCO3, etc. Control experiments would then repeat these observations over a range of pH (at zero CO2/HCO3) and over a range of CO2 (at constant HCO3). Data for zero bicarbonate are not informative, as this will never be a physiological setting (results claim 0-15 mM bicarb to represent physiology). Importantly, there seems to be no significant difference in 2A between 10 v 15 mM bicarb, i.e. the physiological range.

There is also a question on the validity of the model. A rise in respiratory rate will produce more CO2 in the matrix. This may raise matrix HCO3, and stimulate sACs therein, but the authors claim sACs are in the IMS, rather than the matrix. Since HCO3 is impermeable, it is unclear how sACs would detect HCO3 beyond the IMM. CO2 escaping the matrix will enter the continuum of the cytoplasmic space, which has finely controlled pH. Since membranes (including IMM) are highly permeable to CO2, the gradient between matrix and cytoplasm will be small (i.e. you only need a small gradient to drive a big flux, if the permeability is massive). Since CO2 can dissipate over a large volume, it is unlikely to accumulate to any degree. CO2 will be in equilibrium with HCO3 and pH (because there are carbonic anhydrases available). Since the cytoplasm has near-constant pH, [HCO3] must also be close to constancy. It is therefore difficult to imagine how HCO3 could change dramatically to meaningfully affect sACs and hence cAMP. Evidence for major changes in IMS pH in intact cells during swings of respiratory activity would be required to make this point. Indeed, for that reason, it would be more sensible to anchor sACS in the matrix, as there, HCO3 could rise to high levels, as it is impermeable, i.e. could be confined within the mitochondrion. I am therefore not convinced the numbers are favorable to the proposed mechanism to be meaningful physiologically.

Triad dentification exercises 1

Seventh chords identification 1

Crusell Menuetto

Schubert Piano Sonata No. 1 in D.137 in E II Andante

Binary form exercises - audio 1 (Schubert Acht Ecossiasen No 3 (starts at 1:07)

Solfège and scale degree identification 1

Reviewer #1 (Public Review):

Jamge et al. sought to identify the relationships between histone variants and histone modifications in Arabidopsis by systematic genomic profiling of 13 histone variants and 12 histone modifications to define a set of "chromatin states". They find that H2A variants are key factors defining the major chromatin types (euchromatin, facultative heterochromatin, and constitutive heterochromatin) and that loss of the DDM1 chromatin remodeler leads to loss of typical constitutive heterochromatin and replacement of this state with features common to genes in euchromatin and facultative heterochromatin. This study deepens our understanding of how histone variants shape the Arabidopsis epigenome and provides a wealth of data for other researchers to explore.

Strengths:<br /> 1. The manuscript provides convincing evidence supporting the claims that: A) Arabidopsis nucleosomes are homotypic for H2A variants and heterotypic for H3 variants, B) that H3 variants are not associated with specific H2A variants, and C) H2A variants are strongly associated with specific histone post-translational modifications (PTMs) while H3 variants show no such strong associations with specific PTMs. These are important findings that contrast with previous observations in animal systems and suggest differences in plant and animal chromatin dynamics.

2. The authors also performed comprehensive epigenomic profiling of all H2A, H2B, and H3 variants and 12 histone PTMs to produce a Hidden Markov Model-based chromatin state map. These studies revealed that histone H2A variants are as important as histone PTMs in defining the various chromatin states, which is unexpected and of high significance.

3. The authors show that in ddm1 mutants, normally heterochromatic transposable element (TE) genes lose H2A.W and gain H2A.Z, along with the facultative heterochromatin and euchromatin signatures associated with H2A.Z at silent and expressed genes, respectively.

Weaknesses:<br /> 1. Following up on the finding that H2A.Z replaces H2A.W at TE genes in ddm1 mutants, the authors provide in vitro evidence that DDM1 binds to H2A.Z-H2B dimers. These results are taken together to conclude that DDM1 normally removes H2A.Z-H2B dimers from nucleosomes at TE genes and replaces them with H2A.W-H2B dimers. However, the evidence for this model is circumstantial and such a model raises a variety of other questions that are not addressed by the authors. For example: if DDM1 does remove H2A.Z from TE genes, how does H2A.Z normally come to occupy these sites, given that they are highly DNA methylated and that H2A.Z is known to anticorrelate with DNA methylation in plants and animals? Given that H2A.Z does not accumulate in TEs in h2a.w mutants, how would H2A.X and H2A instead become enriched at these sites if DDM1 cannot bind these forms of H2A? Given that there are no apparent regions with common sequence between H2A.Z and H2A.W variants that are not also shared with other H2A classes, how would DDM1 selectively bind to H2A.W-H2B and H2A.Z-H2B dimers to the exclusion of H2A(.X)-H2B dimers?

Reviewer #1 (Public Review):

In this manuscript the authors performed experiments and simulations which showed that substrate evaporation is the main driver of early construction in termites. Additionally, these experiments and simulations were designed taking into account several different works, with independent (and sometimes conflicting) hypotheses, so that the current results shine a light on how substrate evaporation is a sufficient descriptor of most of the results seen previously.

The authors managed through simulations and ingenious experiments to show how curvature is extremely correlated with evaporation, and therefore, how results coming from these 2 environmental factors can most of the time be explained through evaporation alone. The authors have continued to use their expertise of numerical simulations and a previously developed model for termite construction, to highlight and verify their findings. On my first pass of the manuscript I felt the authors were missing an experiment: an array of humidity probes to measure evaporation in the three spatial dimensions and over time. Technologically such an experiment is not out of reach, but the author's alternative (a substrate made with a saline solution and later measuring the salt deposits on the surface) was a very ingenious low tech solution to the problem.

One possible missing experiment (and possibly the explanation of the only inconsistency of their results to previous literature) is to perform similar topographical experiments in high humidity chambers, where no humidity, or very low humidity gradients are present. Previous experiments done by Calovi and collaborators in 2019 showed that termite construction activity (without distinguishing digging from deposition) was focused on high curvature (concave) regions, where here the authors have seen higher depositions on convex structures. Despite the difference of "activity" by Calovi 2019 (clearly acknowledged by the authors), another main difference is that the experiments of the 2019 manuscript were performed in a closed chamber with very high humidity, and smooth transitions between regions of positive and negative curvature. Therefore, it stands to reason that the only missing component of the current article, would have been to perform similar experiments with curvature (positive and negative) but under an environment where gradients are reduced to a minimum.

The results presented here are so far the best attempt on characterizing multiple cues that induce termite construction activity, and that also possibly unifies the different hypothesis presented in the last 8 years into a single factor. More importantly, even if these results come from different species of termites than some of the previous works, they are relatable and seem to be mostly consistent, improving the strength of the author's claims.

Reviewer #1 (Public Review):

This manuscript by Neininger-Castro and colleagues presents a novel automatic image analysis method for assessing sarcomeres, the basic units of myofibrils and validates this tool in a couple of experimental approaches that interfere with sarcomere assembly in iPSC-cardiomyocytes (iPSC-CM).

Automatic quantification of sarcomeres is definitely something that is useful to the field. I am surprised that there is no reference in the manuscript to SarcTrack, published by Toepfer and colleagues in 2019 (PMID 30700234), which has exactly the same purpose. The advantage of the image analysis software presented in the current manuscript appears to me to be that it can cover both mature sarcomeres and nascent sarcomeres in premyofibrils effectively.

When going through the manuscript there were a few issues that should be addressed in a revised version of the manuscript:

1. I am a bit puzzled that they took 1.4 um length as a cutoff length for a mature A-band in their quantifications, since the consensus in the field for thick filament length seems to be 1.6 um?

2. When doing the knockdown for alpha and beta-myosin heavy chain, respectively, why did they not also do a Western blot for the "other" isoform as well (Figure 7)? We know that iPSC-CM express a mixture, so the relatively mild phenotype that they observe in single knockdown experiments may well be due to concomitant upregulation of the expression of the other isoform. In my point of view this should be checked.

3. There seems to be a disconnect between the images for myomesin knockdown shown in Figure 8H and the quantification shown in Figure 8I, which makes me wonder whether the image shown in H middle (MYOM1 (1) KD), where the beta-myosin doublets do not seem to be much affected is really representative?

Reviewer 1 (Public Review):

This is a reasonably good paper and the use of a commonality analysis is a nice contribution to understanding variance partitioning across different covariates. I have some comments that I believe the authors ought to address which mostly relate to clarity and interpretation.

First, from a conceptual point of view, the authors focus exclusively on cognition as a downstream outcome. I would suggest the authors nuance their discussion to provide broader considerations of the utility of their method and on the limits of interpretation of brain-age models more generally. Further, I think that since brain-age models by construction confound relevant biological variation with the accuracy of the regression models used to estimate them, there may be limits to the interpretation of (e.g.) the brain-age gap is as a dimensionless biomarker. This has also been discussed elsewhere (see e.g. https://academic.oup.com/brain/article/143/7/2312/5863667). I would suggest that the authors consider and comment on these issues.

Second, from a methods perspective, there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand how the stacked regression models were constructed. Stacked models can be prone to overfitting when combined with cross-validation. This is because the predictions from the first-level models (i.e. the features that are provided to the second level 'stacked' models) contain information about the training set *and* the test set. If cross-validation is not done very carefully (e.g. using multiple hold-out sets), information leakage can easily occur at the second level. Unfortunately, there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand what was actually done. Please provide more information to enable the reader to better understand the stacked regression models. If the authors are not using an approach that fully preserves training and test separability, they need to do so.

Please also provide an indication of the different regression strengths that were estimated across the different models and cross-validation splits. Also, how stable were the weights across splits?

Please provide more details about the task designs, MRI processing procedures that were employed on this sample in addition to the regression methods, and bias-correction methods used. For example, there are several different parameterisations of the elastic net, please provide equations to describe the method used here so that readers can easily determine how the regularisation parameters should be interpreted.

Reviewer #1 (Public Review):

The study by Korona and colleagues presents a rigorous experimental strategy for generating and maintaining a nearly complete set of monosomic yeast lines, thereby establishing a new standard for studying monosomes. Their careful approach in generating and handling monosome yeast lines, coupled with their use of high-throughput DNA sequencing and RNA sequencing, addresses concerns related to genomic instability and is commendable. However, I would like to express my concerns regarding the second part of the study, particularly the calculation of epistasis and the conclusion that vast positive epistatic effects have been observed. I believe that the conclusion of positive epistasis for fitness might be premature due to potential errors in estimating the expected fitness.

The method used to calculate fitness expectation (1 + sum(di), where di = rDRi - 1) may be inappropriate. By reading Figure 2a, it appears that the authors defined rDR as log(mutant growth rate)/log(wild-type growth rate), but I am unsure about the biological meaning of 1 + sum(di) here. In other words, what does it exactly mean when a negative y-axis value is observed in Figure 2b if it is a relative doubling rate? I would assume that the log transformation should be performed after (rather than before) dividing the mutant growth rate by the wild-type growth rate (i.e., log(mutant growth rate/wild-type growth rate)). I believe the expected growth rate for a monosome should be calculated as exp(sum(log(mutant growth rate i/wild-type growth rate))), which can then be compared with the wild-type (with a value equal to 1). Based on this calculation method, if gene A exhibits a 20% reduction in fitness when halved (A/-) and gene B exhibits a 30% reduction (B/-), the expected fitness of A/- B/- should be 56%. Therefore, it is unclear how exactly the expected fitness without epistasis was calculated and how that would affect the estimation of the sign and quantity of epistasis.

While widespread positive epistasis in yeast has been reported by other studies (e.g., doi: 10.1038/ng.524, but not to the extent reported in this study), the conclusion of the current study might not be sufficiently supported. I recommend that the authors revisit their calculation methods to provide a more convincing conclusion on the presence of positive epistasis for fitness in their dataset. Overall, I appreciate the authors' efforts in this study, but believe that addressing these concerns is essential for strengthening the validity of their findings.

Reviewer #1 (Public Review):

This manuscript is interesting because of the exploration of a novel model organisms utilizing next-generation sequencing approaches, such as single-cell-RNA-seq. Despite the authors' efforts the manuscript lacks a cohesive narrative and suffers from being extremely preliminary in nature. For example, most of the figures are cut and pasted directly from the computational programs with very little formatting or thought to creating new knowledge from the data generated. Essentially the manuscript consists of 2-3 experiments where the authors performed single-cell-RNA-seq on different anatomical locations in the pig and also on a couple of different pig types (The Chenghua and Large White). The authors used standard computational pipelines consisting of Seurat, Monocle, Cell Chat, and others to characterize differences in their data.

There is potential in this manuscript but the authors should improve upon the manuscript by mining the data better and generating a better understanding of anatomical positions of pig skin by evaluating the Hox genes.

Reviewer #1 (Public Review):

Wu et al. provide a powerful cross-species approach to better understand brain cell-type specific responses to mutant tau and aging. Therefore, they use scRNAseq of established Drosophila models that they had previously used for bulk RNAseq (Mangleburg et al., 2020) at 1, 10 and 20 days of age, which thus allows them to study the contribution of pathogenic tau (R406W-mutant) in isolation in an experimentally highly controllable manner. They find a large overlap between tau-induced and aging-induced deregulated genes, however different cell-types were primarily affected, suggesting that expression of tau does not simply induce accelerated aging. When assessing cell number abundance in response to tau expression the authors noted that certain excitatory neurons were preferentially lost. They then examined innate immune pathways downstream of NFkB, which they had already uncovered in their previous bulk studies to be associated with tau expression. Also at the scRNAseq level, they find these pathways to be deregulated after expression of tau. In addition, in control cell types that are lost when tau is expressed, they find an inverse correlation of the expression of these pathways and cellular loss, suggesting they might be predictors of neurodegeneration severity. Finally, they use this finding uncovered in Drosophila and reexamined human Alzheimer's disease snRNAseq datasets, were they also find the NFkB pathway to be deregulated.

This study has several strengths. It demonstrates the power of studying tau-effects in a tractable model and then using the obtained knowledge to pin-point relevant pathways in cross-sectional studies of human tauopathy, which are otherwise not easy to interpret given the overlayed effects of other disease triggers. By examining the single-cell level they uncover cell type specific effects, which would otherwise be hidden. This study also represents a valuable resource. Given that the authors have included multiple time points the dataset provides an opportunity to understand the evolution of cell-type specific tau effects over time. The authors have also included a replication dataset, which confirms the results of the primary analysis of neuronal loss. I also appreciate the efforts to understand the apparent increase in glia cell number after expression of tau. By combining computational and experimental methods the authors reach the well supported conclusion that in fact glial cell numbers remain constant but only appear increased due to the proportional nature of the scRNAseq data and profound loss of some neurons. Overall, it is interesting that the authors nominate the innate immunity and NFkB pathways in tauopathy, based on deregulated genes and also based on vulnerable neurons. Nevertheless, this is a correlative finding and as such does not proof that it is causal.

The authors correctly point out the importance of aging as a risk factor for Alzheimer's disease. However, it is unclear whether their models actually capture age-dependent neurodegeneration. Alternatively, they might represent neurodevelopmental tau toxicity. In Figure 1B it can be seen that all vulnerable cell types are already lost at day 1, most notably a'/b'-KC, a/b-KC and G-KC with a >4-fold decrease. This raises the question whether the lost cells might developmentally have not correctly formed, as suggested by a study that the authors cite (Kosmidis et al., 2010). This distinction is important in order to strengthen the translational value of the study to human tauopathies.

The analysis of tau expression levels relative to its impact across cell types in Figure S8 is interesting, however has caveats. The profound neuronal loss makes the interpretation of the correlation analysis of tau levels vs. neuronal vulnerability difficult - since it might be that the individual surviving a'/b'-KC, a/b-KC and G-KC cells are the ones that expressed little amounts of tau, while those that are missing used to express high tau. In addition, it is unclear from the methods whether the 3' UTR from the transformation vector to generate the models was included in the counting. The majority of reads would be expected to be there.

It would be relevant to know whether the animals were in the same genetic background. I.e. is UAS-TauR406W in the same background of the fly that was crossed to elav-Gal4 to serve as the control. This is not mentioned in the paper and also not in Mangleburg et al., 2020 which the authors refer to. There is a lot of tau-induced DEGs (~1/3 of the detected genes) and it would be relevant to know whether some of them might be due to genetic background.

The finding of the authors that NFkB pathways are higher in cell types that degenerate more is interesting. However, in Figure 4D it is also apparent that multiple cell types that do not degenerate have comparably high expression. Therefore, it is not a sufficient factor to explain why some neurons are vulnerable vs. others are not, but rather predicts amongst the vulnerable neurons how much they will be lost. It would be helpful to make this distinction clear in the text.

Reviewer #1 (Public Review):

The goal of the authors was to understand how the kinase, hpk-1, could regulate and interrogate different aspects of cellular stress resilience. To this end, the authors uncovered that hpk-1 is co-expressed with several transcription factors known to regulate different stress responses and this co-regulation only appears to occur in the nervous system. Taking a deeper dive, they convincingly find that hpk-1 overexpression in either serotonergic of GABAergic neurons can protect animals from heat stress or toxic protein aggregates. Interesting, it appears that hpk1 functions in serotonergic neurons differently from GABAergic neurons in the induction of the heat shock response and autophagy.

Overall, the experiments and results are solid and the conclusions drawn reflect the result. The model suggests that the receiving cell deciphers that either heat shock response or autophagy can be induced in the same cell, but the data suggest otherwise. perhaps the model should be reworked to reflect this point.

Reviewer #1 (Public Review):

Understanding how predators alter the behavior of their prey, a central question in neuroethology, has the potential to provide important insight into the neurobiological basis for behavioral flexibility. In this creative and intriguing work, the authors demonstrate that the predatory nematodes Pacificus pristionchus and P. uniformus can induce long-lasting changes in the behavioral patterns of C. elegans hermaphrodites. Exposure to these predators, probably sensed by the physical damaged caused by a bite, leads C. elegans to spend more time in food-poor environments and to increase their preference for laying eggs in these regions. Interestingly, this behavioral change appears to last for at least 24 hours, indicating that predator exposure induces a longer-term modulation of neural circuit function. The authors convincingly demonstrate that both dopamine and serotonin are required for this behavioral change. They identify specific neurons and receptors important for the effects of dopamine in this process, though whether dopamine signaling is itself modulated by predator exposure remains unclear. Some specific conclusions are not fully supported by the results, including the proposal that the CEM neurons are the key source of dopamine and that injury, rather than chemical cues, triggers the observed behavioral changes. Nevertheless, this paper reports a fascinating and robust behavioral finding, and provides some initial progress toward understanding its underlying neurobiological basis. As such, it will be of interest to those studying neuroethology, behavioral neurogenetics, and the modulation of behavior by monoamines.

Reviewer #1 (Public Review):

The authors have investigated the effect of the toxin mycolactone produced by mycobacterium ulcerans on the endothelium. Mycobacterium ulcerans is involved in Buruli ulcer classified as a neglected disease by WHO. This disease has dramatic consequences on the microcirculation causing important cutaneous lesions. The authors have previously demonstrated that endothelial cells are especially sensitive to mycolactone. The present study brings more insight into the mechanism involved in mycolactone-induced endothelial cells defect and thus in microcirculatory dysfunction. The authors showed that mycolactone directly affected the synthesis of proteoglycans at the level of the golgi with a major consequence on the quality of the glycocalyx and thus on the endothelial function and structure. Importantly, the authors show that blockade of the enzyme involve in this synthesis (galactosyltransferase II) phenocopied the effects of mycolactone. The effect of mycolactone on the endothelium was confirmed in vivo. Finally, the authors showed that exogenous laminin-511 reversed the effects of mycolactone, thus opening an important therapeutic perspective for the treatment of wound healing in patients suffering Buruli ulcer and presenting lesions.

Reviewer #1 (Public Review):

Testosterone modulates a range of adult behaviors, and its signaling contributes to behavioral plasticity. One of the more remarkable examples of this influence can be found in female canaries, who do not normally sing or have elevated levels of testosterone. However, introducing testosterone experimentally causes female canaries to begin singing within days and results in an enlargement of the neural circuitry responsible for song production. This work seeks to characterize the transcriptional responses in a key song brain region, HVC, to testosterone treatment in female canaries. They assay gene expression at a number of time points following testosterone administration and perform analyses characterizing patterns of differential expression using a broad range of approaches. This analysis in particular has a focus on understanding the putative gene regulatory networks that drive the observed testosterone-driven transcriptional responses, with the ultimate aim of understanding how these networks influence neural and behavioral properties.

Strengths

This work is well-focused on a specific question and has a number of excellent qualities. The experimental design of this study is strong, and the fine temporal resolution analysis of testosterone effects on gene expression in female songbirds is a novel and compelling approach to understanding the molecular basis of sex hormone-regulated neural plasticity. The authors have carefully assessed the influence of testosterone on a range of female song features, providing an excellent behavioral reference point for their transcriptional analysis. The gene expression analysis, from differential expression to correlation-based network analysis, appears generally sound and provides a good overview of the effects of testosterone on gene expression in HVC. Combined, the expression, neural, and behavioral data provide a rich resource to better understand the molecular mechanisms underlying testosterone-modulate neural and behavioral plasticity.

Weaknesses

However, I do have several concerns about this work, and these concerns fall into three main areas:

1) At several points, the authors make claims that I believe extend beyond the data presented here. For instance, in the Abstract (line 27), the authors state "the development of adult songs requires restructuring the entire HVC, including most HVC cell types, rather than altering only neuronal subpopulations or cellular components." The gene ontology analyses performed do suggest that there is a progression from cellular transcriptional changes to organ-level changes, however caution should be taken in claiming that "most HVC cell types" exhibit transcriptional changes. In fact, according to Fig. 3D most of the transcriptional changes appear restricted to neurons. As the authors themselves note elsewhere, claims at this resolution are difficult without support from single-cell approaches. I do not suggest that the authors need to perform single-cell RNA-seq for this work, but strong claims like this should be avoided.

2) Similarly the Abstract states that parallel regulation "directly" by androgen and estrogen receptors, as well as the transcription factor SP8, "lead" to the transcriptional and neural changes observed after testosterone treatment of females. However, experiments that demonstrate such a causal role have not been performed. The authors do perform a set of bioinformatic analyses that point in this direction - enrichment of androgen and estrogen receptor binding sites in the promoters of differentially expressed genes, high coexpression of SP8 with other genes, and the enrichment of predicted SP8 binding sites in coexpressed genes. However, further support for direct regulation, at the level that the authors claim, would require some form of transcription factor binding assay, e.g. ChIP-seq or CUT&RUN. I am fully aware that these assays are enormously challenging to perform in this system (and again I don't suggest that these experiments need to be done for this work); however, statements of direct regulation should be tempered. This is especially true for the role of SP8. This does appear to be a compelling target, but without some manipulation of the activity of SP8 (e.g. through knockdowns) and subsequent analysis of gene expression, it is too much to claim that this transcription factor is a regulatory link in the testosterone-driven responses. SP8 does appear to be a highly connected hub gene in correlation network analysis, but this alone does not indicate that it acts as a hub transcription factor in a gene regulatory network.

Along these lines, the in situ hybridizations of ESR2 and SP8 presented in Figure 5 need significant improvement. The signals in the red and green channels, SP8 and ESR2, look suspiciously similar, showing almost identical subcellular colocalization. This signal pattern usually suggests bleed-through during image acquisition, as it's highly unlikely that the mRNA of both genes would show this degree of overlap. I would suggest that control ISHs be run with one probe left out, either SP8 or ESR2, and compare these ISHs with the dual label ISHs to determine if signal intensity and cellular distribution look similar. Furthermore, on lines 354-356 the authors write, "The fact that the two genes were expressed nearby in the same cell may indicate physical interactions between the gene pair and warrant further investigation into the nature of their relationship.". Yet, even if the overlap between ESR2 and SP8 shown in Figure 5 is confirmed, close localization of transcripts does not imply that the protein products physically interact. The STRING bioinformatic analysis is more convincing that there is a putative regulatory interaction between ESR2 and the SP8 locus, and this suggestion of protein-protein interaction is weak and should be omitted. In addition, the authors note that ESR2 has not been detected in the songbird HVC in a previous study. To further demonstrate the expression of ESR2 (and SP8) in HVC, it would be useful to plot their expression from the microarray data across the different testosterone conditions.

3) My final concern lies in the interpretation of these results as generalizable to other sex hormone-modualated behaviors. On lines 452-455, the authors write, "This suggests that the testosterone (or estrogen)-triggered induction of adult behaviors, such as parental behavior and courtship, requires a much more extensive reorganization of the transcriptome and the associated biological functions of the brain areas involved than previously thought.". The experiments and argument likely apply to other neural systems to undergo large seasonal fluctuations in sex hormones and similar morphological changes. However, the authors argue that the large number of transcriptional changes seen here may generalize broadly to sex hormone modulated adult behaviors. I think there are a couple of problems with this argument. First, as described here and in past work, testosterone drives major morphological changes the song system of adult canaries; such dramatic changes are not seen for instance in sex hormone-receptive areas underlying mating behavior in adult mammals. Similarly, the study introduced testosterone into female birds which drives a greater morphological change in HVC relative to similar manipulations in males, which again may account for the large number of differentially expressed genes. I would temper the generality of these results and note how the experimental and biological differences between this system and other sex hormone-responsive systems and behaviors may contribute to the observed transcriptional differences.

Reviewer #1 (Public Review):

The study by Meyer and collaborators is tackling the question of cell type evolution between sea urchins and sea stars. To address this question, they generated single nuclei RNA sequencing libraries originating from early developmental time points of the sea star Patiria miniata. The resulting cell type atlas recapitulated the cell types previously known to exist as indicated by traditional methods in the past and revealed hidden cell type complexity. The authors provide evidence for the existence of previously not described sea star neuronal types and provide a thorough characterization of their molecular signature. Once validating the sea star cell type atlas through means of WMISH they computationally compared the sea star cell types to the sea urchin ones by taking advantage of already available single-cell RNA sequencing data, carried out at equivalent stages of Strongylocentrotus purpuratus development. Using 1-1 orthologs they integrated the sea star and sea urchin datasets and provided evidence for the presence of novel cell types that are not shared between the two animals (at least novel for the specific developmental window analyzed) such as the left coelomic pouch in sea urchin. Moreover, their analysis suggests that sea urchin skeletal cells, a population known to not exist in sea stars, correlate transcriptionally to other mesodermal cell types of the sea star, while sea urchin pigment cells appear to be very similar to sea star immune cells and neurons. Overall, the data of this study demonstrate how single-cell RNA sequencing can be used as a tool to study cell type evolution and provide complete molecular evidence of cell type diversification between the two echinoderm species. Lastly, their P. miniata cell type atlas will be of great importance for the evo-devo field and contribute to a better understanding of the development and evolution of novelties.

Reviewer #1 (Public Review):

This manuscript harnesses recent advances in co-evolution based modeling and computational approaches to provide molecular details about the transport cycles and mechanisms of an entire family of transporters, the sugar porters. The authors evaluate the validity of their approach in a number of ways, including comparison to structurally characterized proteins/states excluded from the training set, comparison to the GLUT5 transport free energy landscape determine through conventional enhanced MD methods in a companion paper, and a global evaluation of RMSDs between models. Based on these structural models, the authors are able to generate a number of interesting insights into the networks of co-evolving contacts that form in different conformational states, and different why certain sugar porters are or are not proton-coupled.

Reviewer #1 (Public Review):

The authors have compiled and analysed a unique dataset of patients with treatment-resistant aggressive behaviours who received deep brain stimulation (DBS) of the posterior hypothalamic region. They used established analysis pipelines to identify local predictors of clinical outcomes and performed normative structural and functional connectivity analyses to derive networks associated with treatment response. Finally, Gouveia et al. perform spatial transcriptomics to determine the molecular substrates subserving the identified circuits. The inclusion of data from multiple centres is a notable strength of this retrospective study, but there are current limitations in the methodology and interpretation of findings that need to be addressed.

1) The validation of findings is heterogeneous and inconsistent across analysis pipelines. While the authors performed non-parametric permutation testing during sweet-spot mapping, structural and functional connectivity were validated using a 'four-fold consistency analysis'. The latter consists of a visual representation of streamlines and peak intensities after randomly dividing data into four groups, the findings were not validated quantitatively. If possible, the authors should apply permutation analysis in alignment with sweet-spot mapping and demonstrate the predictive ability of their identified networks in a LOO or k-fold cross-validation paradigm as carried out by similar studies. Given that the data has been derived from multiple centers, the prediction of left-out cohorts based on models generated by the remaining cohorts could be another means of validation. If validation is not possible, the authors should clearly state the limitations of their approach.

2) In addition to a 'four-fold consistency analysis', functional connectivity was evaluated using LOOCV in a priori identified ROIs. Their network analysis, however, revealed a far more extensive network encompassing cortical, subcortical, and cerebellar structures. To avoid selection bias the authors should incorporate identified structures into their analysis and apply appropriate means of validation.

3) Functional connectivity mapping: how were R-maps generated? The authors mention that patient-specific R-maps were p-thresholded and corrected for multiple comparisons, but it is not clear how group-level maps were generated. How did the authors perform regression on these maps? Were voxels that did not survive thresholding excluded?

4) The authors determined that age was a significant prédictor of the outcome, but it is unclear whether certain age groups presented with distinct etiologies underlying their aggressiveness. For example, aggression in epilepsy may show a better response to DBS as opposed to schizophrenia. How does patient outcome change when stratifying according to etiology? How does model performance change when controlling for etiology? The authors should include the etiology of aggressiveness in Table 1.

5) Stimulation parameters. The authors report average pulse widths of 219 µs and 142µs respectively, which is up to 4-fold higher as compared to DBS settings used conventionally in movement disorders and will significantly alter the volume of activated tissue. Did the authors account for the drastic increases in pulse width during VAT modeling?

6) Imaging transcriptomics. The methods described lack detail: How did the authors account for differences in expression across donors, samples, and regions during preprocessing of the Allen Human Brain Atlas? How was expression data collapsed into regions of interest? Did the authors apply any normalization? Recent publications have introduced reproducible workflows for processing and preparing the AHBA expression data for analysis that is publicly available.

7) 'genes with similar patterns of spatial distribution to the TFCE map were compiled in an extensive list'. It is unclear why authors used TFCE maps for spatial transcriptomics as opposed to the functional connectivity map featured in Figure 5. How was similarity measured between the TFCE map and the AHBA? How were candidate genes identified? Please provide a more comprehensive description of the analysis pipeline.

8) What do the bar plots in Figure 7 (left) represent? P-values? The authors should label the axes to make this clear to the reader.

9) Interprétation of imaging transcriptomics: The authors identify a therapeutic circuit associated with deep brain stimulation of the posterior hypothalamic area, however, it is unclear how to reconcile genes associated with hormones, inflammation, and plasticity in this context. The authors mention and discuss genes implicated in hormonal processing, specifically oxytocin. The results provided in Figure 7, however, do not support this finding and it is unclear how the authors identified genes linked to oxytocin. In addition, the authors identified reductions in the number of microglia and astrocytes, while oligodendrocytes were overexpressed relative to the expected distribution of genes per cell type. These findings were attributed to DBS effects, however, both connectomic and transcriptomic data are acquired from healthy subjects, which suggests a physiological deficit/enrichment in a therapeutic circuit. How do the authors interpret findings given that no electrode implantation and stimulation were performed?

10) Data availability. Code used for data processing should be made openly available or shared as source data along with the Figures that were generated using the code. Sweet-spot, structural, and functional connectivity maps should be shared openly.

Reviewer #1 (Public Review):

The strength of the manuscript is highlighted by the application of fractal formalism, which is commonly used in colloidal systems, in conjunction with MD simulation to study the phase separation of an IDP. The weakness lies in the fact that this study does not provide any discussion on how our understanding of the network structure and dynamical behavior of biomolecular condensates and their biological significance improves through this study. The experimental part remains weak, without any measurements of the dynamics of the condensates. Whether and how the formalism can distinguish between phase-separated condensates (WT) and classical protein aggregates (Y to A variant) remains unclear.

Reviewer #1 (Public Review):

This article describes the application of a computational model, previously published in 2021 in Neuron, to an empirical dataset from monkeys, previously published in 2018 in eLife. The 2021 modeling paper argued that the model can be used to determine whether a particular task depends on the perirhinal cortex as opposed to being soluble using ventral visual stream structures alone. The 2018 empirical paper used a series of visual discrimination tasks in monkeys that were designed to contain high levels of 'feature ambiguity' (in which the stimuli that must be discriminated share a large proportion of overlapping features), and yet animals with rhinal cortex lesions were unimpaired, leading the authors to conclude that perirhinal cortex is not involved in the visual perception of objects. The present article revisits and revises that conclusion: when the 2018 tasks are run through the 2021 computational model, the model suggests that they should not depend on perirhinal cortex function after all, because the model of VVS function achieves the same levels of performance as both controls and PRC-lesioned animals from the 2018 paper. This leads the authors of the present study to conclude that the 2018 data are simply "non-diagnostic" in terms of the involvement of the perirhinal cortex in object perception.

The authors have successfully applied the computational tool from 2021 to empirical data, in exactly the way the tool was designed to be used. To the extent that the model can be accepted as a veridical proxy for primate VVS function, its conclusions can be trusted and this study provides a useful piece of information in the interpretation of often contradictory literature. However, I found the contribution to be rather modest. The results of this computational study pertain to only a single empirical study from the literature on perirhinal function (Eldridge et al, 2018). Thus, it cannot be argued that by reinterpreting this study, the current contribution resolves all controversy or even most of the controversy in the foregoing literature. The Bonnen et al. 2021 paper provided a potentially useful computational tool for evaluating the empirical literature, but using that tool to evaluate (and ultimately rule out as non-diagnostic) a single study does not seem to warrant an entire manuscript: I would expect to see a reevaluation of a much larger sample of data in order to make a significant contribution to the literature, above and beyond the paper already published in 2021. In addition, the manuscript in its current form leaves the motivations for some analyses under-specified and the methods occasionally obscure.

Reviewer #1 (Public Review):

In this manuscript, Yong and colleagues link perturbations in lysosomal lipid metabolism with the generation of protein aggregates resulting from proteosome inhibition. The main tool used is the ProteoStat stain to assess protein aggregate burden in native cells (i.e. cells under no exogenous or endogenous stress). They initially use CRISPR-based genome-wide screens to identify several genes that affect this aggregate burden. Interestingly, knockdown of genes involved in lysosomal acidification was a major signature which led to identification of other culprit lysosome-associated genes that included ones involved in lipid metabolism. Subsequent CRISPR screen focused on lipidomic analysis led to identification of sphingolipid and cholesterol esters as lipid classes with effects on proteostasis. Despite using various tools of lysosomal function, acidity, permeability, etc, the authors couldn't identify the link between lysosomal lipid metabolism and protein aggregate formation. Nevertheless, the interrelationship of these two processes was the overall conclusion of this manuscript.

Although this work is interesting and thought-provoking, their approach to identify novel pathways involved in proteostasis is limited and this weakens the contribution of the paper in its current form.

Reviewer #1 (Public Review):

A typical path from preprocessed data to findings in systems neuroscience often includes a set of analyses that often share common components. For example, an investigator might want to generate plots that relate one time series (e.g., a set of spike times) to another (measurements of a behavioral parameter such as pupil diameter or running speed). In most cases, each individual scientist writes their own code to carry out these analyses, and thus the same basic analysis is coded repeatedly. This is problematic for several reasons, including the waste of time, the potential for errors, and the greater difficulty inherent in sharing highly customized code.

This paper presents Pynapple, a python package that aims to address those problems.

Strengths:

The authors have identified a key need in the community - well-written analysis routines that carry out a core set of functions and can import data from multiple formats. In addition, they recognized that there are some common elements of many analyses, particularly those involving timeseries, and their object-oriented architecture takes advantage of those commonalities to simplify the overall analysis process.

The package is separated into a core set of applications and another with more advanced applications, with the goal of both providing a streamlined base for analyses and allowing for implementations/inclusion of more experimental approaches.

Weaknesses:

There are two main weaknesses of the paper in its present form.

First, the claims relating to the value of the library in everyday use are not demonstrated clearly. There are no comparisons of, for example, the number of lines of code required to carry out a specific analysis with and without Pynapple or Pynacollada. Similarly, the paper does not give the reader a good sense of how analyses are carried out and how the object-oriented architecture provides a simplified user interaction experience. This contrasts with their GitHub page and associated notebooks which do a better job of showing the package in action.

Second, the paper makes several claims about the values of object-oriented programming and the overall design strategy that are not entirely accurate. For example, object-oriented programming does not inherently reduce coding errors, although it can be part of good software engineering. Similarly, there is a claim that the design strategy "ensures stability" when it would be much more accurate to say that these strategies make it easier to maintain the stability of the code. And the authors state that the package has no dependencies, which is not true in the codebase. These and other claims are made without a clear definition of the properties that good scientific analysis software should have (e.g., stability, extensibility, testing infrastructure, etc.).

There is also a minor issue - these packages address an important need for high-level analysis tools but do not provide associated tools for preprocessing (e.g., spike sorting) or for creating reproducible pipelines for these analyses. This is entirely reasonable, in that no one package can be expected to do everything, but a bit deeper account of the process that takes raw data and produces scientific results would be helpful. In addition, some discussion of how this package could be combined with other tools (e.g., DataJoint, Code Ocean) would help provide context for where Pynapple and Pynacollada could fit into a robust and reliable data analysis ecosystem.

Public Review:

This paper presents two new tools for investigating GLP-1 signaling. The genetically encoded sensor GLPLight1 follows the plan for other GPCR-based fluorescent sensors, inserting a circularly permuted GFP into an intracellular loop of the GPCR. The light-uncaged agonist peptide, photo-GLP1, has no detectable agonist activity (as judged by the GLPLight1 sensor) until it is activated by light. However, based on the current characterization, it is unclear how useful either of these tools will be for investigating native GLP-1 signaling.

The GLPLight1 sensor has a strong fluorescent response to GLP-1 with an EC50 of ~10 nM, and its specificity is high, as shown by lack of response to ligands of related class B GPCRs. However, the native GLP1R enables biological responses to concentrations that are ~1000-fold lower than this (as shown, for instance, in a supplemental figure of this paper). This makes it difficult to see how the sensor will be useful for in vivo detection of GLP-1 release, as claimed; although there may be biological situations where the concentration is adequate to stimulate the sensor, this is not established. Data using a GLP-1 secreting cell line suggest that the sensor has bound some of the released GLP-1, but it is difficult to have confidence without seeing an actual fluorescence response to stimulated release.

Alternatively, the sensor might be used for drug screening, but it is unclear that this would be an improvement over existing high-throughput methods using the cAMP response to GLP1R activation (since those are much more sensitive and also allow detection of signaling through different downstream pathways).

The utility of the caged agonist PhotoGLP1 is similarly unclear. The data demonstrate a substantial antagonism of GLP-1 binding by the still-caged compound, and it is therefore unclear whether the kinetics of the response to PhotoGLP1 itself would mimic the normal activation by GLP-1 in the absence of the caged compound. A further concern is that the light-dependence of the agonist effect of PhotoGLP1 was evaluated only with the GLPLight1 sensor and not with GLP1R signaling itself, which is 1000x more sensitive and which would be the presumed target of the tool. In addition, PhotoGLP1 is based upon native GLP-1, which is rapidly truncated and inactivated by the peptidase DPPIV, expressed in most cell types, and expressed at very high levels in the plasma. The utility of PhotoGLP1 is therefore limited to acute (minutes) in vitro experiments.

Reviewer #1 (Public Review):

Mature mammalian olfactory sensory neurons (OSN) express only one of the hundreds of possible odor receptors (ORs) encoded in the genome. The process of selecting this OR in each OSN is the consequence of both deterministic developmental processes involving transcription factors, and more stochastic processes. How this balance is implemented is a major problem in molecular neuroscience, one whose solution has significant systems-level implications for odor coding. In Bashkirova et al the authors substantially revise the canonical view of how this process works. By querying single cell transcriptomes and genetic architecture across OSN development, the authors demonstrate that OSN progenitors express ORs for their zone and for more dortsal zones, and that the degree of heterochromatinization of non-expressed ORs varies as a function of which zone a given OSN resides in. Through additional genetic experiments (including knockouts of transcription factors that seem to be associated with zonal identity, and the clever use of OR transgenes) they synthesize these findings into a model in which progenitors co-express many ORs - both ORs that are appropriate for their zone and ORs that are dorsal to their zone - and that this expression both facilitates heterochromatinzation of non-selected and extra-zonal ORs, and enables singular OR selection. The experiments are careful and the data are novel, and definitely revise our simplistic current view of how this process works; as such this work will have significant impact on the field. As presented the model requires additional experiments to fully flesh it out, and to definitively demonstrate that i.e., precocious expression leads to gene silencing, but with some additional clarifications in the discussion this paper both breaks new ground and sets the stage for future work exploring mechanisms of OSN development and OR selection.

Reviewer #1 (Public Review):

The initial goal of this work was to study how the activity of the C. trachomatis effector Cdu1 impacts on the number and nature of ubiquitinated proteins in infected host cells, and how this is related to a previously described function of Cdu1 in promoting Golgi distribution around the Chlamydia vacuole, known as inclusion.

The authors generated a cdu1-null mutant in C. trachomatis and used proteomics to analyse ubiquitinated proteins in cells infected with Cdu1-producing and Cdu1-deficient chlamydiae, by comparison to mock-infected cells. It was found that among the four proteins specifically ubiquitinated after infection with Cdu1-deficient chlamydiae there were three other C. trachomatis effectors (InaC, IpaM and CTL0480). These three proteins are part of a large family of Chlamydia effectors, known as Incs, that insert in the inclusion membrane.

Based on these observations, the authors then focused in understanding how Cdu1 protects InaC, IpaM and CTL0480 from ubiquitination, and what are the consequences of this protection for the protein levels of these Incs and for their functions during infection. It is shown that Cdu1 can bind InaC, IpaM and CTL0480, and protects these Incs and itself from ubiquitination and proteasomal degradation. This protective function of Cdu1 depends on its acetylation, but not on its deubiquitinating activity, and host cells infected by the cdu1 null mutant show defects that phenocopy those of cells infected by inaC, ipaM or ctl0480 null-mutants.

Finally, it was previously shown that CLT0480 controls/inhibits a pathway of chlamydial egress from host cells involving extrusion of the entire inclusion. The authors show that InaC and IpaM also control/promote extrusion of C. trachomatis inclusion and that the cdu1 null mutant also shows a defect in this process. This leads to the conclusion stated in the title that Cdu1 regulates chlamydial exit from host cells by protecting specific C. trachomatis effectors from degradation.

This is an excellent and impressive work, both from technical and conceptual perspectives, which accomplishes the goal of providing mechanistic insights on the mode of action of Cdu1. Overall, the data provides solid evidence for the proposed model by which the acetylation activity of Cdu1 protects itself and three Incs (InaC, IpaM and CTL0480) from degradation.

I agree that (all together) the data provides a solid support for the idea that the multiple phenotypes displayed by cells infected with the cdu1 null mutant are related to the decreased levels of InaC, IpaM and CTL0480. However, to some extent, these Incs can still be detected in cells infected with the cdu1 null mutant and it cannot be formally excluded that Cdu1 directly promotes assembly of F-actin and Golgi repositioning around the inclusion, MYPT1 recruitment to the inclusion, and extrusion of the inclusion.

Still, I think the major significance of this work comes from the combined use of proteomics and chlamydial genetics to disclose a unique a mechanism by which one effector controls the levels of other effectors. This further emphasizes that for a single bacterium injecting dozens of effectors into host cells, the function of one bacterial effector can control, and be controlled by other effectors.

Reviewer #1 (Public Review):

In the manuscript titled "Vangl2 suppresses NF-κB signaling and ameliorates sepsis by targeting p65 for NDP52-mediated autophagic degradation" by Lu et al, the authors show that Vangl2, a planner cell polarity component, plays a direct role in autophagic degradation of NFkB-p65 by facilitating its ubiquitination via PDLIM2 and subsequent recognition and autophagic targeting via the autophagy adaptor protein NDP52. Conceptually it is a wonderful study with excellent execution of experiments and controls. The concerns with the manuscript are mainly on two counts - First issue is the kinetics of p65 regulation reported here, which does not fit into the kinetics of the mechanism proposed here, i.e., Vangl2-mediated ubiquitination followed by autophagic degradation of p65. The second issue is more technical- an absolute lack of quantitative analyses. The authors rely mostly on visual qualitative interpretation to assess an increase or decrease in associations between partner molecules throughout the study. While the overall mechanism is interesting, the authors should address these concerns as highlighted below:

Major points:

1) Kinetics of p65 regulation by Vangl2: As mentioned above, authors report that LPS stimulation leads to higher IKK and p65 activation in the absence of Vangl2. The mechanism of action authors subsequently work out is that- Vangl2 helps recruit E3 ligase PDLIM to p65, which causes K63 ubiquitination, which is recognised by NDP52 for autophagic targeting. Curiously, peak p65 activation is achieved within 30 minutes of LPS stimulation. The time scale of all other assays is way longer. It is not clear that in WT cells, p65 could be targeted to autophagic degradation in Vangl2 dependent manner within 30 minutes. The HA-Myc-Flag-based overexpression and Co-IP studies do confirm the interactions as proposed. However, they do not prove that this mechanism was responsible for the Vangl2-mediated modulation of p65 activation upon LPS stimulation. Moreover, the Vangl2 KO line also shows increased IKK activation. The authors do not show the cause behind increased IKK activation, which in itself can trigger increased p65 phosphorylation.<br /> 2) The other major concern is regarding the lack of quantitative assessments. For Co-IP experiments, I can understand it is qualitative observation. However, when the authors infer that there is an increase or decrease in the association through co-IP immunoblots, it should also be quantified, especially since the differences are quite marginal and could be easily misinterpreted.<br /> 3) Figure 4E and F: It is evident that inhibiting Autolysosome (CQ or BafA1) or autophagy (3MA) led to the recovery of p65 levels and inducing autophagy by Rapamycin led to faster decay in p65 levels. Did the authors also note/explore the possibility that Vangl2 itself may be degraded via the autophagy pathway? IB of WCL upon CQ/BAF/3MA or upon Rapa treatment does indicate the same. If true, how would that impact the dynamics of p65 activation?<br /> 4) Autophagic targeting of p65 should also be shown through alternate evidence, like microscopy etc., in the LPS-stimulated WT cells.

Limitation: The mechanism behind enhanced activation of IKK in the absence of Vangl2 remains unclear. It is possible there is an autophagy-independent mechanism also involved in this regulation.

Summary: 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):

This manuscript features a key technical advance in single-molecular force spectroscopy. The critical advance is to employ a click chemistry (DBCO-cycloaddition) for making a stable covalent connection between a target biomacromolecule and solid support in place of conventional antigen-antibody binding. This tweak dramatically improves the mechanical stability of the pulling system such that the pulling/relaxation can be repeated up to a thousand times (the previous limit was a few hundred cycles at best). This improvement is broadly applicable to various molecular interactions and other types of single-molecule force spectroscopy allowing for more statistically reliable force measurements. Another strength of this method is that all conjugation steps are chemically orthogonal (except for Spy-catcher conjugation to the termini of a target molecule) such that the probability of side reactions could be reduced.

The reliability of kinetic and thermodynamic parameters obtained from single-molecule force spectroscopy depends on statistics, that is, the number of pulling measurements and their distribution. By extending the number of measurements, this robust method enables fundamental/critical statistical assessment of those parameters. That is, it is an important and interesting lesson from this study that ~200 repeats can yield statistically reasonable parameters.

The authors carried out carefully designed optimization steps and inform readers of the critical aspects of each. The merit, quality, and rigor as a method-oriented manuscript are impressive. Overall, this is an excellent study.

I think Google search page is doing a good job. People can search by voice and photo search. The whole page is very clean and easy to understand. There are no extra buttons on the page, which makes even first-time users understand the usage quickly.

Reviewer #1 (Public Review):

In the manuscript " Cell Rearrangement Generates Pattern Emergence as a Function of Temporal Morphogen Exposure" by Fulton et al., the authors set out to link cell dynamics and single-cell gene expression states, in order to understand the dynamics of cell differentiation. This important challenge is tackled by studying somitogenesis in the zebrafish embryo and combining reverse-engineering gene regulatory networks (GRNs) with cell tracking data. The differentiation of the presomitic cells is evaluated by the differential tbx marker expression through in situ HCR and antibody staining, and live imaging of reporters. Through mathematical modelling taking into consideration the HCR tbx data, live reporter data of the morphogen activity, and the 3D tracking data at different stages, the authors find a candidate model of a gene regulatory network that recapitulates both in vivo and in vitro patterns of the dynamics of cell differentiation. Using this live-modelling approach, the authors move on to question the impact of cell movement on gene expression and conclude that pattern emerges as a function of cell rearrangements tuning the temporal exposure of the cells to the morphogen gradients.

The major strength of the manuscript is the development of a unique method for addressing cell differentiation dynamics by combining static gene expression data with live cell dynamics. Bridging spatiotemporal information is key to understanding tissue and embryo development and this work provides a great basis for it. A potential weakness is how one selects which of the GRNs predicted from the live-modelling is physiologically relevant to the system of interest, since it requires fitting techniques.

The major goal of the paper is mostly achieved. This is evident by the proposed model predicting well the dynamics of differentiation both in vivo and in vitro. To fully support the conclusion that cell rearrangements are necessary for patterning, the addition of functional experiments targeted in this direction might be beneficial.

Overall, this live-modelling approach has the potential of being relevant to various model systems where gene expression and migration are changing simultaneously (e.g. organoids and embryos) and it is thus important to a wide audience including the fields of developmental, stem cell, and quantitative biology.

Reviewer #1 (Public Review):

In this study, authors examine immune signatures from patients that experienced mild, moderate, or severe COVID-19 symptoms and followed them for months to evaluate whether there was a correlation between their immune activation phenotypes, disease severity, and long COVID. Authors observed higher T cell activation/proliferation marker expression in blood samples of patients with severe disease whereas other cell types were more or less unchanged. The authors also examined the cytokine profile of the patient's serum samples to determine the potential drivers of T cell activation phenotypes. Authors then perform T-cell responses to viral peptides to determine the differences in activation phenotypes with disease severity.

The major strengths of the paper appear in the evaluation of the appropriate cohort of human samples and following them over a period of months. Additionally, the authors perform detailed T-cell analysis in an unbiased way to determine any possible activation correlations with disease severity. The authors also perform antigen-specific T-cell analysis via peptide stimulation which adds to the overall findings. However, there are a number of drawbacks that need to be mentioned. Firstly, the phenotypes of T cells prior to the 3-month time-point are not known. Hence, there is no information on baseline or during the early phase of infection. Secondly, the response is largely obtained from blood. How much information about T cells in blood correlate with lung disease is a matter of concern. Analysis of lungs, where actual disease manifestation is ideal, however close to impossible in the human cohort. Alternatively, analysis of local lymph node aspirate or nasal swabs could be useful. Thirdly, the claim that bystander T cell activation plays a role seems loose, specifically the IL-15 in vitro data. Moreover, the analysis of T cells seems very focused on activation/proliferation phenotypes. Alternative T cell phenotypes such as regulatory, IL-10 producing, or FoxP3 expression are not extensively analyzed.

Major points

1) In Figure 1, the CD4 T cell activation phenotypes do not seem consistent across the groups. Why does moderate vs. severe show increases in CXCR3 expression but not mild vs. severe? The same goes for other markers. Performing T cell stimulation with class II peptides specific for CoV-2 and looking at IFN etc. to determine antigen-specific T cells and then gating on these activation/proliferation markers may be a better way to observe differences.

2) One major drawback is the control patients. It would have helped to include a batch of samples from uninfected patients. Or to have the plasma/blood from patients before COVID-19 symptoms. This way there is a baseline for each group that could be compared. It is difficult to draw broad conclusions across the group at 3 months if we do not know their baseline phenotypes.

3) Although the authors focused on activating/proliferating markers to correlate with disease severity, this analysis does not consider alternate T cell phenotypes such as the ones with regulatory or anti-inflammatory phenotypes. Did authors detect differences in T cells with regulatory profiles such as expression of IL-10, FoxP3, etc. in their unsupervised UMAP analysis or otherwise flow experiments?

Reviewer #1 (Public Review):

The authors introduce a computational model that simulates the dendrites of developing neurons in a 2D plane, subject to constraints inspired by known biological mechanisms such as diffusing trophic factors, trafficked resources, and an activity-dependent pruning rule. The resulting arbors are analyzed in terms of their structure, dynamics, and responses to certain manipulations. The authors conclude that 1) their model recapitulates a stereotyped timecourse of neuronal development: outgrowth, overshoot, and pruning 2) Neurons achieve near-optimal wiring lengths, and Such models can be useful to test proposed biological mechanisms- for example, to ask whether a given set of growth rules can explain a given observed phenomenon - as developmental neuroscientists are working to understand the factors that give rise to the intricate structures and functions of the many cell types of our nervous system.

Overall, my reaction to this work is that this is just one instantiation of many models that the author could have built, given their stated goals. Would other models behave similarly? This question is not well explored, and as a result, claims about interpreting these models and using them to make experimental predictions should be taken warily. I give more detailed and specific comments below.

Line 109. After reading the rest of the manuscript, I worry about the conclusion voiced here, which implies that the model will extrapolate well to manipulations of all the model components. How were the values of model parameters selected? The text implies that these were selected to be biologically plausible, but many seem far off. The density of potential synapses, for example, seems very low in the simulations compared to the density of axons/boutons in the cortex; what constitutes a potential synapse? The perfect correlations between synapses in the activity groups is flawed, even for synapses belonging to the same presynaptic cell. The density of postsynaptic cells is also orders of magnitude of, etc. Ideally, every claim made about the model's output should be supported by a parameter sensitivity study. The authors performed few explorations of parameter sensitivity and many of the choices made seem ad hoc.

Many potentially important phenomena seem to be excluded. I realize that no model can be complete, but the choice of which phenomena to include or exclude from this model could bias studies that make use of it and is worth serious discussion. The development of axons is concurrent with dendrite outgrowth, is highly dynamic, and perhaps better understood mechanistically. In this model, the inputs are essentially static. Growing dendrites acquire and lose growth cones that are associated with rapid extension, but these do not seem to be modeled. Postsynaptic firing does not appear to be modeled, which may be critical to activity-dependent plasticity. For example, changes in firing are a potential explanation for the global changes in dendritic pruning that occur following the outgrowth phase.

Line 167. There are many ways to include activity -independent and -dependent components into a model and not every such model shows stability. A key feature seems to be that larger arbors result in reduced growth and/or increased retraction, but this could be achieved in many ways (whether activity dependent or not). It's not clear that this result is due to the combination of activity-dependent and independent components in the model, or conceptually why that should be the case.

Line 183. The explanation of overshoot in terms of the different timescales of synaptic additions versus activity-dependent retractions was not something I had previously encountered and is an interesting proposal. Have these timescales been measured experimentally? To what extent is this a result of fine-tuning of simulation parameters?

Line 203. This result seems at odds with results that show only a very weak bias in the tuning distribution of inputs to strongly tuned cortical neurons (e.g. work by Arthur Konnerth's group). This discrepancy should be discussed.

Line 268. How does the large variability in the size of the simulated arbors relate to the relatively consistent size of arbors of cortical cells of a given cell type? This variability suggests to me that these simulations could be sensitive to small changes in parameters (e.g. to the density or layout of presynapses).

The modeling of dendrites as two-dimensional will likely limit the usefulness of this model. Many phenomena- such as diffusion, random walks, topological properties, etc - fundamentally differ between two and three dimensions.

The description of wiring lengths as 'approximately optimal' in this text is problematic. The plotted data show that the wiring lengths are several deviations away from optimal, and the random model is not a valid instantiation of the 2D non-overlapping constraints the authors imposed. A more appropriate null should be considered.

It's not clear to me what the authors are trying to convey by repeatedly labeling this model as 'mechanistic'. The mechanisms implemented in the model are inspired by biological phenomena, but the implementations have little resemblance to the underlying biophysical mechanisms. Overall my impression is that this is a phenomenological model intended to show under what conditions particular patterns are possible. Line 363, describing another model as computational but not mechanistic, was especially unclear to me in this context.

Reviewer #1 (Public Review):

MCM8 and MCM9 are paralogues of the eukaryotic MCM2-7 proteins. MCM2-7 form a heterohexameric complex to function as a replicative helicase while MCM8-9 form another hexameric helicase complex that may function in homologous recombination-mediated long-tract gene conversion and/or break-induced replication. MCM2-7 complex is loaded during the low Cdk period by ORC, CDC6, and Cdt1, when the origin DNA may intrude into the central channel via the MCM2-MCM5 entry "gate". In the S phase, MCM2-7 complex is activated as CMG helicase with the help of CDC45 and GINS complex. On the other hand, it still remains unclear how MCM8-9 complex is loaded onto DNA and then activated.

In this study, the authors first investigated the cryo-EM structure of chicken MCM8-9 (gMCM8-9) complex. Based on the data obtained, they suggest that the observed gMCM8-9 structure might represent the structure of a loading state with possible DNA entry "gate". The authors further investigated the cryo-EM structure of human MCM8-9 (hMCM8-9) complex in the presence of the activator protein, HROB, and compared the structure with that obtained without HROB1, which the authors published previously. As a result, they suggest that MCM8-9 complex may change the conformation upon HROB binding, leading to helicase activation. Furthermore, based on the structural analyses, they identified some important residues and motifs in MCM8-9 complex, mutations of which actually impaired the MCM8-9 activity in vitro and in vivo.

Overall, the data presented would support the authors' conclusions and would be of wide interest for those working in the fields of DNA replication and repair. One caveat is that most of the structural data are shown only as ribbon model without showing the density map data obtained by cryo-EM, which makes accurate evaluation of the data somewhat difficult.

Reviewer #1 (Public Review):

This manuscript presents a model in which combined action of the transporter-like protein DISP and the sheddases ADAM10/17 promote shedding of a mono-cholesteroylated Sonic Hedgehog (SHH) species following cleavage of palmitate from the dually lipidated precursor ligand. The authors propose that this leads to transfer of the cholesterol-modified SHH to HDL for solubilization. The minimal requirement for SHH release by this mechanism is proposed to be the covalently linked cholesterol modification because DISP could promote transfer of a cholesteroylated mCherry reporter protein to serum HDL. The authors used an in vitro system to demonstrate dependency on DISP/SCUBE2 for release of the cholesterol modified ligand. These results confirm previously published results from other groups (PMC3387659 and PMC3682496). In vivo support for these activities is provided by data from previously published studies from this group. It is unclear whether new in vivo experiments were conducted for this study.

A strength of the work is the use of a bicistronic SHH-Hhat system to consistently generate dually-lipidated ligands to determine the quantity and lipidation status of SHH released into cell culture media.

A critical shortcoming of the study is that the experiments showing SHH secretion/export by western blot of media fractions do not include a SHH(-) control condition. This is an essential control because SHH media blots can be dirty. Without demonstration that the bands being analyzed are specific for SHH(+) conditions, these experiments cannot be appropriately evaluated. Further, it appears that SHH is transiently transfected/expressed for each experimental condition. A stably expressing SHH/HHAT cell line would reduce condition to condition and experiment to experiment variability. Unusual normalization strategies are used for many experiments, and quantification/statistical analyses are missing for several experiments. Due to these shortcomings, the data do not justify the conclusions. The significance of the data provided is overstated because many of the presented experiments confirm/support previously published work. The study provides a modest advance in the understanding of the complex issue of SHH membrane extraction.

Reviewer #1 (Public Review):

Park et al demonstrate that cells on either side of a BM-BM linkage strengthen their adhesion to that matrix using a positive feedback mechanism involving a discoidin domain receptor (DDR-2) and integrin (INA-1 + PAT-3). In response to its extracellular ligand (Collagen IV/EMB-9), DDR-2 is endocytosed and initiates signaling that in turn stabilizes integrin at the membrane. DDR-2 signaling operates via Ras/LET-60. This work's strength lies in its excellent in vivo imaging, especially of endogenously tagged proteins. For example, tagged DDR-2:mNG could be seen relocating from seam cell membranes to endosomes. I also think a second strength of this system is the ability to chart the development of BM-BM linkage over time based on the stages of worm larval development. This allows the authors to show DDR signaling is needed to establish linkage, rather than maintain it. It likely is relevant to many types of cells that use integrin to adhere to BM and left me pondering a number of interesting questions. For example: (1) Does DDR-2 activation require integrin? Perhaps integrin gets the process started and DDR-2 positively reinforces that (conversely is DDR-2 at the top of a linear pathway)? (2) In ddr-2(qy64) mutants, projections seem to form from the central portion of the utse cell. Does this reveal a second function for DDR-2, regulating perhaps the cytoskeleton? And (3) can you use the forward genetic tools available in C. elegans to find new genes connecting DDR-2 and integrin?

I do see two areas where the manuscript could be improved. First, the authors rely on imprecise genetic methods to reach their conclusions (i.e. systemic RNAi, or expression of dominant negative constructs.) I think their conclusion would be stronger if they used tissue specific degradation to block ddr-2 function specifically in the utse or seam cells. Methods to do this are now regularly used in C. elegans and the authors have already developed the necessary tissue-specific promoters. Second, the manuscript is presented in the introduction as a study on formation and function of BM-BM linkage. The authors start the discussion in a similar manner. But their results are about adhesion between cells and BM. In fact they show the BM-BM linkage forms normally in ddr-2 mutants. Thus it seems like what they have really uncovered is an adhesion mechanism that works in parallel to the BM-BM linkage. Since ddr-2 appears to function equally in both utse + seam cells (based on their dominant negative data), there are likely three layers of adhesion (utse-BM, BM-BM, BM-seam) and if any of those break down, you get a partially penetrant rupture phenotype.

These concerns do not undercut the significance of this work, which identifies an interesting mechanism cells use to strengthen adhesion during BM linkage formation. In fact, I am excited to read future papers detailing the connection between DDR-2 and integrin. But before undertaking those experiments the authors should be certain which cells require DDR-2 activity, and that should not be determined based solely on mis expression of a dominant negative.

Reviewer #1 (Public Review):

The present study examined the physiological mechanisms through which impaired TG storage capacity in adipose tissues affects systemic energy homeostasis in mice. To accomplish this, the authors deleted DGAT1 and DGAT2, crucial enzymes for TG synthesis, in an adipocyte-specific manner. The authors found that ADGAT DKO mice substantially lost the adipose tissues and developed hypothermia when fasted; however, surprisingly, ADGAT KO mice were metabolically healthy on a high-fat diet. The authors found that it was accompanied by elevated energy expenditure, enhanced glucose uptake by the BAT, and enhanced browning of white adipose tissues. This unique animal model provided exciting opportunities to identify new mechanisms to maintain systemic energy homeostasis even in a compromised energy storage capacity. Overall, the data are compelling and well support the conclusion of this paper. The manuscript is clearly written.

Reviewer #1 (Public Review):

This study uses single-cell genomics and gene pathway analysis to characterize the transcriptional effects of influenza H1N1 infection on cell types of the lateral hypothalamus and dorsomedial hypothalamus. The authors use droplet-based single-nuclei RNA-seq to profile single-cell gene expression at 3, 7, and 23 days post intranasal infection with H1N1 influenza virus. Through state-of-the-art and rigorous computational methods, the authors find that many hypothalamic cell types, including glia and neurons, are transcriptionally altered by respiratory infection with a non-neurotropic influenza virus, and that these alterations can persist for weeks and potentially affect cell type interactions that disrupt function. Their thorough discussion of the findings raises interesting questions and hypotheses about the functional implications of the molecular changes they observed, including the physiological changes that can persist long after acute viral infection. Given the role of the hypothalamus in homeostasis, this work sheds light on potential mechanisms by which the H1N1 virus can disrupt cell function and organismal homeostasis beyond the cells that it directly infects.

Despite its strengths, there are several points in the manuscript lacking sufficient evidence or clarity, which need to be addressed through revision. For instance, the conclusion that neurons but not non-neurons show persistent changes in gene expression may be alternatively explained by differences in the number of neuron and non-neuronal cells and transcripts. Also, the authors highlight the connection between influenza infection and loss of appetite and sleepiness but do not explore whether the influenza infection affected the cell types in their dataset previously associated with appetite and sleepiness, or whether differences in weight loss among the influenza-infected subjects correspond to any differences in gene expression.

Joint Public Review:

In the current paper, Jones et al. describe a new framework, named coccinella, for real-time high-throughput behavioral analysis aimed at reducing the cost of analyzing behavior. In the setup used here each fly is confined to a small circular arena and able to walk around on an agar bed spiked with nutrients or pharmacological agent. The new framework, built on the researchers' previously developed platform Ethoscope, relies on relatively low-cost Raspberry Pi video cameras to acquire images at ~0.5 Hz and pull out, in real time, the maximal velocity (parameter extraction) during 10 second windows from each video. Thus, the program produces a text file, and not voluminous videos requiring storage facilities for large amounts of video data, a prohibitive step for many behavioral analyses. The maximal velocity time-series is then fed to an algorithm called Highly Comparative Time-Series Classification (HCTSA)(which itself is based on a large number of feature extraction algorithms) developed by other researchers. HCTSA identifies statistically salient features in the time-series which are then passed on to a type of linear classifier algorithm called support vector machines (SVM). In cases where such analyses are sufficient for characterizing the behaviors of interest this system performs as well as other state-of-the-art systems used in behavioral analysis (e.g., DeepLabCut).

In a pharmacobehavior paradigm testing different chemicals, the authors show that coccinella can identify specific compounds as effectively as other more time-consuming and resource-consuming systems.<br /> The new paradigm should be of interest to researchers involved in drug screens, and more generally, in high-throughput analysis focused on gross locomotor defects in fruit flies such as identification of sleep phenotypes. By extracting/saving only the maximal velocity from video clips, the method is fast. However, the rapidity of the platform comes at a cost--loss of information on subtle but important behavioral alterations. When seeking subtle modifications in animal behavior, solutions like DeepLabCut, which are admittedly slower but far superior in terms of the level of details they yield, would be more appropriate.

The manuscript reads well, and it is scientifically solid.

1- The fact that Coccinella runs on Ethoscopes, an open source hardware platform described by the same group, is very useful because the relevant publication describes Ethoscope in detail. However, the current version of the paper does not offer details or alternatives for users that would like to test the framework, but do not have an Ethoscope. Would it be possible to overcome this barrier and have coccinella run with any video data (and, thus, potentially be used to analyze data obtained from other animal models)?

2- Readers who want background on the analytical approaches that the platform relies on following maximal velocity extraction, will have to consult the original publications. In particular, the current manuscript does not provide much information on Highly Comparative Time-Series Classification (HCTSA) or SVM; this may be reasonable because the methods were developed earlier by others. While some readers may find that the lack of details increases the manuscript's readability, others may be left wanting to see more discussion on these not-so-trivial approaches. In addition, it is worth noting that the same authors who published the HCTSA method also described a shorter version named catch22, that runs faster with a similar output. Thus, explaining in more detail how HCTSA operates, considering that it is a relatively new method, will make the method more convincing.

Reviewer #1 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

This study presents an important finding on human m6A methyltransferase complex (including METTL3, METTL14 and WTAP). The evidence supporting the claims of the authors is convincing, although the model and assays need to be further modified. The work will be of interest to biologists working on RNA epigenetics and cancer biology.

In mammals, a large methyltransferase complex (including METTL3, METTL14 and WTAP) deposits m6A across the transcriptome, and METTL3 serves as its catalytic core component. In this manuscript, the authors identified two cleaved forms of METTL3 and described the function of METTL3a (residues 239-580) in breast tumorigenesis. METTL3a mediates the assembly of METTL3-METTL14-WTAP complex, the global m6A deposition and breast cancer progression. Furthermore, the METTL3a-mTOR axis was uncovered to mediate the METTL3 cleavage, providing potential therapeutic target for breast cancer. This study is properly performed and the findings are very interesting; however, some problems with the model and assays need to be modified. It is widely known that METTL3 and METTL14 form a stable heterodimer with the stoichiometric ratio of 1:1 (Wang X et al. Nature 534, 575-578 (2016), Su S et al. Cell Res 32(11), 982-994 (2022), Yan X et al. Cell Res 32(12), 1124-1127 (2022)), the numbers of METTL3 and METTL14 in the model of Fig 7P are not equivalent and need to be modified.

Reviewer #1 (Public Review):

Masson et al. leveraged the natural genetic diversity presented in a large cohort of the Diversity Outbred in Australia (DOz) mice (n=215) to determine skeletal muscle proteins that were associated with insulin sensitivity. The hits were further filtered by pQTL analysis to construct a proteome fingerprint for insulin resistance. These proteins were then searched against Connectivity Map (CMAP) to identify compounds that could modulate insulin sensitivity. In parallel, many of these compounds were screened experimentally alongside other compounds in the Prestwick library to independently validate some of the compound hits. These two analyses were combined to score for compounds that would potentially reverse insulin resistance. Thiostrepton was identified as the top candidate, and its ability to reverse insulin resistance was validated using assays in L6 myotubes. The mechanism of action was also partially investigated. The concept of this work is certainly interesting, and the reviewer appreciates the amount of work the authors put into this study.

(1) What's the rationale of trypsinizing the tissue prior to mitochondrial isolation? This is not standard for subsequent proteomics analysis. This step will inevitably cause protein loss, especially for the post mitochondrial fractions (PMF). Treating samples with 0.01ug/uL trypsin for 37oC 30 min is sufficient to partially digest a substantial portion of the proteome. If samples from different subjects were not of the same weight, then this partial digestion step may introduce artificial variability as variable proportions of proteins from different subjects would be lost during this step. In addition, the mitochondrial protein enrichment in the mito fraction, despite statistically significant, does not look striking (Figure 1E, ~30% mitochondrial proteins in the mito fraction). As a comparison, Williams et al., MCP 2018 seem to have obtained high mitochondrial protein content in the mito fraction without trpsinizing the frozen quadriceps using a similar SWATH-MS-based approach.

(2) The authors mentioned that the proteomics data were Log2 transformed and median-normalized. Would it be possible to provide a bit more details on this? Were the subjects randomized?

(3) In Figure 1D, what were the numbers of mice the authors used for the CV comparisons in each group? Were they of similar age and sex? Were the differences in CV values statistically significant?

(4) The authors stated in lines 155-157 that proteins negatively associated with the Matsuda index were further filtered by presence of their cis-pQTLs. Perhaps more explanations would be needed to justify this filtering criterion? Having a cis-pQTL would mean the protein abundance variation is explained by the variation in its coding gene, this however conceptually would not be relevant to its association with the Matsuda index. With the data that the authors have in hand, would it not be natural to align the Matsuda index QTL with the pQTLs (cis and trans if available), and/or to perform mediation analysis to examine causal relationships with statistical significance?

(5) It seems a bit odd that the first half of the paper focused extensively on the authors' discoveries in the mitochondrial proteome, and how proteins involved in mitochondrial processes (such as complex I) were associated with Matsuda Index, but the final fingerprint list of insulin resistance, which contained 76 proteins, only had 7 mitochondrial proteins. Was this because many mitochondrial proteins were filtered out due to no cis-pQTL presenting?

(6) The authors found that thiostrepton-induced insulin resistance reversal effects were not through insulin signalling. It activated glycolysis but the mechanism of action was not clear. What are the proteins in the fingerprint list that led to identification of thiostrepton on CMAP? Is thiostrepton able to bind or change the expression of these proteins? Since thiostrepton was identified by searching the insulin resistance fingerprint protein list against CMAP, it would be rational to think that it exerts the biological effects by directly or indirectly acting on these protein targets.

Reviewer #1 (Public Review):

The hippocampus is a structure in the cerebral cortex known to be compartmentalised into regions with different functions. Dorsal hippocampus is involved in cognitive functions such as declarative memory and spatial navigation and interconnects chiefly with the neocortex. Ventral hippocampus interconnects with limbic structures such as amygdala and hypothalamus and is involved in affective states and anxiety. What specifies this functional regionalisation during development is not well understood. The present study focuses on the role of transcription factors COUPTFI and COUPTFII, confirming a previously observed dorsal to ventral gradient of expression of COUPTFI in both embryonic and adult mouse hippocampus, and reporting that expression of COUPTFII is strongest in ventral hippocampus. The aim of the authors was then to probe the role of these transcription factors with the use of conditional knockout of one or both factors using RxCre+ mice (sometimes Emx1Cre+ for comparison). As predicted, COUPTFI insufficiency resulted in failure of the CA1 subregion of the dorsal hippocampus to develop properly (with concomitant loss of performance in a spatial memory task) COUPTFII knockdown had even more marked effects upon the ventral hippocampus with ectopic CA1/CA3 domains forming, while a double knockout lead to a drastic reduction in size of the hippocampus with subsequent effects upon the appearance of hippocampal synaptic circuitry and the capacity for adult neurogenesis (a feature of rodent hippocampus). In order to help explain the role of COUPTFI/II a role in regulating expression of two transcription factors LHX2 and LHX5, known to be crucial to hippocampal development, was tested by examining gene and protein expression. Changes in LHX2 and LHX5 was observed and a role for COUPTFI/II in regulating expression of these genes was postulated.

I believe the authors have largely achieved their aims and the results mostly support the conclusions, but, as discussed further below, there are some weaknesses in the data and some areas that could be expanded upon and improved. The methods are mostly appropriate. The use of the transgenic mice and the application of histological methods, especially tyramide amplified immunohistochemistry, is exemplary. However, I'm not sure a wide enough range of tests to explore the phenotype of the transgenic mice was employed to back the conclusions drawn by the authors. The introduction and discussion are nicely written and explain the general concepts and conclusions well. The work makes an important contribution to our understanding of brain development in general and hippocampal development in particular.

Turning to more specific comments, I must first point out that specification of the ventral hippocampus by expression of COUPTFII is not an entirely original finding, as it was suggested for the developing human hippocampus following immunohistochemical experiments illustrating COUPTFII expression to be confined to the ventral hippocampal structures of the medial temporal cortex (doi: 10.1093/cercor/bhx185). Of course, this study, unlike the present study, was restricted to fetal cortex, not adult, and also reported expression of COUP-TFI throughout dorsal and ventral hippocampal structures but without observing any dorsal to ventral gradient, however I feel its contribution to the field has been overlooked by the present study, and should be incorporated into the introduction and/or discussion.

More information about Rx-cre mice would be informative and could help explain the different phenotype observed when EMX1-cre mice were used to conditionally knock down COUPTFI/II expression.

The demonstration of antagonistic gradients of COUP-TFI and -TFII across the hippocampus is more convincing in the immunohistochemical preparations than in the western blots. The qualitative data presented in Fig.1p does not convincingly represent the quantitative data presented in Fig.1q. There seem to be multiple bands for COUP-TFII and I wonder exactly how quantifying this was approached?

Behavioural testing is limited to one test of dorsal hippocampus function. other tests for non-spatial memory, e.g. novel object recognition, or ventral hippocampus function, e.g. step through passive avoidance, might have lead to some interesting discriminations between the various knock down animals (see doi: 10.3389/fnagi.2018.00091).

Abnormalities in the trisynaptic circuit. No studies of actual synapses, either physiological or morphological, were carried out. I wonder to what extent these immunohistochemical studies just further reflect the abnormalities in hippocampal morphology presented earlier in the manuscript without specifically telling us about synaptic circuits? Although the immunohistochemical preparations are beautiful, they are inadequate on their own in telling us much about what sort of synaptic circuitry exists in the transgenic animals.

LHX2/LHX5 interaction. The immunohistochemical study, which shows clear differences in LHX5 and LHX2 protein expression at E14.5 in double knockdown mice is more convincing than the qPCR study at E11.5, which show surprisingly small differences in mRNA expression. Could the authors expand upon whether this is due to stage of development, or differences between mRNA and protein expression? Why hasn't both mRNA and protein expression data at both time points been presented?

Reviewer #1 (Public Review):

In this paper, the interocular/binocular combination of temporal luminance modulations is studied. Binocular combination is of broad interest because it provides a remarkable case study of how the brain combines information from different sources. In addition, the mechanisms of binocular combination are of interest to vision scientists because they provide insight into when/where/how information from two eyes is combined.

This study focuses on how luminance flicker is combined across two eyes, extending previous work that focused mainly on spatial modulations. The results appear to show that temporal modulations are combined in different ways, with additional differences between subcortical and cortical pathways.

1. Main concern: subcortical and cortical pathways are assessed in quite different ways. On the one hand, this is a strength of the study (as it relies on unique ways of interrogating each pathway). However, this is also a problem when the results from two approaches are combined - leading to a sort of attribution problem: Are the differences due to actual differences between the cortical and subcortical binocular combinations, or are they perhaps differences due to different methods. For example, the results suggest that the subcortical binocular combination is nonlinear, but it is not clear where this nonlinearity occurs. If this occurs in the final phase that controls pupillary responses, it has quite different implications.

At the very least, this work should clearly discuss the limitations of using different methods to assess subcortical and cortical pathways.

2. Adding to the previous point, the paper needs to be a better job of justifying not only the specific methods but also other details of the study (e.g., why certain parameters were chosen). To illustrate, a semi-positive example: Only page 7 explains why 2Hz modulation was used, while the methods for 2Hz modulation are described in detail on page 3. No justifications are provided for most of the other experimental choices. The paper should be expanded to better explain this area of research to non-experts. A notable strength of this paper is that it should be of interest to those not working in this particular field, but this goal is not achieved if the paper is written for a specialist audience. In particular, the introduction should be expanded to better explain this area of research, the methods should include justifications for important empirical decisions, and the discussion should make the work more accessible again (in addition to addressing the issues raised in point 1 above). The results also need more context. For example, why EEG data have overtones but pupillometry does not?

Joint Public Review:

Barlow et al performed a viral insertion screen in larval zebrafish for sleep mutants. They identify a mutant named dreammist (dmist) that displayed defects in sleep, namely, decreased sleep both day and night, accompanied by increased activity. They find that dmist encodes a previously uncharacterized single-pass transmembrane protein that shows structural similarity to Fxyd1, a Na+K+-ATPase regulator. They go on to show that genetic manipulations of either FXYD1 or the Na/K pump also reduce sleep. They use pharmacology and sleep deprivation experiments to provide further evidence that the NA/K pump regulates intracellular sodium and rebound sleep.

This study provides additional evidence for the important role of membrane excitability in sleep regulation. The conclusions of this paper are mostly well supported by data, with the following strengths and weaknesses as described below.

Strengths:<br /> Elegant use of CRISPR knockout methods to disrupt multiple genes that help establish the importance of regulating Na+K+-ATPase function in sleep.<br /> Data are mostly clearly presented.<br /> Double mutant analysis of dmist and atp1a3a help establish an epistatic relationship between these proteins.

Weaknesses:<br /> The authors emphasize the role of increased cellular sodium. It will be interesting to also see the consequences of perturbating potassium. The potassium channel shaker has been previously identified as a critical sleep regulator in Drosophila.

Reviewer #1 (Public Review):

The authors used a meta-mask based on previous LC structural studies to delineate the LC on functional scans within two large public datasets (3T CamCAN and 7T HCP).

The rostral part of the LC was characterized by connections to the posterior and anterior cingulate cortices, medial temporal lobe, hippocampus, amygdala and striatum, while the caudal part projected to the parietal cortex, occipital cortex, precentral and postcentral regions, and thalamus. Older ages were associated with less rostral-like connectivity and increased asymmetry. The gradient explained variance above the effects of age, sex and education on some emotional and cognitive measures. In particular, the old-like functional gradient (loss of rostral-like connectivity and more clustered functional organization) was associated with worse performance on emotional memory and emotion regulation tasks but not to executive functioning or self-rated sleep quality.

Participants with higher anxiety and depression also showed less rostral-like connectivity and more asymmetry. Both the aging and the anxiety/depression asymmetry manifested as less rostral-like connectivity in the left LC than the right LC.

A strength of this study is that it is the first to attempt a voxel-based approach to quantifying functional connectivity in the LC. The results finding differences between rostral and caudal LC connectivity patterns are broadly consistent with prior work indicating differences between rostral/caudal LC and should help advance understanding of the LC's connectivity patterns with cortical regions.

A limitation of the study is the challenge of assessing activity not only from the small LC brainstem nucleus but also within it. Given the current spatial limitations of whole-brain functional imaging, the current findings are bolstered by including the 7T 1.6mm isotropic data. Spatial smoothing was applied with a 3mm FWHM isotropic kernel which may have reduced precision.

Another limitation was that the authors made conclusions about clustered functional organization but it was not clear how clustering was quantified.

Reviewer #1 (Public Review):

People can perform a wide variety of different tasks, and a long-standing question in cognitive neuroscience is how the properties of different tasks are represented in the brain. The authors develop an interesting task that mixes two different sources of difficulty, and find that the brain appears to represent this mixture on a continuum, in the prefrontal areas involved in resolving task difficulty. While these results are interesting and in several ways compelling, they overlap with previous findings and rely on novel statistical analyses that may require further validation.

Strengths<br /> 1. The authors present an interesting and novel task for combining the contributions of stimulus-stimulus and stimulus-response conflict. While this mixture has been measured in the multi-source interference task (MSIT), this task provides a more graded mixture between these two sources of difficulty

2. The authors do a good job triangulating regions that encoding conflict similarity, looking for the conjunction across several different measures of conflict encoding

3. The authors quantify several salient alternative hypothesis and systematically distinguish their core results from these alternatives

4. The question that the authors tackle is of central theoretical importance to cognitive control, and they make an interesting an interesting contribution to this question

Concerns<br /> 1. It's not entirely clear what the current task can measure that is not known from the MSIT, such as the additive influence of conflict sources in Fu et al. (2022), Science. More could be done to distinguish the benefits of this task from MSIT.

2. The evidence from this previous work for mixtures between different conflict sources make the framing of 'infinite possible types of conflict' feel like a strawman. The authors cite classic work (e.g., Kornblum et al., 1990) that develops a typology for conflict which is far from infinite, and I think few people would argue that every possible source of difficulty will have to be learned separately. Such an issue is addressed in theories like 'Expected Value of Control', where optimization of control policies can address unique combinations of task demands.

3. Wouldn't a region that represented each conflict source separately still show the same pattern of results? The degree of Stroop vs Simon conflict is perfectly negatively correlated across conditions, so wouldn't a region that *just* tracks Stoop conflict show these RSA patterns? The authors show that overall congruency is not represented in DLPFC (which is surprising), but they don't break it down by whether this is due to Stroop or Simon congruency (I'm not sure their task allows for this).

4. The authors use a novel form of RSA that concatenates patterns across conditions, runs and subjects into a giant RSA matrix, which is then used for linear mixed effects analysis. This appears to be necessary because conflict type and visual orientation are perfectly confounded within the subject (although, if I understand, the conflict type x congruence interaction wouldn't have the same concern about visual confounds, which shouldn't depend on congruence). This is an interesting approach but should be better justified, preferably with simulations validating the sensitivity and specificity of this method and comparing it to more standard methods.

A chief concern is that the same pattern contributes to many entries in the DV, which has been addressed in previous work using row-wise and column-wise random effects (Chen et al., 2017, Neuroimage). It would also be informative to know whether the results hold up to removing within-run similarity, which can bias similarity measures (Walther et al., 2016, Neuroimage).

Another concern is the extent to which across-subject similarity will only capture consistent patterns across people, making this analysis very similar to a traditional univariate analysis (and unlike the traditional use of RSA to capture subject-specific patterns).

5. Finally, the authors should confirm all their results are robust to less liberal methods of multiplicity correction. For univariate analysis, they should report the effects from the standard p < .001 cluster forming threshold for univariate analysis (or TFCE). For multivariate analyses, FDR can be quite liberal. The authors should consider whether their mixed-effects analyses allow for group-level randomization, and consider (relatively powerful) Max-Stat randomization tests (Nichols & Holmes, 2002, Hum Brain Mapp).

Reviewer #1 (Public Review):

Microglia are increasingly recognized as playing an important role in shaping the synaptic circuit and regulating neural dynamics in response to changes in their surrounding environment and in brain states. While numerous studies have suggested that microglia contribute to sleep regulation and are modulated by sleep, there has been little direct evidence that the morphological dynamics of microglia are modulated by the sleep/wake cycle. In this work, Gu et al. applied a recently developed miniature two-photon microscope in conjunction with EEG and EMG recording to monitor microglia surveillance in freely-moving mice over extended period of time. They found that microglia surveillance depends on the brain state in the sleep/wake cycle (wake, non-REM, or REM sleep). Furthermore, they subjected the mouse to acute sleep deprivation, and found that microglia gradually assume an active state in response. Finally, they showed that the state-dependent morphological changes depend on norepinephrine (NE), as chemically ablating noradrenergic inputs from locus coeruleus abolished such changes; this is in agreement with previous publications. The authors also showed that the effect of NE is partially mediated by β2-adrenergic receptors, as shown with β2-adrenergic receptor knock-out mice. Overall, this study is a technical tour de force, and its data add valuable direct evidence to the ongoing investigations of microglial morphological dynamics and its relationship with sleep. However, there are a number of details that need to be clarified, and some conclusions need to be corroborated by more control experiments or more rigorous statistical analysis. Specifically:

1. The number of branch points per microglia shown here (e.g., Fig. 2g) is much lower than the values of branch points in the literature, e.g., Liu T et al., Neurobiol. Stress 15: 100342, 2021 (mouse dmPFC, IHC); Liu YU et al., Nat. Neurosci. 22: 1771-81, 2019 (mouse S1, in vivo 2P imaging). The authors need to discuss the possible source of such discrepancy.<br /> 2. Microglia process end-point speed (Fig. 2h, o): here the authors show that the speed is highest in the wake state and lowest in NREM, which agrees with the measurement on microglia motility during wakefulness vs NREM in a recent publication (Hristovska I et al., Nat. Commun. 13: 6273, 2022). However, Hristovska et al. also reported lower microglia complexity in NREM vs wake state, which seems to be the opposite of the finding in this paper. The authors need to discuss the possible source of such differences.<br /> 3. Fig. 3: the authors used single-plane images to analyze the morphological changes over 3 or 6 hours of SD, which raises the concern that the processes imaged at the baseline may drift out of focus, leading to the dramatic reduction in process lengths, surveillance area, and number of branch points. In fact, a previous study (Bellesi M et al., J. Neurosci. 37(21): 5263-73, 2017) shows that after 8 h SD, the number of microglia process endpoints per cell and the summed process length per cell do not change significantly (although there is a trend to decline). The authors may confirm their findings by either 3D imaging in vivo, or 3D imaging in fixed tissue.<br /> 4. Fig. 4b: the EEG and EMG signals look significantly different from the example given in Fig. 2a. In particular, the EMG signal appears completely flat except for the first segment of wake state; the EEG power spectrum for REM appears dark; and the wake state corresponds to stronger low frequency components (below ~ 4 Hz) compared to NREM, which is the opposite of Fig. 2a. This raises the concern whether the classification of sleep stage is correct here.<br /> 5. Fig. 4 NE dynamics. How long is a single continuous imaging session for NE? When monitoring microglia surveillance, the authors were able to identify wake or NREM states longer than 15 min, and REM states longer than 5 min. Here the authors selected wake/NREM states longer than 1 min and REM states longer than 30 s. What makes such a big difference in the time duration selected for analysis? Also, the definition of F0 is a bit unclear. Is the same F0 used throughout the entire imaging session, or is it defined with a moving window?<br /> 6. Fig. 5b: how does the microglia morphology in LC axon ablation mice compare with wild type mice under the wake state? The text mentioned "more contracted" morphology but didn't give any quantification. Also, the morphology of microglia in the wake state (Fig. 5b) appears very different from that shown in Fig. S3C1 (baseline). What is the reason?<br /> 7. The relationship between NE level and microglia dynamics. Fig. 4C shows that the extracellular NE level is the highest in the wake state and the lowest in REM. Previous studies (Liu YU et al., Nat. Neurosci. 22(11):1771-1781, 2019; Stowell RD et al., Nat. Neurosci. 22(11): 1782-1792, 2019) suggest that high NE tone corresponds to reduced microglia complexity and surveillance. Hence, it would be expected that microglia process length, branch point number, and area/volume are higher in REM than in NREM. However, Fig. 2l-n show the opposite. How should we understand this?

Reviewer #1 (Public Review):

This study demonstrates that vitamin D-bound VDR increased the expression of SIRT1 and that vitamin D-bound VDR interacts with SIRT1 to cause auto-deacetylation on Lys610 and activation of SIRT1 catalytic activity. This is an important finding that is relevant to the actions of VDR on colorectal cancer. The data presented to support the presented conclusion is convincing.

A strength of the study is that it is focused on a narrow group of conclusions.

The major weakness of the study is that the site of SIRT1 regulatory lysine acetylation is defined by mutational analysis rather than by direct biochemical analysis. This issue is partially mitigated by previous reports of K610 acetylation using mass spec (https://www.phosphosite.org/proteinAction.action?id=5946&showAllSites=true). However, Fig. 4E is reassuring because it shows that the apparent acetylation of the K610 mutant SIRT1 appears to be lower than WT SIRT1

A second weakness of the study relates to the use of shRNA-mediated knockdown of VDR for some studies in which a previously reported cell line was employed. The analysis presented would be more compelling if similar data was obtained using more than one shRNA. Similarly, only a single siRNA for SIRT1 is presented in Table 1.

A third weakness of the study is that the conclusion that the VDR interaction with SIRT1 is the cause of auto-deacetylation rather than an associated event mediated by another mechanism would be more strongly supported by mutational analysis of SIRT1 and VDR residues required for the binding interaction. Will VDR increase SIRT1 activity when mutations are introduced to block the interaction? While the finding that catalytically inactive SIRT1 does not interact with VDR is helpful, this does not address the role of the binding surface.

A fourth weakness of the study is that it would be improved by testing the proposed hypothesis through in vitro reconstitution with purified proteins. Does VDR cause auto-deacetylation and activation of Sirt1 in vitro?

Reviewer #1 (Public Review):

In this study, Jiamin Lin et al. investigated the potential positive feedback loop between ZEB2 and ACSL4, which regulates lipid metabolism and breast cancer metastasis. They reported a correlation between high expression of ZEB2 and ACSL4 and poor survival of breast cancer patients, and showed that depletion of ZEB2 or ACSL4 significantly reduced lipid droplets abundance and cell migration in vitro. The authors also claimed that ZEB2 activated ACSL4 expression by directly binding to its promoter, while ACSL4 in turn stabilized ZEB2 by blocking its ubiquitination. While the topic is interesting, there are several major concerns with the study and its conclusions are not convincing.

1. Figure 1A, the clinical relevance or biological significance of drug-resistant luminal breast cancer cell lines with metastatic cancer is questionable. Additionally, the RNA-seq analysis lacked multiple test correction for differential gene expression analysis, and no fold-change cut-off was used, leading to incorrect thresholds and wrongly identified significant signals.

2. Figure 1D-E, the clinical associations between ACSL4 and ZEB2 overexpression and poor patient survival are not justified. The authors used an old web tool, the Kaplan-Meier plotter database, based on microarray data, to perform the analysis. The reviewer repeated the analysis and found that multiple microarray probes for ZEB2 were available, leading to opposite results when different probes were selected. The reviewer also repeated the analysis using more reliable TCGA RNA-seq data and found no correlation between ASCL4 or ZEB2 expression and post-progression survival.

3. Figure 1I relied on IHC to support the negative correlation between ACSL4 and Erα expression, but the small sample size limits the power to establish the relationship and the results are not definitive without further replication or biological investigation. The authors should provide more detailed and comprehensive analysis, including appropriate statistical tests, to ensure the findings are robust and reliable.

4. Figure 3B-C lacks justification of the differences by showing only one field without any internal control for exposure. The reviewer suggests to show additional fields where cells with both efficiently and inefficiently knocked-down are present, to justify the robustness of the results. This can also be achieved by mixing control and knockdown cells.

5. Figure 4A-D, oleate-induced cell migration is a well-documented feature across different cancer types. To make it more relevant to the current study, the authors should examine multiple cell lines with high and low ZEB2/ACSL4 expression to determine the underlying relevance.

6. Figure 4E, it is difficulty to conclude that cancer cells utilize stored lipids during migration to fuel metastasis based on current data. Do you see any evidence of lipid signal decreasing in the leading edge of the scratch wound-healing migration assay? The authors should also compare signals between unmigrated and migrated cells in the transwell assay.

7. Figure 6 warrants a genome-wide ChIP-seq to justify direct regulation of ASCL4 promoter by ZEB2. The reviewer's analysis of publicly available ZEB2 ChIP-seq in multiple cell types detected no ZEB2 binding signaling within {plus minus} 5 kb of ASCL4 promoter.

8. Figure 7 presents a series of self-contradictory results. Figure 7C, why no significant change in ZEB2-MYC expression was observed in the presence of ACSL4 and/or HA-Ubi? In Figure 7 E&G, why robust ACSL4 expression is present in the control group in (E) but not in (G)? Additionally, why there is no degradation in ZEB2 baseline level over time in the shACSL4 group in (E)? These raise severe concerns about the data quality.

9. Figure 7D, the IP result of ACSL4 is not justified as there is no enrichment of ACSL4 in the IP compared to input. With the current data, it is hard to justify that there is any direct interaction. Moreover, based on IF data in Figure 3B-C, ACSL4 is exclusively localized in the cytoplasm, while ZEB2 is exclusively localized in the nucleus. It is hard to believe there is any direct interaction and mutual regulation.

Reviewer #1 (Public Review):

The authors present a study of visuo-motor coupling primarily using wide-field calcium imaging to measure activity across the dorsal visual cortex. They used different mouse lines or systemically injected viral vectors to allow imaging of calcium activity from specific cell-types with a particular focus on a mouse-line that expresses GCaMP in layer 5 IT (intratelencephalic) neurons. They examined the question of how the neural response to predictable visual input, as a consequence of self-motion, differed from responses to unpredictable input. They identify layer 5 IT cells as having a different response pattern to other cell-types/layers in that they show differences in their response to closed-loop (i.e. predictable) vs open-loop (i.e. unpredictable) stimulation whereas other cell-types showed similar activity patterns between these two conditions. They analyze the latencies of responses to visuomotor prediction errors obtained by briefly pausing the display while the mouse is running, causing a negative prediction error, or by presenting an unpredicted visual input causing a positive prediction error. They suggest that neural responses related to these prediction errors originate in V1, however, I would caution against over-interpretation of this finding as judging the latency of slow calcium responses in wide-field signals is very challenging and this result was not statistically compared between areas. Surprisingly, they find that presentation of a visual grating actually decreases the responses of L5 IT cells in V1. They interpret their results within a predictive coding framework that the last author has previously proposed. The response pattern of the L5 IT cells leads them to propose that these cells may act as 'internal representation' neurons that carry a representation of the brain's model of its environment. Though this is rather speculative. They subsequently examine the responses of these cells to anti-psychotic drugs (e.g. clozapine) with the reasoning that a leading theory of schizophrenia is a disturbance of the brain's internal model and/or a failure to correctly predict the sensory consequences of self-movement. They find that anti-psychotic drugs strongly enhance responses of L5 IT cells to locomotion while having little effect on other cell-types. Finally, they suggest that anti-psychotics reduce long-range correlations between (predominantly) L5 cells and reduce the propagation of prediction errors to higher visual areas and suggest this may be a mechanism by which these drugs reduce hallucinations/psychosis.

This is a large study containing a screening of many mouse-lines/expression profiles using wide-field calcium imaging. Wide-field imaging has its caveats, including a broad point-spread function of the signal and susceptibility to hemodynamic artifacts, which can make interpretation of results difficult. The authors acknowledge these problems and directly address the hemodynamic occlusion problem. It was reassuring to see supplementary 2-photon imaging of soma to complement this data-set, even though this is rather briefly described in the paper. Overall the paper's strengths are its identification of a very different response profile in the L5 IT cells compared other layers/cell-types which suggests an important role for these cells in handling integration of self-motion generated sensory predictions with sensory input. The interpretation of the responses to anti-psychotic drugs is more speculative but the result appears robust and provides an interesting basis for further studies of this effect with more specific recording techniques and possibly behavioral measures.

Reviewer #1 (Public Review):

The paper by Dr. Ter-Ovanesyan et. all discussing a very important topic in the field of extracellular vesicles: how to enrich EVs compare to more abundant other circulating particles like lipoproteins, especially VLDL and LDL, which overlap in size and density with EVs and make the purification process challenging. The authors discussed several approaches, including size exclusion chromatography, density-gradient centrifugation, and methods combining charge and size separation. They also proposed the Tri-Mode Chromatography (TMC) method as a good alternative to conventional SEC separation. However, the results provided for the TMC method do not fully support the claim. TEM images provided show the presence of lipoprotein particles at a higher rate than EVs. In addition, proteomics data suggest that lipoproteins and free proteins are still overrating ones associated with EVs.

The importance of this paper is the code available for an automated device for simultaneous fraction collection, which can be very useful for researchers with limited resources since commercial devices are quite expensive.

Reviewer #1 (Public Review):

In this manuscript, the authors use purified human proteins to assess the factors required for the reglucosylation of MHC-I and describe an elegant, mass-spectrometry-based assay to assess reglucosylation. This process is an essential quality-control step for peptide-MHC-I complexes before they are trafficked to the cell surface. Earlier studies have established TAPBPR as a tapasin-like peptide editor of MHC-I outside the peptide loading complex. The ER chaperone UGGT1 has also been shown to interact with MHC-I loaded with a low-affinity peptide, reglucosylating it to allow re-interaction with the peptide loading complex via calreticulin. That TAPBPR facilitates the interaction of UGGT1 with MHC-I was described by Boyle and co-workers in 2017. In that study, a free cysteine on TAPBPR was shown to be essential for the interaction between TAPBPR and UGGT1, although there was no inter-molecular disulfide linkage formed. The data in the current in vitro study suggests that while TAPBPR is an essential facilitator of reglucosylation of the HLA-A*68:02 allele, the free Cys on TAPBPR is not required to bridge the interaction between MHC-I and UGGT1.

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the role of Elg1 in the regulation of telomere length. The main role of the Elg1/RLC complex is to unload the processivity factor PCNA, mainly after completion of synthesis of the Okazaki fragment in the lagging strand. They found that Elg1 physically interacts with the CST (Cdc13-Stn1-Ten1) and propose that Elg1 negatively regulates telomere length by mediating the interaction between Cdc13 and Stn1 in a pathway involving SUMOylation of both PCNA and Cdc13. Accumulation of SUMOylated PCNA upon deletion of ELG1 or overexpression of RAD30 leads to elongated telomeres. On the other hand, the interaction of Elg1 with Sten1 is SIM-dependent and occurs concurrently with telomere replication in late S phase. In contrast Elg1-Cdc13 interaction is mediated by PCNA-SUMO, is independent on the SIM of Elg1 but still dependent on Cdc13 SUMOylation. The authors present a model containing two main messages 1) PCNA-SUMO acts as a positive signal for telomerase activation 2) Elg1 promotes Cdc13/Stn1 interaction at the expense of Cdc13/Est1 interaction thus terminating telomerase action.

The manuscript contains a large amount of data that make a major inroad on a new type of link between telomere replication and regulation of the telomerase. Nevertheless, the detailed choreography of the events as well as the role of PCNA-SUMO remain elusive and the data do not fully explain the role of the Stn1/Elg1 interaction. The data presented do not sufficiently support the claim that SUMO-PCNA is a positive signal for telomerase activation.

Reviewer #1 (Public Review):

This manuscript describes the identification of influential organisms on rice growth and an attempt of validation. The analysis of eDNA on rice pot and mimic field provides rice growth promoting organisms. This approach is novel for plant ecology field. However current results did not fully support whether eDNA analysis-based detection of influencing organism.

The strength of this manuscript is to attempt application of eDNA analysis-based plant growth differentiation. The weakness is too preliminary data and experimental set-up to make any conclusion. The trials of authors experiments are ideal. However, the process of data analysis did not meet certain levels. For example, eDNA analysis of different time points on rice growth stages resulted in two influential organisms for rice growth. Then they cultivate two species and applied rice seedlings. Without understanding of fitness and robustness, how we can know the effect of the two species on rice growth.

The authors did not check the fate of two species after introducing into rice. If this is true, it is difficult to link between the rice gene expression after treatments and the effectiveness of two species. I think the validation experiment in 2019 needs to be re-conducted.

Reviewer #1 (Public Review):

This study investigates the context-specificity of facial expressions in three species of macaques to test predictions for the 'social complexity hypothesis for communicative complexity'. This hypothesis has garnered much attention in recent years. A proper test of this hypothesis requires clear definitions of 'communicative complexity' and 'social complexity'. Importantly, these two facets of a society must not be derived from the same data because otherwise, any link between the two would be trivial. For instance, if social complexity is derived from the types of interactions individuals have, and different types of signals accompany these interactions, we would not learn anything from a correlation between social and communicative complexity, as both stem from the same data.

The authors of the present paper make a big step forward in operationalising communicative complexity. They used the Facial Action Coding System to code a large number of facial expressions in macaques. This system allows decomposing facial expressions into different action units, such as 'upper lid raiser', 'upper lip raiser' etc.; these units are closely linked to activating specific muscles or muscle groups. Based on these data, the authors calculated three measures derived from information theory: entropy, specificity and prediction error. These parts of the analysis will be useful for future studies.

The three species of macaque varied in these three dimensions. In terms of entropy, there were differences with regard to context (and if there are these context-specific differences, then why pool the data?). Barbary and Tonkean macaques showed lower specificity than rhesus macaques. Regarding predicting context from the facial signals, a random forest classifier yielded the highest prediction values for rhesus monkeys. These results align with an earlier study by Preuschoft and van Schaik (2000), who found that less despotic species have greater variability in facial expressions and usage.

Crucially, the three species under study are also known to vary in terms of their social tolerance. According to the highly influential framework proposed by Bernard Thierry, the members of the genus Macaca fall along a graded continuum from despotic (grade 1) to highly tolerant (grade 4). The three species chosen for the present study represent grade 1 (rhesus monkeys), grade 3 (Barbary macaques), and grade 4 (Tonkean macaques).

The authors of the present paper define social complexity as equivalent to social tolerance - but how is social tolerance defined? Thierry used aggression and conflict resolution patterns to classify the different macaque species, with the steepness of the rank hierarchy and the degree of nepotism (kin bias) being essential. However, aggression and conflict resolution are accompanied by facial gestures. Thus, the authors are looking at two sides of the same coin when investigating the link between social complexity (as defined by the authors) and communicative complexity. Therefore, I am not convinced that this study makes a significant advance in testing the social complexity for communicative complexity hypothesis. A further weakness is that - despite the careful analysis - only three species were considered; thus, the effective sample size is very small.

Reviewer #1 (Public Review):

This study aims to address the mechanism of eccDNA generation during spermatogenesis in mice. Previous efforts for cataloging eccDNA in mammalian germ cells have provided inconclusive results, particularly in the correlation between meiotic recombination and the generation of eccDNA. The authors employed an established approach (Circle-seq) to enrich and amplify eccDNA for sequencing analyses and reported that sperm eccDNA is not associated with miotic recombination hotspots. Rather, the authors reported that eccDNAs are widespread, and oligonucleosomal DNA fragments from sperm undergoing apoptosis, with the ligation of DNA ends by microhomology-mediated end-joining, would be a major source of eccDNA.

The strength of the study includes evaluating the eccDNA contents not only in sperm but also from earlier stages of cells in spermatogenesis. The differences in eccDNA size peaks between sperm and other progenitors, in particular, the unique peak in sperm around 360 bp, are intriguing. Results from sequencing data analysis were presented elegantly.

I also have critiques. First, the lack of eccDNA quality control step is a concern. Previous studies employed electron microscopy to ensure that DNA species are mostly circular before rolling-circle amplification. Phi29 polymerase is widely used for DNA amplification, including whole genome amplification of linear chromosomal DNA. Phi29 polymerase has a high processivity and strand displacement activity. When those activities occur within a molecule, it creates circular DNA from linear DNA in vitro. In vitro-created eccDNA from linear DNA would be randomly distributed in the genome, which may explain the low incidence of common eccDNA between replicates. Therefore, it will be crucial to show that DNA prior to amplification is dominantly circular. Electron microscopy would be challenging for the study because the relatively small number of cells were processed to enrich eccDNA. An alternative method for quality controls includes spiking samples with linear and circular exogenous DNA and measuring the ratios of circular/linear control DNA before and after column purification/exonuclease digestion. eccDNA isolation procedures can be validated by a very high circular/linear control DNA ratio.

Another critique is regarding the limitation of the study. It is important to remind the readers of the limitations of the study. As the authors mentioned, rolling circle amplification preferentially increases the copy numbers of smaller eccDNA. Therefore, the native composition of eccDNA is skewed. In addition, the candidate eccDNAs are identified by split reads or discordant read pairs. The details of the mapping process are unclear from the methods, but such a method would require reads with high mapping quality; the identification of eccDNA is expected to require sequencing reads that are mapped to genomic locations uniquely with high confidence, and reads mapped to more than one genomic location, such as highly similar repeat sequences or duplications, are eliminated. Such identification criteria would favor eccDNA formed by little or no homology at the junction sequences, and eliminate eccDNA formed by long homologies at the ends, such as eccDNA formed exclusively by satellite DNA. Therefore, it is not surprising that the authors found the dominance of microhomology-mediated eccDNA. It remains to be determined whether small eccDNA with microhomologies are the dominant species of eccDNA in the native composition. In this regard, it is noted that similar procedures of eccDNA enrichment (column purification, exonuclease digestion, and rolling circle amplification ) revealed variable sizes and characteristics of eccDNA in sperm (human from Henriksen et al. or mice from this study), dependent on the methods of sequencing (long-read or short-read sequencing). Considering these limitations, the last sentence of the introduction, "We conclude that germline eccDNAs are formed largely by microhomology mediated ligation of nucleosome protected fragments, and barely contribute to de novo genomic deletions at meiotic recombination hotspots" needs to be revised.

Small eccDNA (microDNA) data from various mouse tissues are available from the study by Dillion et al., (Cell Reports 2015). Authors are encouraged to examine whether the notable findings in this study (oligonucleosomal-sized eccDNA peaks and the association with apoptotic cell death) are unique to sperm or common in the eccDNA from other tissues.

Reviewer #1 (Public Review):

In their manuscript "Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes," Farrell and colleagues employ carefully chosen approaches to assay mitotic timeliness in the absence of centrosomes in mammalian culture cells, namely the mechanism behind it and its function. The authors acknowledge prior work well and present their data succinctly, clearly, and with a clear logical flow of experiments. The experiments are thoughtfully designed and presented, with appropriate controls in place.

The authors' model whereby centrosome separation and its early definition of poles mediates timely mitosis without relying on a SAC-dependent delay is compelling, and the authors' data are consistent with it. They show using two different MPS1 inhibitors that acentrosomal cell division fails, supporting their claims that a SAC-dependent delay is required in these instances. Furthermore, they show that reintroducing a time delay is sufficient to restore cell division, but inhibiting a different aspect of SAC function does not restore cell division. Next, the authors rule out polyploidy as a potential confounding factor for requiring a SAC-dependent delay, and instead demonstrate that inhibiting centrosome separation by Eg5 inhibition is sufficient to require this delay for mitotic progression. The authors' findings overall support their proposed model.

Probing what aspects of centrosomes protect against a requirement for SAC-dependent delays would strengthen the work and specifically the conclusion that centrosomes provide "two-ness". For example, the authors could examine division in a population of cells with only one centrosome. Seeing some restoration of mitotic progression in the absence of SAC-dependent delays would suggest that even one centrosome with uninhibited Eg5 is sufficient to negate SAC-dependent delays, and would limit models for what exactly centrosomes contribute. This would help disentangle the roles of actual centrosomes and their biochemical cues, Eg5-driven centrosome separation, and early definition of poles on mitotic progression in the absence of SAC-dependent delays. Making a high fraction of cells with one centrosome could be achieved by using centrinone for a shorter time.

Reviewer #1 (Public Review):

Based on a recent report of spontaneous and reversible remapping of spatial representations in the enthorhinal cortex (Low et al 2021), this study sets out to examine possible mechanisms by which a network can simultaneously represent a positional variable and an uncorrelated binary internal state. To this end, the authors analyse the geometry of activity in recurrent neural networks trained to simultaneously encode an estimate of position in a one-dimensional track and a transiently-cued binary variable. They find that network activity is organised along two separate ring manifolds. The key result is that these two manifolds are significantly more aligned than expected by chance, as previously found in neural recordings. Importantly, the authors show that this is not a direct consequence of the design of the model, and clarify scenarios by which weaker alignment could be achieved. The model is then extended to a two-dimensional track, and to more than two internal variables. The latter case is compared with experimental data that had not been previously analysed.

Strengths:<br /> - rigorous and careful analysis of activity in trained recurrent neural networks<br /> - particular care is taken to show that the obtained results are not a necessary consequence of the design of the model<br /> - the writing is very clear and pleasant to read<br /> - close comparison with experimental data<br /> - extensions beyond the situations studied in experiments (two-dimensional track, more than two internal states)

Weaknesses:<br /> - no major weaknesses<br /> - (minor) the comparison with previous models of remapping could be expanded

Altogether the conclusions claimed by the authors seem to be strongly supported and convincing.

Reviewer #1 (Public Review):

Meta-cognition, and difficulty judgments specifically, is an important part of daily decision-making. When facing two competing tasks, individuals often need to make quick judgments on which task they should approach (whether their goal is to complete an easy or a difficult task).

In the study, subjects face two perceptual tasks on the same screen. Each task is a cloud of dots with a dominating color (yellow or blue), with a varying degree of domination - so each cloud (as a representation of a task where the subject has to judge which color is dominant) can be seen an easy or a difficult task. Observing both, the subject has to decide which one is easier.

It is well-known that choices and response times in each separate task can be described by a drift-diffusion model, where the decision maker accumulates evidence toward one of the decisions ("blue" or "yellow") over time, making a choice when the accumulated evidence reaches a predetermined bound. However, we do not know what happens when an individual has to make two such judgments at the same time, without actually making a choice, but simply deciding which task would have stronger evidence toward one of the options (so would be easier to solve).

It is clear that the degree of color dominance ("color strength" in the study's terms) of both clouds should affect the decision on which task is easier, as well as the total decision time. Experiment 1 clearly shows that color strength has a simple cumulative effect on choice: cloud 1 is more likely to be chosen if it is easier and cloud 2 is harder. Response times, however, show a more complex interactive pattern: when cloud 2 is hard, easier cloud 1 produces faster decisions. When cloud 2 is easy, easier cloud 1 produces slower decisions.

The study explores several models that explain this effect. The best-fitting model (the Difference model is the paper's terminology) assumes that the decision-maker accumulates evidence in both clouds simultaneously and makes a difficulty judgment as soon as the difference between the values of these decision variables reaches a certain threshold. Another potential model that provides a slightly worse fit to the data is a two-step model. First, the decision maker evaluates the dominant color of each cloud, then judges the difficulty based on this information.

Importantly, the study explores an optimal model based on the Markov decision processes approach. This model shows a very similar qualitative pattern in RT predictions but is too complex to fit to the real data. It is hard to judge from the results of the study how the models identified above are specifically related to the optimal model - possibly, the fact that simple approaches such as the Difference model fit the data best could suggest the existence of some cognitive constraints that play a role in difficulty judgments.

The Difference model produces a well-defined qualitative prediction: if the dominant color of both clouds is known to the decision maker, the overall RT effect (hard-hard trials are slower than easy-easy trials) should disappear. Essentially, that turns the model into the second stage of the two-stage model, where the decision maker learns the dominant colors first. The data from Experiment 2 impressively confirms that prediction and provides a good demonstration of how the model can explain the data out-of-sample with a predicted change in context.

Overall, the study provides a very coherent and clean set of predictions and analyses that advance our understanding of meta-cognition. The field would benefit from further exploration of differences between the models presented and new competing predictions (for instance, exploring how the sequential presentation of stimuli or attentional behavior can impact such judgments). Finally, the study provides a solid foundation for future neuroimaging investigations.

Reviewer #1 (Public Review):

This paper performed a functional analysis of the poorly characterized pseudo-phosphatase Styxl2, one of the targets of the Jak/Stat pathway in muscle cells. The authors propose that Styxl2 is essential for de novo sarcomere assembly by regulating autophagic degradation of non-muscle myosin IIs (NM IIs). Although a previous study by Fero et al. (2014) has already reported that Styxl2 is essential for the integrity of sarcomeres, this study provides new mechanistic insights into the phenomenon. In vivo studies in this manuscript are compelling; however, I feel the contribution of autophagy in the degradation of NM IIs is still unclear.

Major concerns:

1) The contribution of autophagy in the degradation of Myh9 is still unclear to this reviewer. It has been reported that autophagy is dispensable for sarcomere assembly in mice (Cell Metab, 2009, PMID; 1994508). In Fig. 7A, the authors showed that overexpressed Styxl2 downregulated the amount of ectopically expressed Myh9 in an ATG5-dependent manner in C2C12 cells; however, the experiment is far from a physiological condition. Therefore, the authors should test ATG5 knockdown and the genetic interaction between Styxl2 and ATG5 in vivo. That is, 1) loss of ATG5 on sarcomere assembly in zebrafish, and 2) the genetic interaction between Styxl2 and ATG5; co-injection of Styxl2 mRNA and ATG5-MO into the zebrafish embryos.

2) As referenced, Yamamoto et al. reported that Myh9 is degraded by autophagy. Mechanistically, Nek9 acts as an autophagic adaptor that bridges Atg8 and Myh9 through interactions with both. Inconsistent with the model, the authors mentioned on page 12, lines 365-367, "A recent report showed that Myh9 could also undergo Nek9-mediated selective autophagy (Yamamoto et al., 2021), suggesting that Myh9 is ubiquitinated". I think it is not yet explored whether autophagic degradation of Myh9 requires its ubiquitination. Moreover, I cannot judge whether Myh9 is ubiquitinated in a Styxl2-dependent manner from the data in Fig. 7C. The author should test whether Nek9 is required for Myh9 degradation in muscles. If Nek plays a role in the Myh9 degradation, it would be better to remove Fig. 7C.

3) In Fig. 5F, the protein level of Styxl2 and Myh10 should be checked because the efficiency of Myh10-MO was not shown anywhere in this manuscript.

Reviewer #1 (Public Review):

C. elegans is a pre-eminent model for developmental genetics, and its invariant lineage makes it possible in theory to define molecular features such as gene expression comprehensively and at single cell resolution across the organism.

Previously published single-cell RNA-seq studies have mapped gene expression across the lineage through the 16-cell stage (Tintori et al 2017, Hashimshony et al 2016), and at later stages (Packer et al 2019, with good coverage starting at the 100-cell stage and some coverage at the ~50-cell stage). This left the critical period around gastrulation (~28-cell and ~50-cell) without comprehensive transcriptome data. This study covers this gap with a heroic effort involving the manual isolation and analysis of over 800 cells from embryos of known stage, combined with painstaking curation using known markers from small scale studies and larger imaging-based expression atlases. Importantly, the dataset overlaps at early and late stages with data from prior studies.

The data quality and overlap with Tintori and Packer datasets both appear high, but to make this inference required additional analysis from Supplemental Table 6 by this reviewer as it is not explored or described in the manuscript. Analyses demonstrating continuity with these datasets would greatly increase the value of the resource.

The authors show that specific lineages and stages preferentially express TFs with different classes of DNA binding domains. This extends previous work implicating homeodomains as preferentially involved in nervous system patterning and as enriched in neural and muscle progenitors in mid-stage embryos.

They also show that C. elegans homologs of Drosophila early embryonic regulators (which function based on spatial position in that system) tend to also be patterned in early C. elegans embryos, but with lineage-specific patterns. This conserved use of regulators would be fairly remarkable given the dramatically different developmental modes in these two species, although this observation is not backed up by quantitative analyses.

Finally, there is an argument that combinations of TFs expressed in lineage-specific patterns give rise to "stripe" patterns. This section is also not based on statistical analyses but suggests the possibility that lineage and positional regulation may be more convoluted than was previously thought.

Reviewer #1 (Public Review):

The authors of this well-described publication provided strong evidence that current DNA-based microbial genomics methodologies have an inherent constraint. These approaches cannot detect the source of sequenced DNA, and they fail to demonstrate the origin of sequenced DNA from live or non-viable bacteria. Moreover, scientists proved in people and mice that live bacteria for the most part remained within hair follicles rather than on the skin's surface. Overall, this study is of excellent quality and has broad implications beyond a particular subject.

Strengths:

The study is well-designed, and the experimental methods are well-described.<br /> The results are presented clearly and are supported by statistical analyses.<br /> The study's findings are novel and have important implications for understanding the skin microbiome and the biology of the skin.

Weakness:

RNA-based NGS could parallelly study the results of this DNA-based microbiome study. The bulk RNA-Seq can sequence thousands of transcripts from each viable bacterium and match them with the bacterial genome and transcriptome references. It is one of the best confirmatory methods for showing the diversity of viable cutaneous bacteria.

Reviewer #1 (Public Review):

Previous reports suggested an association between ceramide accumulation in skeletal muscle and disruption of insulin signaling and metabolic dysregulation. Mechanistically, however, how intracellular ceramide attenuates insulin action and reduces metabolism is not fully understood. It was suggested that insulin receptor (IR) signaling to PI3-K/AKT is inhibited by elevated intracellular ceramide. However, other studies failed to demonstrate an inhibitory effect of ceramide on PI3K/AKT. More recently, a study was published describing that intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle (PMID: 29415895). In the present study, Diaz-Vegas and colleagues used an in vitro system to investigate this topic further and better understand how intracellular ceramide accumulation causes cellular insulin resistance and metabolic dysregulations in cultured myocytes.

The authors applied multiple methods to achieve this goal. Among these procedures are:

1) The overexpression of enzymes involved in mitochondrial ceramide synthesis and degradation;

2) Treatments of myocytes with different pharmacological tools to validate their findings;

3) Mitochondrial proteomics and lipidomics analyses.

The effects of these experimental conditions and treatment on intracellular lipids contents, mitochondrial functions, and insulin signaling in myocytes were then evaluated.

Findings:

The authors' findings indicate that incubation of myocytes with palmitate increases mitochondrial ceramide and reduces the insulin-stimulated GLUT4-HA translocation to the myocyte surface without affecting AKT activation. The elevation in mitochondrial ceramide lowers the coenzyme Q levels e depletes the electron transport chain (ETC) components, impairing mitochondrial respiration. Such mitochondrial dysfunction appears to attenuate the translocation of GLUT4-HA to the plasma membrane of the L6-myotubule. Also, mitochondrial proteomic analysis revealed an association of insulin sensitivity with mitochondrial ceramide and ETC expression levels in human muscle.

Based on these findings, the authors propose a mechanism whereby the building up of ceramide inside mitochondria depletes CoQ and compromise mitochondrial respiratory complexes, raising ROS. The resulting mitochondrial dysfunction causes insulin resistance in cultured myocytes. They postulate that CoQ depletion links ceramides with insulin resistance and define the respirasome as a critical connection between ceramides and mitochondrial dysfunction.

Relevance and critiques:

This original study provides direct evidence that mitochondrial ceramide accumulation depletes CoQ and downregulates multiple ETC components in myocytes. Consequently, elevation in the levels of reactive oxygen species (ROS) and mitochondrial dysfunctions occur. The authors proposed that such mitochondrial dysregulation attenuates insulin-stimulated GLUT4 translocation to the plasma membrane of L6-myotubules. Moreover, mitochondrial ceramide accumulation does not affect insulin action on AKT activation.

Overall, this is a well-done study, showing that in obesity, elevated mitochondrial ceramide suppresses mitochondrial function and attenuates insulin action on glucose transporter GLUT4 translocation into the myocyte surface. The main conclusion is supported by the results presented. The study also applied multiple methods and described several experiments designed to test the author's central hypothesis.

Importantly, these new findings shed light on possible cellular mechanisms whereby ectopic fat deposition in skeletal muscle drives insulin resistance and metabolism dysregulation. The results demonstrating that alterations in mitochondrial ceramide are sufficient to attenuate insulin-stimulated GLUT4 trafficking in cultured myocytes are very interesting. Well-done.

Comments for further discussion and suggestions:

Although the authors' results suggest that higher mitochondrial ceramide levels suppress cellular insulin sensitivity, they rely solely on a partial inhibition (i.e., 30%) of insulin-stimulated GLUT4-HA translocation in L6 myocytes. It would be critical to examine how much the increased mitochondrial ceramide would inhibit insulin-induced glucose uptake in myocytes using radiolabel deoxy-glucose.

Another important question to be addressed is whether glycogen synthesis is affected in myocytes under these experimental conditions. Results demonstrating reductions in insulin-stimulated glucose transport and glycogen synthesis in myocytes with dysfunctional mitochondria due to ceramide accumulation would further support the authors' claim.

In addition, it would be critical to assess whether the increased mitochondrial ceramide and consequent lowering of energy levels affect all exocytic pathways in L6 myoblasts or just the GLUT4 trafficking. Is the secretory pathway also disrupted under these conditions?

Reviewer #1 (Public Review):

In this manuscript, Elkind et al. use a deep learning segmentation algorithm trained on detecting putative cell nuclei in mouse brains to count cells in the Allen Mouse Brain Connectivity Atlas. The Allen Mouse Brain Connectivity Atlas is a dataset compromising hundreds of mice brains. The authors use this increased statistical power for detecting differences in volume, cell count, and cell density between strains (C57BL/6J and FVB.CD1) as well as sex differences.

Both volume, cell count, and cell density are regularly used in neuroanatomy to normalize or benchmark results so having a large available dataset for others to compare their data would be a useful resource. The trained segmentation algorithm might also find utility in assays where investigators for one reason or another can't dedicate an entire labeled channel to count cell nuclei.

Nevertheless, because of technical reasons, I find the current work problematic.

Major:

The authors make use of the "red" channel from the Allen Mouse Brain Connectivity Project (AMBCP). The AMBCP was acquired using two-photon tomography with the TissueCyte 1000 system (http://help.brain-map.org/download/attachments/2818171/Connectivity_Overview.pdf?version=2&modificationDate=1489022310670&api=v2). The sample is illuminated at 925 nm wavelength and the channel the authors describe as autofluorescence is collected through a 593/40 nm bandpass filter. The authors go on to describe their rationale for using this channel for quantifying cell nuclei:<br /> "We noticed that the red (background) channel of STPT images, taken for the purpose of atlas alignment, typically features dark, round-like objects resembling cell nuclei. We had observed this phenomenon in our own imaging of mouse brains but found little more than anecdotal mentions of it in the literature8,9,10,11".<br /> The authors here cite a Scientific Reports paper from 2021 with 11 citations, a Journal of Clinical Pathology paper from 2005 with 87 citations, and lastly a paper in Laboratory Investigation from 2016 with 41 citations. The authors completely fail to cite the work from Watt Webb's group (co-inventor of 2p microscopy) in PNAS from 2003 that entirely described the phenomena of native fluorescence by multiphoton-excitation (https://www.pnas.org/doi/10.1073/pnas.0832308100 ), citations so far: 1959 citations. This is either indicative of poor scholarship or an attempt to describe something as novel. Either way, the native fluorescence and second harmonic generation from multiphoton illumination are perfectly characterized by Webb and colleagues and they clearly show the differential effect on nucleosides, retinol, indoleamines, and collagen. This is also where the authors should have paid more attention to discrepancies in their own data when correlated to well-established cell nuclei markers (Murakami et al). The authors will note "black large spots" in the data at specific anatomical regions and structures, like the fornix and stria medullaris:<br /> https://connectivity.brain-map.org/projection/experiment/siv/263780729?imageId=263780960&imageType=TWO_PHOTON,SEGMENTATION&initImage=TWO_PHOTON&x=15702&y=18833&z=5

which is not reproduced in for example the Allen Reference Atlas H&E staining:<br /> http://atlas.brain-map.org/atlas?atlas=1&plate=100960284#atlas=1&plate=100960284&resolution=4.19&x=5507.4000244140625&y=5903.39990234375&zoom=-2

In connection here notice the poor signal in the 2p "autofluorescence" within the paraventricular nucleus:<br /> https://connectivity.brain-map.org/projection/experiment/siv/263780729?imageId=263780960&imageType=TWO_PHOTON,SEGMENTATION&initImage=TWO_PHOTON&x=15702&y=17833&z=6

and then compare it to the H&E staining:<br /> http://atlas.brain-map.org/atlas?atlas=1&plate=100960280#atlas=1&plate=100960276&resolution=1.50&x=5342.476283482143&y=5368.023856026786&zoom=0

These multiphoton-specific signals are especially pronounced in the pons and medulla which makes quantification especially dubious, which is even apparent simply from looking at Figure 1c in the manuscript. The authors here use the correlation on log-log coordinates between their data and that of Murakami et al to argue that the method has validity. However, the variance explained here is R^2 = 0.74 which is very poor given the log-log coordinates. A more valid metric would use linear coordinates and computing the ICC and interpret it according to established guidelines (e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913118/).

In addition to the above concern, the authors argue that the large sample size of the AMBCP is what would enable them to find statistically significant small effect sizes that might have gone undetected in the literature. However, this argument falls flat once we examine some of the main findings the authors report. Although the authors do not directly report measures of dispersion we can estimate it from the figures and then arrive at the sample size needed to find the reported effect size. For example, the effect that describes ORBvl2/3 volume is larger in female mice compared to males would only require n=13 mice at the desired power of 0.8. Likewise, the sample size needed to detect the increased BST volume in male mice looks to be roughly n=16 mice at the desired power of 0.8. Both of these estimates are well within what is a reasonable sample size to expect in an ordinary study. This begs the question: why did the authors simply not verify some of their main findings in an independent sample obtained through traditional ways to quantify volume and cell density since it is well within reach? Such validation would strengthen the arguments of the paper.

Reviewer #1 (Public Review):

Alignment between high dimensional data which express their dynamics in a subspace is a challenge which has recently been addressed both with analytic-based solutions like the Procrustes transformation, and, most interestingly, via deep learning approaches based on adversarial networks. The authors have previously proposed an adversarial network approach for alignment which relied on first dimensionally-reducing the binned neural spikes using an autoencoder. Here, they use an alternative approach to align data without use of an initial dimensional-reduction step.

The results are fairly clear - the Cycle-GAN approach works better than their previous ADAN approach and one based on dimensionality reduction followed by the Procrustes transform. In general, a criticism of this entire field is to understand what alignment teaches us about the brain or how it specifically will be used in a BCI context.

There are a few issues with the paper.

1.) To increase the impact of their work, the investigators have now used it to align data in multiple types of tasks. There was an unanswered question about this related to neuroscience - does alignment in one task predict alignment for another?

2) Investigators use decoding as a way of comparing alignment performance. The description of the cycle GAN was not super detailed, and it wasn't clear whether there was any dynamic information stored in the network that might create questions of causality in actual use. It seems that input is simply the neural activity at a current time point rather than neural activity across the trial, which would alleviate this concern. However, they mention temporal alignment but never describe in detail whether all periods of spikes are properly modeled by the system or if only subsets of data (specific portions of task or non-task time) will work. Perhaps this is more a question of the Wiener filter, for which precise details are missing.

3) In general, precise details of the algorithms should have been provided.

4) Cross validation for day-0 alignment is not explained.

5) Details of statistical tests is not provided.

6) (minor) The idea that for neurons that have disappeared that the CycleGAN can "infer their response properties", seems an incorrect description. A proper description should be that it "hallucinates" their response properties?

Reviewer #1 (Public Review):

The study by Yang et al. reports a new mechanistic role of vinculin in inhibiting the Mef2c nuclear translation and sclerostin expression in osteocytes and promoting bone formation. The authors showed the reduction of vinculin in aged bone human bone samples. A 10kb DMP-1-Cre mouse model was generated that deleted vinculin in osteocytes. They found that vinculin deletion caused bone loss and decreased bone formation associated with increased sclerostin expression. This increase does not affect the protein level of transcription factor Met2c but interestingly enhances nuclear translocation. Vinculin is interested in Mef2c and appears to retain Mef2c in the cytosol. As expected, as a component of the mechanosensory focal adhesion complex, bone formation via tibial loading was decreased in vinculin deletion. Intriguingly, the bone loss associated with estrogen deficiency through ovariectomy was attenuated. Overall, the study unveiled an important role concerning a key player of focal adhesion and the study was well designed and executed. The paper would be strengthened by including a more thorough discussion including variables such as male vs. female, and cortical vs. trabecular bone as the vinculin deletion appeared to primarily affect trabecular bone while mechanical loading exerts anabolic effects on both bone types. The effect of estrogen deficiency effect is interesting and is worth some discussion.

Strengths:<br /> The paper shows a novel mechanism that vinculin retains Mef2c in the cytosol via protein interaction to prevent it from migrating to the nucleus and increases transcription of sclerostin, an inhibitory factor for Wnt/β-catenin signaling, a critical pathway for osteoblast activity and bone formation.<br /> They employed various in vivo and in vitro models as well as human tissue samples including generating conditional knockout of vinculin in osteocytes in vivo and vinculin gene knockdown in MLO-Y4 cells. They also used physiological/pathological relevant models, tibial loading, and ovariectomy to study the role of vinculin under mechanical loading and estrogen deficiency. The adopted standard techniques to study bone properties include microCT, bone formation, bone histomorphometry, histochemistry as well as biochemical assays such as immunoprecipitation, ChIP assays, etc.

The study is comprehensive and thorough and the noticeable uniqueness is that after observing the phenotypes from in vivo data, they further explored the underlying mechanisms using cell models. The experiments in general are well-designed and presented with adequate repeats and statistical analysis. The paper is also logically written and the figures were clearly labeled.

Minor weaknesses:<br /> More discussion is necessary concerning the potential difference in responses between male and female. Most of the studies were conducted in male mice except ovariectomy mice.<br /> It is interesting that the cKO of vinculin in osteocytes primarily affects trabecular bones with limited effect on cortical bones. However, sclerostin is increased in cortical bones. The promotion of bone formation by mechanical loading appears to affect both cortical and trabecular bones. If focal adhesion is a key mechanosensory complex, how to reconcile the different responses in the cKO model?<br /> The OVX response is interesting and it is worthwhile to elaborate more regarding the potential underlying mechanism and what's the relationship between estrogen and mechanical loading and if the action of estrogen on vinculin shares any similar mechanisms with mechanical loading, etc.

Reviewer #1 (Public Review):

This study represents in exciting collaboration between two young independent scientists in Uruguay and Japan. Trigo and Kawaguchi provide evidence for the presynaptic modulation of the opening-probability of calcium channels as a major mechanism of digital-analog coupling in immature cerebellar molecular layer interneurons (MLI). Applying a combination of electrophysiological methods including direct axonal whole-cell patch-clamp recordings and glutamate photolysis in acute brain slices and dissociated cultured neurons, the authors provide the following empirical findings: 1) Spontaneous and evoked EPSPs are reliably transmitted into the presynaptic compartment. The amplitude of the spontaneous EPSPs decayed with a length constant of 180 µm in the axon. 2) Physiologically relevant short and subthreshold (< 10 mV) depolarizations before action potentials ('pre-AP') increase the release probability and subsequently short-term depression at the MLI-Purkinje cell synapse without changing the duration of APs and just a minor reduction in amplitude of APs (< 10%). 3) The pre-AP subthreshold depolarizations subsequently increase the amplitude of AP-induced presynaptic calcium currents and GABAergic postsynaptic currents. 4) A short interval of only 3 ms duration between the pre-AP depolarization and the AP blocks the analog coupling. 5) A biophysical model of presynaptic calcium channel gating is proposed, which involves depolarization-induced intermediate gating steps that increase the probability of activating the channels during the AP.

A particular strength of this study is the large data set of technically very challenging direct recordings from small presynaptic terminals. The proposed mechanism provides an innovative explanation for the experimental findings. The most innovative experiments might be those with a 3-ms-gap between the pre-APs and APs. At this synapse, elevated residual intracellular calcium concentration was previously shown to mediate analog coding (https://doi.org/10.1523/JNEUROSCI.5127-10.2011). However, the elevated residual calcium cannot explain the surprising block of analog coding by a 3-ms-gap in the depolarization, because intracellular calcium signals decay with kinetics in the range of 100 ms. Both mechanisms (residual calcium and priming of calcium channels) are probably operating in parallel and future studies should resolve the exact interplay of both mechanisms. A potential weakness of the study is that the proposed priming of calcium channels is not shown explicitly to be able to explain the experimental data. Quantitive simulations of calcium channel gating states were only performed in steady-state but not in a time-dependent manner during pre-APs and APs.

Reviewer #1 (Public Review):

This manuscript investigates the mechanisms of 'summiting disease' using a previously characterised Drosophila model. The authors also show that E. muscae infiltrates the brain likey through a defective blood-brain barrier and populates regions of the brain in the medial protocerebrum. It likely releases metabolites into the haemolymph of summiting flies that has the ability to induce summiting in uninfected flies. They also show that a burst of locomotor activity precedes death. To understand the circuit basis of this, they perform a screen of more than a hundred neuronal lines and genes to identify an active DPN1>pars intercerebralis neurons> corpora allata>JH axis as being invovled in the summiting behaviour while not affecting death.

Reviewer #1 (Public Review):

The manuscript by Gochman and colleagues reports the discovery of a very strong sensitization of TRPV2 channels by the herbal compound cannabidiol (CBD) to activation by the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB). Using patch-clamp electrophysiology the authors show that the ~100-fold enhancement by micromolar CBD of TRPV2 current responses to low concentrations of 2-APB reflects a robust increase in apparent affinity for the latter agonist. Cryo-EM structures of TRPV2 in lipid nanodiscs in the presence of both drugs report two-channel conformations. One conformation resembles previously solved structures whereas the second conformation reveals two distinct CBD binding sites per subunit, as well as changes in the conformation of the S4-S5 linker. Interestingly, although TRPV1 and TRPV3 are highly homologous to TRPV2 and both CBD binding sites are relatively conserved, the CBD-induced sensitization towards 2-APB is observable only for TRPV3 but not for TRPV1. Moreover, the simultaneous substitution of non-conserved residues in the CBD binding sites and the pore region of TRPV1 with the amino acids present in TRPV2 fails to confirm strong CBD-induced sensitization. The authors conclude that CBD-dependent sensitization of TRPV2 channels depends on structural features of the channel that are not restricted to the CBD binding site but involve multiple channel regions.

These are important findings that promote our understanding of the molecular mechanisms of TRPV family channels, and the data provide convincing evidence for the conclusions.

Reviewer #1 (Public Review):

The authors' objectives were to identify the features of uORFs that determine their effects on the translation of the main ORF found in the same transcript. The major strengths of the paper are the creative and powerful experimental platforms used to measure translation, the computational approaches used to identify the key features that determine the effect of uORFs on translation and the comparative analysis of two closely related species to understand how uORF activity evolves. The authors successfully and convincingly identify features associated with the regulatory effects of uORFs and have results suggesting that uORFs that would have strong repressive effects would be selected against. Although these insights regarding evolution are very interesting and may contribute to our understanding of regulatory evolution, at a level that is rarely explored, this section could benefit from additional analyses of existing data to fully support the conclusions. Another aspect that would need to be considered is the possible interaction between the uORFs and the main ORFs. Here, all experiments are performed with the same main ORFs (YFP) for practical and essential reasons, but it would be useful to know whether some uORF features would have effects whose sign and magnitude may depend on which main ORFs they associate with. Overall, there are several areas in which the authors' claims or conclusions are not fully justified and require either additional statistical analysis or new experimentation.

Reviewer #1 (Public Review):

The manuscript by Muthana et al. describes the effect of injection of an antibody specific for human CTLA4 conjugated to a cytotoxic molecule (Ipi-DM1) in knock-in mice expressing human CTLA4. The authors show that Ipi-DM1 administration causes a partial decrease (about 50% in absolute number) of mature B cells in blood and bone marrow 9-14 days after the beginning of treatment. Ipi-DM1 also results in a partial decrease in Foxp3+ Tregs (about 40% in absolute number) and a slight increase in activation of conventional T cells (Tconvs) in the blood at D9. Tconv depletion, CTLA4-Ig or anti-TNF mAb partially prevents the effect of ipi-DM1 on B cells. This work is interesting but has the following major limitations:

1- This work could have been of more interest if the Ipi-DM1 molecule would be used in the clinic. As this is not the case, the intimate mechanism of the effect of this molecule in mice is of reduced interest.<br /> 2- The fact that a partial deletion of Tregs is associated with activation of Tconvs and a decrease in B cells has been published several times and is therefore not new. According to the authors, their work would be the first to show that activation of Tconvs would lead to B cell depletion. However, this is shown in an indirect way and the mechanisms are not really elucidated. Indeed, this work shows a correlation between an increase in Tconv activation and a decrease in the number of B cells in the blood. The experiments to try to show a causal link are of 2 types: deletion of T cells (Fig 4) and blocking T cell activation with CTLA4-Ig (Fig 5) (neutralization of TNF addresses another question). Neither of these 2 experiments is totally convincing. Indeed, the absence of B cell depletion when T cells are deleted can be explained by other mechanisms than the preservation of B cell destruction by activated T cells. The phenomenon could be explained by B cell recirculation to lymphoid tissues or an effect of massive T cell death for example. The experiment shown in Fig. 5 with Belatacept is more convincing because this time the effect is targeted to activated T cells only. However, the prevention of B cell ablation is only partial. Again, since only blood is analyzed, other mechanisms could explain the B cell loss, such as their recirculation in lymphoid tissues.<br /> 3- It is disappointing that only the blood (and sometimes the bone marrow) was studied in this work. The interest of doing experiments in mice is to have access to many tissues such as the spleen, lymph nodes, colon, lung, and liver. To conclude that there is B cell deletion without showing lymphoid organs (where the majority of B cells reside) is insufficient. As discussed above, the drop in B cells in the blood could be due to their recirculation in lymphoid organs. In addition, there is no measurement of functional B cells activity. Do mice treated with Ipi-DM1 have a decreased ability to develop an antibody response following immunization?<br /> 4- Although it is difficult to study in vivo, there is not a single evidence of increased B cell death after injection of Ipi-DM1.<br /> 5- In most of the experiments, B cells are quantified with the B220 marker alone, but this marker, in some cases, can be expressed by other cells. It would have been preferable to use a marker more specific to B cells such as CD19 for example.

In conclusion, the concept that T cell activation can lead to B cell deletion is interesting but this study shows it only in an indirect and incomplete way.

Reviewer #1 (Public Review):

This study was designed to examine the bypass of Ras/Erk signaling defects that enable limited regeneration in a mouse model of hepatic regeneration. The authors show that this hepatocyte proliferation is marked by expression of CD133 by groups of cells. The CD133 appears to be located on intracellular vesicles associated with microtubules. These vesicles are loaded with mRNA. The authors conclude that the CD133 vesicles mediate an intercellular signaling pathway that supports cell proliferation. These are new observations that have broad significance to the fields of regeneration and cancer.

The primary observation is that the limited regeneration observed in livers with Ras/Erk signaling defects is associated with CD133 expression by groups of cells. The functional significance of CD133 was tested using Prom1 KO mice - the data presented are convincing.

The major weakness of the study is that some molecular mechanistic details are unclear - this is, in part, due to the extensive new biology that is described. Nevertheless, the data used to support some key points in this study are unclear:

a) What is the evidence that the observed CD133 groups of cells are not due to clonal growth. Is this conclusion based on the time course (the groups appear more rapidly than proliferation) or is this based on the GFP clonal analysis?

b) What is the evidence that the CD133 vesicles mediate intercellular communication. This is an exciting hypothesis, but what is the evidence that this happens? Is this inferred from IEG mRNA diversity? or some other data. Is there direct evidence of transfer - for example, the does the GFP clonal analysis show transfer of GFP that is not mediated by clonal proliferation? Moreover, since the hepatocytes are isogenic, what distinguishes the donor and recipient cells?

Increased clarity concerning what is hypothesis and what is directly supported by data - would improve the presentation of this study.

Reviewer #1 (Public Review):

This is an interesting manuscript that proposes a new approach to for accounting for viral diversity within hosts in phylogenetic analyses of pathogens. Concretely, the authors consider sites for which a minor allele exist as an additional base in the substitution model. For example, if at a particular site 60% of reads have an C and 40% have a G, then this site is assigned Cg, as opposed to an C which is typical of analysing consensus sequences. Because we typically model sequence evolution as a Markovian process, as is the case here, the data become naturally more informative, given that there are more states in the Markov chain when adding these bases. As a result, phylogenetic trees estimated using these data are better resolved than those from consensus sequences. The branches of the trees are probably also longer, which is why temporal signal becomes more apparent.

I commend the authors on their rigorous simulation study and careful empirical data analyses. However, I strongly suggest they consider whether treating minor alleles as an additional base is biologically realistic and whether this may have implication for other analyses, particularly when there is very high within-host diversity and the number of states in becomes very large.

Reviewer #1 (Public Review):<br /> <br /> Beta-hemoglobinopathies, such as sickle cell disease and beta-thalassemia, are common and debilitating genetic diseases caused by mutations in the adult beta-globin gene. Many in the field are pursuing various strategies to therapeutically upregulate fetal gamma-globin to treat these diseases. In this paper, the authors aimed to instead edit the promoter of the delta-globin gene to cause upregulation of delta-globin expression. Delta-globin is highly homologous to adult beta-globin and is pan-cellularly expressed in adult red blood cells, albeit at low levels due to the low activity of its promoter. Gene editing to activate the promoter of delta-globin could allow delta-globin expression to be elevated which could compensate for defective beta-globin in patients with beta-hemoglobinopathies. This is an underexplored and very interesting approach, and this study represents the first time that delta-globin upregulation has been attempted using gene editing in adult-like human erythroid immortalised and primary cells.

The first major finding from this study was that gene editing to insert KLF1, beta-DRF, and TFIIb sites into the delta-globin promoter was sufficient to cause upregulation of delta-globin expression at the mRNA and protein levels in immortalized HUDEP-2 cells. Modest upregulation was seen in pooled populations of HUDEP-2 cells (where ~25% of cells were HDR edited). Robust expression of delta-globin was seen in homozygously edited clonal populations of HUDEP-2 cells, with delta-globin constituting ~25% of total beta-like globin expression at the mRNA level in these cells. The results presented thus strongly support this finding.

The second major finding was that gene editing to insert KLF1, beta-DRF, and TFIIb sites into the delta globin promoter was sufficient to cause upregulation of delta-globin in primary human CD34+ cells. Despite HDR editing efficiencies of ~25% in these primary cells, and possibly due to only two donor cell populations being used, significant upregulation of delta-globin was not detected in pooled populations of edited primary CD34+ cells. Encouraging evidence of upregulation was seen in the clonal population of edited cells from the two donors. As such the results provide moderate support for this finding.

In combination, the HUDEP-2 cell and CD34+ cell data provide compelling evidence that gene editing of the delta-globin promoter is a promising line of enquiry for the treatment of beta-hemoglobinopathies.

This important study establishes and provides a proof-of-principle for this alternative therapeutic approach for those with beta-hemoglobinopathies. Future studies based on this work may enable delta-globin to be upregulated to therapeutically relevant levels in patient cells, including in cells from patients with beta-hemoglobinopathies. The therapeutic benefits of delta-globin upregulation will then be able to be assessed. This finding will be of interest to those in the globin switching and gene editing fields.

Reviewer #1 (Public Review):

In this interesting manuscript, Nasser et al explore long-term patterns of behavior and individuality in C. elegans following early-life nutritional stress. Using a rigorous, highly quantitative, high-throughput approach, they track patterns of motor behavior in many individual nematodes from L1 to young adulthood. Interestingly, they find that early-life food deprivation leads to decreased activity in young larvae and adults, but that activity between these times, during L2-L4, is largely unaffected. Further, they show that this "buffering" of stress requires dopamine signaling, as L2-L4 activity is significantly reduced by early-life starvation in cat-2 mutants. The paper also provides evidence that serotonin signaling has a role in modulating sensitivity to stress in L1 larvae and adults, but the size of these effects is modest. To evaluate patterns of individuality, the authors use principal components analysis to find that three temporal patterns of activity account for much of the variation in the data. While the paper refers to these as "individuality types," it may be more reasonable to think of these as "dimensions of individuality." Further, they provide evidence that stress may alter the strength and/or features of these dimensions. Though the circuit mechanisms underlying individuality and stress-induced changes in behavior remain unknown, this paper lays an important foundation for evaluating these questions. As the authors note, the behaviors studied here represent only a small fraction of the behavioral repertoire of this system. As such, the findings here are an interesting and very promising entry point for a deeper understanding of behavioral individuality, particularly because of the cellular/synaptic-level analysis that is possible in this system. This paper should be of interest to those studying C. elegans behavior and also more generally to those interested in behavioral plasticity and individuality.

Reviewer #1 (Public Review):

The DNA damage checkpoint is a cellular signalling pathway that responds to DNA damage and replication stress. This manuscript by Ho et al. systematically investigates an aspect of the checkpoint response in budding yeast that has been previously understudied, namely which proteins change subcellular and how these changed depend on the checkpoint kinases Mec1 and Rad53. By nice detective work the authors find a new mode of activation of Rad53, which is Mec1-independent, but rather depends on factors of so called retrograde signalling. Currently, we view checkpoint signalling as hierarchical, with Mec1 and Tel1 activating Rad53, despite both Mec1 and Rad53 having independent targets. This manuscript challenges that view by finding a Mec1 (and Tel1) independent mode of activation. It is very clear from survival and mass spectrometry data that in the absence of Mec1 this activation pathway and Rtg3 has a key role in activating Rad53. In the current form of the manuscript, it remains however difficult to assess what is the contribution of these factors on Rad53 activation in an otherwise WT background.

Reviewer #1 (Public Review):

In this paper, Reato, Steinfeld et al. investigate a question that has long puzzled neuroscientists: what features of ongoing brain activity predict trial-to-trial variability in responding to the same sensory stimuli? They record spiking activity in the auditory cortex of head-fixed mice as the animals performed a tone frequency discrimination task. They then measure both overall activity and the synchronization between neurons, and link this 'baseline state' (after removing slow drifts) of cortex to decision accuracy. They find that cortical state fluctuations only affect subsequent evoked responses and choice behavior after errors. This indicates that it's important to take into account the behavioral context when examining the effects of neural state on behavior.

Strengths of this work are the clear and beautiful presentation of the figures, and the careful consideration of the temporal properties of behavioral and neural signals. Indeed, slowly drifting signals are tricky as many authors have recently addressed (e.g. Ashwood, Gupta, Harris). The authors are well aware of the difficulties in correlating different signals with temporal and cross-correlation (such as in their 'epoch hypothesis'). To disentangle such slow trends from more short-lived state fluctuations, they remove the impact of the past 10 trials and continue their analyses with so-called 'innovations' (a term that is unusual, and may more simply be replaced with 'residuals').

I do wonder if this throws out the baby with the bathwater. If the concern is statistical confound, the 'session permutation' method (Harris) may be better suited. If the concern is that short-term state fluctuations are more behaviorally relevant (and obscured by slow drifts), then why are the results with raw signals in the supplement (Suppfig 8) so similar?

While the authors are correct that go-nogo tasks have drawbacks in dissociating sensitivity from response bias, they only cursorily review the literature on 2AFC tasks and cortical state. In particular, it would be good to discuss how the specific method - spikes, EEG (Waschke), widefield (Jacobs) and algorithm for quantifying synchronization may affect outcomes. How do these population-based measures of cortical state relate to those described extensively with slightly different signals, notably LFP or EEG in humans (e.g. work by Saskia Haegens, Niko Busch, reviewed in https://doi.org/10.1016/j.tics.2020.05.004)? This review also points out the importance of moving beyond simple measures of accuracy and using SDT, which would be an interesting improvement for this paper too.

Public Review:

Barreat and Katzourakis analyze the evolutionary history of eukaryotic viruses (and related mobile elements) in the Bamfordvirae kingdom, and discuss potential scenarios regarding the origin of different viral taxa in this group. This version of their manuscript includes a larger number of sequences to better represent diversity in these viral groups, and explored new evolutionary scenarios, including a "virophage-first" hypothesis now presented as the one best supported by phylogenetic analyses. The authors also present compelling analyses suggesting that the "nuclear escape" hypothesis in which these different viral groups separately "escaped" from nuclear (integrated) elements is not consistent with the current genomic and phylogenetic information available.

This work is thus an important step in our collective understanding of the ancient evolutionary history of eukaryotic viruses, and more generally of the constraints and main drivers of virus evolution.

Reviewer #1 (Public Review):

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

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

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

The authors could have been more precise in the manuscript title and abstract to emphasize that these findings apply to human cells, as indeed there is no demonstration yet that the findings presented here can be transposed to mouse cells.

The manuscript's main conceptual advance is that the authors propose a novel model of gene regulation whereby transcriptional repressors of transposable elements could regulate genes at distance by modulating the local chromatin environment within TADs. Additional experiments would be needed to strengthen this model. For example the authors could have performed TRIM28 ChIP in ZMYM2-kd cells to test if ZMYM2 favors the recruitment of TRIM28 to its genomic targets, as well as ChIP-seq of repressive chromatin marks (such as H3K9me3) in ZMYM2-kd cells to investigate if the loss of ZMYM2 leads to reduced H3K9me3 in ERVs and over large regions surrounding the ERVs.

Reviewer #1 (Public Review):

In the manuscript there is not much comparison between the crystal and cryoEM structures provided, and on inspection they appear to be very similar. The crystal structures also reveal parts of the CC domains in Las1, which is not present in the cryoEM structures. It is interesting the CC domains in Sc and Cj are quite different as illustrated in Figure 4B. They also seem to be somewhat disconnected from the rest of the complex (more so for Cj), even though that's not apparent in Figures 2-4. Despite this, it would be very useful to show the cryoEM densities when describing the catalytic site and C-terminal domain interactions, for example, as this can be very useful to increase confidence in the model and proposed mechanisms.

The description of the complex as a butterfly is engaging, and from a certain angle it can be made to look as such; this was also described previously in (Pillon et al., 2019, NSMB) for the same complex from a different organism (Ct). However, it is a bit misleading, because the complex is actually C2 symmetric. Under this symmetry, the 'body' would consist of two 'heads' one pointing up, one down facing towards the back, and one wing would have its back toward the viewer, the other the front. The structures presented here in Sc and Cj seem quite similar to the previous structure of the same complex in Ct, though the latter was only solved with cryoEM, and was also lacking the structure of the CC domain in Las1.

For the model suggested in Figure 8, perhaps in the 'weak activity' state, the LCT in Las1 could still be connected to Grc3, via the LCT, rather than disconnected as shown. This could facilitate faster assembly of the 'high activity' state. The complex is described as 'compact and stable', but from the structure and this image, it appears more dynamic, which would serve its purpose and the illustrated model better. The two copies of HEPN appear to have more connective area, meaning they are indeed more likely to remain assembled in the 'weak activity' state. On the other hand, HEPN in one protein appears to have less binding surface with PNK in Grc3, and even less so with the CTD (both PNK and CTD being from the other associated protein), meaning these bindings could release easily to form the 'weak activity' state.

There is also the potential to speculate that the GCT is bound to HEPN near the catalytic area in the 'weak activity' state. The reduced activity when the GCT residues are replaced by Alanine could then be explained by the complex not being able to assemble as quickly upon binding of the substrate, as it could if the GCT remained bound, rather than by a conformational change that it induces upon binding. The conformational change is also likely to be influenced by the combined binding of PNK and CTD in the assembled state, which also contact HEPN, rather than by GCT alone.

When comparing the structure of the HEPN domain in the lone Las1 protein to the structure of Las1-HEPN in the Las1-Grc3 complex, it is mentioned that 'large conformational changes are observed'. These could be described a bit better. The conformational change is ~3-4Å C-alpha RMSD across all ~150 residues in the domain (~90 residues forming a stable core that only changes by ~1Å). There is also a shift in the associated HEPN domain in Las1B domain compared to the bound HEPN in the Las1-Grc3 complex, as shown in Figure 7D: ~1Å shift and ~12degrees rotation. This does point to the conformation of HEPN changing upon complex formation, as does the relative positions of the HEPN domains in Las1A and Las1B. The conformational change and relative shift could indeed by key for the catalysis of the substrate as mentioned.

Overall, the structures presented should be very useful in further study of this system, even though the exact dynamics and how the substrate is bound are aspects that are perhaps not fully clear yet. The addition of the structures of the CC domain in two different organisms and the Las1 HEPN domain (not in complex with Grc3) as new structural information should allow for increasing our understanding of the overall complex and its mechanism.

Reviewer #1 (Public Review):

Membrane receptor guanylyl cyclases are important for many physiological processes but their structures in full-length and their mechanism are poorly understood. Caveney et al. determined the cryo-EM structure of a highly engineered GC-C in a complex with endogenous HSP90 and CDC37. The structural work is solid and the structural information will be useful for the membrane receptor guanylyl cyclases field and the HSP90 field. However, a detailed characterization of the protein sample is lacking. Moreover, the physiological significance of this structure is not fully exploited by supporting experiments and the mechanistic insight is currently limited.

1. The characterization of the protein sample is lacking. SDS-PAGE would be useful to identify potential proteolysis, leading to the dissociation of GC dimer. Further size-exclusion chromatography would be helpful to estimate the molecular weight of the complex and to determine if only GC-C monomer is purified.

2. The orientation distribution of the particles is not homogenous in Fig. S1D. It would be helpful to present the 3DFSC curve to evaluate the effect of preferred orientation on the reconstruction.

3. Description of protein expression details is lacking. Did the author use transient transfection, stable cell line or virus-mediated transduction?

4. HSP90 binds ATP and is often co-purified with endogenous ATP/ADP. Is there ATP or ADP present in the sample/cryo-EM maps? Is the conformation of NBD similar to ATP-bound HSP90? The author needs to include the description/figures about the nucleotide state of HSP90.

5. The catalytic domains of GC have to be dimerized to perform cyclase function. The presence of only one GC-PK monomer in the cryo-EM structure indicates the structure does not represent an active state of GC. These results suggest the GC expressed in this way is not functional. The authors need to explain why most of the GC protein is trapped in this inactive form.

6. The GC-C construct used here is a highly engineered "artificial" construct, which has not been fully characterized in this work. Does this construct have similar activity as the activated wt GC-C? Does the protein (this engineered construct) expressed in CHO cells show activity?

7. Are the residues on the interface between GC and HSP conserved in other members of membrane receptor guanylyl cyclases? Would mutations on this interface affect the activity of GC?

8. The authors propose that targeting HSP90 would tune the activity of GC. Is there any experimental data supporting this idea?

9. The model in Fig. S3 is largely speculative due to the lack of supporting functional data. In addition, it would be better to change the title to "structure of the protein kinase domain of guanylyl cyclase receptor in complex with HSP90 and cdc37" because the mechanistic insight is limited.

Reviewer #1 (Public Review):

This manuscript conducts a classic QTL analysis to identify the molecular basis of natural variation in disease resistance. This identifies a pair of glycosyltransferases that contribute to steroidal glycoalkaloid production. Specifically altering the final hexose structure of the compound. This is somewhat similar to the work in tomatine showing that the specific hexose structure mediates the final potential bioactivity. Using the resulting transgenic complementation lines that show that the gene leads to a strong resistance phenotype to one isolate of Alternaria solani and the Colorado potato beetle. This is solid work showing the identification of a new gene and compound influencing plant biotic interactions. While the experiments are solid, the introduction, discussion and associated claims don't accurately reflect my reading of what is known and said in the current literature.

The sentence on line 53-54 is misleading. It provides only three citations on specific links between specialized metabolism and disease resistance. However, there are actually at least 40 on specific links of camalexin and indolic phytoalexins to disease resistance. Similarly there are dozens of uncited papers on benzoxazinoids, indolic glucosinolates, aliphatic glucosinolates and tomatine to both non-host and host based resistance mechanisms. This even goes as far as showing how the pathogens resist an array of these compounds. The choices in the introduction make it appear that little is known about specialized metabolism to disease resistance but I would suggest that this is not an allusion supported by the literature. I would agree that given the breadth of specialized metabolism we have a lot of knowledge about a set of them but that there are hundreds to thousands of untested compounds but to indicate that little is known is unfair to the specialized metabolism community. This is especially true as the introduction and discussion give no image of the large body of literature on specialized metabolism to insect interactions even though this is a major component of this manuscript.

I would also agree that specialized metabolism is not a conscious target of breeding programs but the work on benzoxazinoids in maize and glucosinolates in the Brassica's has shown that these compounds have been influenced by breeding programs. Similarly work on de novo domestication of multiple crops is focused on the adjustment of specialized metabolism in these crops.

I would disagree with the hint on line 49-50 and again on lines 236-239 that specialized metabolism may have less pleiotropy. This is not supported by recent work on benzoxazinoids and glucosinolates showing that they have numerous regulatory links to the plant and can be highly pleiotropic. Even the earliest avenicin work in oat showed that the deficient lines had altered root development.

My main message from the above three paragraphs is to point out that there are a number of places in the manuscript where the current state of the specialized metabolite literature is not accurately portrayed. To properly place the manuscript in the broader context, I would suggest a more even handed introduction and discussion that takes into account the current state of the specialized metabolism literature.

Is it accurate to say complete resistance to A. solani if only a single isolate of the pathogen is used? Is there evidence that I am unaware of that there are no isolates of this pathogen with saponin resistance? There are pathogens with natural tomatine resistance and this is a common feature of plant pathogens that they have genetic variation in the resistance to specialized metabolism. For example, it should be noted that Botrytis BO5.10 is a tomatine sensitive isolate and the van Kan and Hahn groups have published on isolates that are resistant to saponins. I would suggest caveating across the manuscript that this is a single isolate and that it is possible that there may be isolates with natural resistance to the steroidal glycoalkaloid?

In Figure 4b, is the infection site about 3.5 mm in size such that 3.5 mm means absolutely no infection? If not, that would mean there is some outgrowth by Alternaria and the resistance isn't complete.

Reviewer #1 (Public Review):

In this article, Cacioppo et al., report on a previously unappreciated mechanism of the regulation of Aurora Kinase A (AURKA) protein levels that is orchestrated via coordinated action of alternative polyadenylation of AURKA mRNA and hsa-let-7a miRNA. Moreover, it is proposed that this mechanism may play a major role in neoplasia. In support of their model, the authors demonstrate that short-to-long 3'UTR AURKA mRNA isoform ratio is elevated in triple negative breast cancer patients where it correlates with poor prognosis. The authors further generated reporters suitable for single cell live imaging that express different 3'UTR variants, which revealed highly variable ratios of short and long 3'UTR AURKA isoforms across different cell lines. This was followed by actinomycin D chase and nascent chain immunoprecipitation assays in U2OS osteosarcoma cells to demonstrate that while short and long 3'UTR AURKA isoforms have comparable stability, short 3'UTR AURKA isoforms appear to exhibit higher ribosome association which is indicative of higher translation activity. Furthermore, using an additional reporter assay which takes advantage of trimethoprim-based stabilization of highly unstable E. Coli dihydrofolate reductase mutants Cacioppo et al., provide evidence that in contrast to the short 3'UTR AURKA mRNA isoform which appears to be constitutively translated throughout the cell cycle, long 3'UTR AURKA mRNA isoform is preferentially translated in the G2 phase. Further evidence is provided that suppression of long 3'UTR AURKA mRNA isoform is at least in part mediated by hsa-let-7a miRNA. Finally, the authors provide evidence that disrupting the expression of long 3'UTR AURKA mRNA isoform using CRISPR-based strategy, leads to overexpression of AURKA driven by the short 3'UTR isoform which is paralleled by an increase in cancer-related phenotypes.

Strengths: Overall it was thought that this study is of potentially broad interest inasmuch as it delineates a hitherto unappreciated mechanisms of regulation of AURKA protein levels, whereby AURKA is emerging as one of the major factors in neoplasia, including resistance to anti-cancer treatments. In general, it was thought that the author's conclusions were sufficiently supported by provided data. It was also thought that this study incorporates innovative methodology including single-cell expression sensors coupled with live cell microscopy and an assay to study translation in different phases of cell cycle without need for cell synchronization.

Weaknesses: Several relatively minor issues were observed regarding methodology and data interpretation. Namely, some inconsistencies between the models and/or cell lines that were used throughout the manuscript were noted. For instance, key experiments were performed almost exclusively in U2OS osteosarcoma cells, whereby triple negative breast cancer patient data were used to set the scientific foundation of the study. Considering potential differences in alternative polyadenylation between cell and tissue types, it was thought that investigation across the broader compendium of cell lines may be required for generalization of findings observed in U2OS cells. It was also found that the precise mechanisms underpinning the role of hsa-let-7a miRNA in regulation of AURKA protein levels remain largely obscure.

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to compare, from testis tissues at different ages from mice in vivo and after culture, multiple aspects of Leydig cells. These aspects included mRNA levels, proliferation, apoptosis, steroid levels, protein levels, etc. A lot of work was put into this manuscript in terms of experiments, systems, and approaches. However, as written the manuscript is incredibly difficult to follow. The Introduction and Results sections contain rather loosely organized lists of information that were altogether confusing. At the end of reading these sections, it was unclear what advance was provided by this work. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation but fail to appeal to a broader readership. This may be best exemplified by the statements at the end of both the Abstract and Discussion which state that more work needs to be done to improve this system.

Reviewer #1 (Public Review):

In this study the authors sought to address the issue of whether the Steller's sea cow -- a massive extinct sirenian ("sea cow") species that differs from its living relatives (manatees and dugongs) not only in body mass but also in having inhabited cold climates in the northern Pacific -- had hemoglobin adaptations that enhanced the species' thermoregulatory capacities relative to those of the extant species, which are restricted to relatively warm waters. To do so, the authors synthesized recombinant hemoglobin proteins of all the major sea cow lineages and used these data to assess differences in O2 binding, Hb solubility, responses to allosteric effectors, and thermal sensitivity. The work presented is very innovative and in my opinion convincingly demonstrates that the Steller's sea cow had remarkable hemoglobin adaptations that allowed for an extreme range extension into cool waters despite several physiological constraints that are inherent to the sirenian (and paenungulate, afrotherian, etc.) clade. I did not detect any obvious weaknesses of the paper, whereas the use of ancient DNA to resurrect 'extinct' hemoglobins, and the various analyses of these extinct hemoglobins alongside those of extant relatives is very exciting and are major strengths of the paper that make this study a very important advance for our understanding of Steller's sea cow's paleophysiology, as well as our understanding of the potential for extreme hemoglobin phenotypes that have not been documented among living species. Moving forward, these methods can be used to study aspects of the paleophysiology of other recently extinct mammals. I applaud the authors on an excellent and innovative study that significantly augments our understanding of the Steller's sea cow.

Reviewer #1 (Public Review):

This study utilizes scRNA-seq to generate a detailed map of transcriptional changes that occur in asynchronously replicating the Trypanosoma brucei insect (PCF) and mammalian (BSF) stages. The analyses were performed on both fresh and cryo-preserved parasites, and transcriptional changes in PCF compared to existing proteomic datasets at the same stage. This is the first study to comprehensively map cell cycle-related transcriptional changes in T. brucei BSF and to undertake a side-by-side analysis of the two major parasite developmental stages. The study identified >1,500 transcripts that exhibit dynamic changes during the cell cycle across the two stages, substantially increasing the number of cell cycle-regulated (CCR) genes compared to previous analyses. Analysis of the data revealed common as well as stage-specific CCR transcripts and identified transcripts with known/suspected functions in cell cycle regulation as well as hypothetical proteins. The findings also support and quantify previous observations suggesting that most transcript changes (83-86% of CCR transcripts) are not reflected by similar changes in corresponding proteins, and where there is a correlation, protein expression levels expectedly lag behind transcripts. Overall, the study provides the most comprehensive transcriptome atlas of the T. brucei cell cycle undertaken to date, highlighting a large number of genes and cellular processes that are linked to cell cycle progression, while further confirming the importance of post-transcriptional regulatory processes in these divergent eukaryotes. The work represents a significant technical advance, particularly in the validation of the use of cryo-preserved parasites for single-cell RNS-seq, and nicely integrates results from previous proteomics and gene-knockout studies.

Reviewer #1 (Public Review):

The nuclear receptor Nurr1 is a target of interest in neurodegenerative diseases like Parkinson's and Alzheimer's, but its mechanism of activation on NBRE-containing promoters and potential druggability is unknown. A heterodimer of Nurr1 with RXRa can be activated by a subset of ligands that bind to the RXRa ligand binding domain (LBD). Here, the authors provide evidence that transcriptional activation occurs through ligand-induced dissociation of the heterodimer, leading to an active Nurr1 monomer.

NMR spectroscopy and other biophysical, biochemical, and cell-based assays provide a strong foundation for the work. The manuscript is well-written and easy to follow, and for the most part, it thoughtfully addresses experimental results and data interpretation with reasonable caveats. However, a reliance on simple correlative analyses, including some with rather modest correlations (R2 values {less than or equal to} 0.5), may fail to account for some potentially interesting outlier ligands and oversimplify conclusions. Despite this possible oversimplification, this manuscript provides solid evidence of their discovery of an interesting mechanism by which a subset of RXRɑ ligands leads to transcriptional activation of Nurr1 at NBRE promoters--this is an exciting finding that could be potentially relevant in the development of neuroprotective therapies.

Reviewer #1 (Public Review):

In this manuscript, the authors describe a novel HA20-causing missense mutation, p.(Leu236Pro), in three patients from one family with periodic fevers, GI symptoms, urogenital ulcers, arthritis, and pustular rash. The patients had elevated levels of multiple proinflammatory cytokines, including IL-1b, IL-6, and TNFa. Patients had reduced A20 expression levels, and in silico analysis suggested that the p.Leu236Pro mutation destabilized the A20 protein. Using transfection assays, the authors determined that steady state protein expression of mutant A20 protein was lower, and that the half-life of the mutant protein was shorter. Treatment with MG132 increased the half life of mutant A20, suggesting that the mutant protein underwent degradation through the proteasome. Further experiments in the transfection system revealed that mutant A20 failed to suppress TNFα-induced NF-κB activity.

This paper will be of great interest to the field. HA20 is a novel disease (first described in 2016), and although the effects of frameshift/truncating mutations are quite evident, there is quite a lot of debate about the potential effects of missense mutations. It is not really clear which missense mutations cause disease and why, and clinicians who treat this disease are frequently faced with the dilemma of evaluating a patient with a rare missense variant of unknown significance. Thus, a paper that can explain the potential mechanisms by which missense mutations cause disease is highly relevant -- and this is an area of active investigation by several groups.

The strengths of this study include the thorough functional assessment of this novel mutation: the authors have collected quite a lot of data to show the effects of their mutation on protein stability and function. Another strength is the comparison with other similar mutations in the OTU and other domains. However, the data are not currently sufficient to support the conclusions of the authors about the effects of their mutation on protein folding. Similarly, the data do not sufficiently support the generalizability of this mechanism to other mutations in the OTU domain.

Reviewer #1 (Public Review):

Yamanaka et al.'s research investigates into the impact of volatile organic compounds (VOCs), particularly diacetyl, on gene expression changes. By inhibiting histone acetylase (HDACs) enzymes, the authors were able to observe changes in the transcriptome of various models, including cell lines, flies, and mice. The study reveals that HDAC inhibitors not only reduce cancer cell proliferation but also provide relief from neurodegeneration in fly Huntington's disease models. Although the findings are intriguing, the research falls short in providing a thorough analysis of the underlying mechanisms.

HDAC inhibitors have been previously shown to induce gene expression changes as well as control cell division and demonstrated to work on disease models. The authors demonstrate diacetyl as a prominent HDAC inhibitor. Though the demonstration of diacetyl is novel, several similar molecules have been used before.

Reviewer #1 (Public Review):

Asthma is a syndromic disease with multiple subtypes with different pathogenetic paths to a final wheezing phenotype. This limits the insights gleaned from genetic investigations of asthma. One of the most important phenotypes is early life onset wheezing, which persists. Here, the authors use data from multiple birth cohorts and by coupling latent class analysis of clinical phenotypic data with GWAS discovery, identify a novel locus close to annexin 1 (ANXA1) associated exclusively with early-onset persistent wheeze. The methodology is a major strength of the work and highlights the importance of acquiring and analysing phenotypic over simple use of doctor labels for complex diseases.

The authors went on to demonstrate a putative mechanism such that the risk allele (T) may confer a reduction in ANXA1 expression. Altered ANXA1 expression was additionally recapitulated in a murine model of house dust mite (HDM)-induced allergic airway disease. In this model, ANXA1 increased, rather than decreased, which may be attributable to its role in resolving inflammation. ANXA1-deficient mice had a more severe phenotype. This strengthens the evidence for causality in the novel link between ANXA1 and asthma and opens the door for further investigations. While novel for this link, the finding is well supported by prior knowledge about ANXA1-related pathways and inflammation. ANXA1 is known to participate in phospholipase A2-dependent reduction of inflammatory mediator production. Glucocorticoids increase ANXA1 levels. ANXA1 deficiency leads to airway hyperreactivity in mice. Overall, ANXA1 appears to be suitable as a therapeutic target and this may spur further investigations into the pathway.

Reviewer #1 (Public Review):

In their study, Osorio-Valeriano and colleagues seek to understand how bacterial-specific polymerizing proteins called bactofilins contribute to morphogenesis. They do this primarily in the stalked budding bacterium Hyphomonas neptunium, with supporting work in a spiral-shaped bacterium, Rhodospirillum rubrum. Overall the study incorporates bacterial genetics and physiology, imaging, and biochemistry to explore the function of bactofilins and cell wall hydrolases that are frequently encoded together within an operon. They demonstrate an important, but not essential, function for BacA in morphogenesis of H. neptunium. Using biochemistry and imaging, they show that BacA can polymerize and that its localization in cells is dynamic and cell-cycle regulated. The authors then focus on lmdC, which encodes a putative M23 endopeptidase upstream of bacA in H. neptunium, and find that is essential for viability. The purified LmdC C-terminal domain could cleave E. coli peptidoglycan in vitro suggesting that it is a DD-endopeptidase. LmdC interacts directly with BacA in vitro and co-localizes with BacA in cells. To expand their observations, the authors then explore a related endopeptidase/bactofilin pair in R. rubrum; those observations support a function for LmdC and BacA in R. rubrum morphogenesis as well.

An overall strength of this study is the breadth and completeness of approaches used to assess bactofilin and endopeptidase function in cells and in vitro. The authors establish a clear function for BacA in morphogenesis in two bacterial systems, and demonstrate a physical relationship between BacA and the cell wall hydrolase LmdC that may be broadly conserved. The eventual model the authors favor for BacA regulation of morphogenesis in H. neptunium is that it serves as a diffusion barrier and limits movement of morphogenetic machinery like the elongasome into the elongating stalk and/or bud. However, there is no data presented here to address that model and the role of LmdC in H. neptunium morphogenesis remains unclear.

The data presented illuminate aspects of bacterial morphogenesis and the physical and functional relationship between polymerizing proteins and cell wall enzymes in bacteria, a recurring theme in bacterial cell biology with a variety of underlying mechanisms. Bactofilins in particular are relatively recently discovered and any new insights into their functions and mechanisms of action are valuable. The findings presented here are likely to interest those studying bacterial morphogenesis, peptidoglycan, and cytoskeletal function.

Reviewer #1 (Public Review):

The manuscript provides analyses on a very complete dataset on weight and length growth, as well as several physiological markers related to growth, in bonobos. Moreover, there is a good overview of the presence of adolescent growth spurts in non-human primates, by reviewing published data, in comparison to their own dataset. They discuss the need to consider scaling laws when interpreting and comparing growth curves of different species and variables.

The manuscript is very well written, the sample is large, and the methods are well explained. It seems they have analyzed a very complete dataset. Also, the discussion and the references supporting the findings are complete.

The main weakness of this manuscript is that they do not provide a direct comparison with previous analyzed datasets in other species, using their own method (in part maybe because there is not available data, but just published figures).

On the other side, conclusions are well supported by the results, and the previous published datasets are discussed in the manuscript, although not in detail.

Reviewer #1 (Public Review):

For many years it has been understood that transposable elements (TEs) are an important source of natural variation. This is because, in addition to simple knockouts of genes, TEs carry regulatory sequences that can, and sometimes do, affect the expression of genes near the TEs. However, because TEs can be difficult to map to reference genomes, they have generally not been used for trait mapping. Instead, single nucleotide polymorphisms are widely used because they are easy to detect when using short reads. However, improvements in sequencing technology, as well as an increased appreciation of the importance of TEs to both linked to favorable alleles and are more likely to be causing the changes that make those alleles beneficial in a given environment. Further, because TE activity can occur after bottlenecks, they can provide polymorphisms in the absence of variation in point mutations.

In this manuscript, the authors carefully examine insertion polymorphisms in rice and demonstrate linkage to differences in expression. To do this, they used expression quantitative trait locus (eQTL) GWAS using TIPs as genetic markers to examine variation in 208 rice accessions. Because they chose to focus on genes that were expressed in at least 10% of the accessions, presumably because more rare variants would end up lacking statistical power. This is an understandable decision, but it says that recent insertions, such as the MITE elements detailed in a previous paper, would not be included. Importantly, although TIPs associated with differentially expressed genes are far less common than SNPs' traditional eQTLs, there were a significant number of eQTLs that showed linkage to TIPs but not to QTL.

The authors then show that of the eQTLs associated with both TIPs and SNPs, TIPs are more tightly linked to the eQTL, and are more likely to be associated with a reduction in expression, with variation in the effects of various TEs families supporting that hypothesis. Here and throughout, however, the distance of the TEs could be an important variable. It is also worth noting the relative numbers in order to assess the claim in the title of the paper. The total number of eQTL-TIPs is ten-fold less than the number of eQTL-SNPs, and, of the eQTLs that have both, there are a significant number of eQTL-TIPs that are not more tightly linked to the expression differences than the eQTL.

The authors show that eQTL-TIPs are more likely to be in the promoter-proximal region, but this may be due to insertion bias, which is well documented in DNA-type elements. Here and throughout the authors are careful to state that the data is consistent with the hypothesis that TEs are the cause of the change, but do not claim that the data demonstrate that they are.

Throughout the rest of the manuscript, the authors systematically build the case for a causal role for TEs by showing, for instance, that eQTL-TIPs show much stronger evidence for selection, with increased expression being more likely to be selected than decreased expression. The authors provide examples of genes most likely to have been affected by TE insertions.

Overall, the authors build a convincing case for TEs being an important source of regulatory information. I don't have any issues with the analysis, but I am concerned about the sweeping claims made in the title. Once you get rid of eQTLs that could be altered by either SNPs or TIPs and include only those insertions that show strong evidence of selection, the number of genes is reduced to only 30. And even in those cases, the observed linkage is just that, not definitive evidence for the involvement of TEs. Although clearly beyond the scope of this analysis, transgenic constructs with the TEs present or removed, or even segregating families, would have been far more convincing.

The fact that many of the eQTL-TIPs were relatively old is interesting because it suggests that selection in domesticated rice was on pre-existing variation rather than new insertions. This may strengthen the argument because those older insertions are less likely to be purged due to negative effects on gene expression. Given that the sequence of these TEs is likely to have diverged from others in the same family, it would have been interesting to see if selection in favor of a regulatory function had caused these particular insertions to move away from more typical examples of the family.

Reviewer #1 (Public Review):

With this work, the authors address a central question regarding the potential consequences of post-translational modifications for the pathogenesis of neurodegenerative diseases. Phosphorylation and mislocalization of the RNA binding protein TDP43 are characteristic of ~50% of frontotemporal lobar degeneration (FTLD), as well as >95% of amyotrophic lateral sclerosis (ALS). To determine if acetylation is a primary, disease-driving event, they generated a TDP-43 mutant harboring an acetylation-mimicking mutation (K145Q). Animals carrying the acetylation-mimic mutation (K145Q) displayed key pathological features of disease, including more cytoplasmic TDP43 and impaired TDP43 splicing activity, together with behavioral phenotypes reminiscent of FTLD.

This is a well-written and well-illustrated manuscript, with clear and convincing findings. The observations are significant and emphasize the importance of post-translational modifications to TDP-43 function and disease phenotypes. In addition, the TDP43(K145Q) mice may prove to be a valuable model for studying TDP-43-related mechanisms of neurodegeneration and therapeutic strategies.

However, as it stands it is challenging to determine if any or all of the phenotypes are a direct consequence of interrupted RNA binding by TDP-43, rather than acetylation per se. Furthermore, all the results are obtained using an acetylation-mimic mutation that may simply be disrupting a key residue involved in RNA binding by TDP43, instead of mirroring acetylation itself, which in theory is a reversible modification. Lastly, it remains unknown why TDP43(K145Q) mice developed features of FTLD, but not ALS, despite the fact that TDP-43 acetylation was found in ALS tissue and not FTLD.

Reviewer #1 (Public Review):

This manuscript reports the unique finding that specific ligands and receptors in the natriuretic peptide signaling pathway act during early embryogenesis to discriminate between neural crest (NC) and cranial placode (CP) fates. This is a significant finding for two reasons: 1) the developmental role of this pathway has not been studied in any detail; and 2) how cells located in the border between the neural ectoderm and non-neural ectoderm decide on NC versus CP fates is of broad interest and being actively pursued by a number of laboratories. The authors present logical and experimentally convincing support for their conclusions. They report the expression patterns by in situ hybridization and qPCR of the various ligands and receptors of the natriuretic peptide signaling pathway, clearly demonstrating that several of these molecules are expressed in the right place at the right time to influence NC and/or CP formation. They establish that Npr3 is a target of Pax3 and Zic1, two transcription factors previously shown to be required for NC and CP formation, further illustrating that it is part of the appropriate regulatory network. Next, the authors use morpholino knock down of Npr3 to show that the resulting embryos have deficient expression of two NC genes and two CP genes. The controls used for the knock-down are the correct ones and were confirmed by treatment with a high-affinity and selective Npr3 antagonist. The function of Npr3 was further explored by discriminating between its known two functions - clearance of natriuretic peptides and inhibition of adenylyl cyclase - by expressing either WT or mutant versions of human NPR3 in Npr3 morphant embryos. That WT rescued both NC and CP genes but the mutant version only rescued NC genes leads to the appropriate conclusion that Npr3 regulates NC and CP fates via different mechanisms. This conclusion was confirmed by treating Npr3 morphants with a specific adenylyl cyclase inhibitor, which restored CP gene expression, and treating CP promoting explants with an adenylyl cyclase activator, which repressed CP gene expression. Using similar knock-down approaches the authors convincingly demonstrate that Npr2 does not participate in NC/CP formation, but Npr1 does; again, the knock-down results were confirmed by treating embryos with a specific Npr1 antagonist. Finally, the authors complete the story by determining by equally well-controlled knock-down experiments which of the three natriuretic peptides participate in this process. In short, the many different experiments strongly support the conclusions, and the experiments are well controlled and include large numbers of embryos to provide exceptional rigor.

Reviewer #1 (Public Review):

Rapan et al. analyzed the cytoarchitectonic of the prefrontal cortex based on observer-independent analysis, confirming previous parcellations based on cyto-, myelo-, and immunoarchitectonic approaches, but also defining novel subdivisions of areas 10, 9, 8B, and 46 and identified the receptor density "fingerprint" of each area and subdivision. Furthermore, they analyzed the functional connectivity of the prefrontal cortex with caudal frontal, cingulate, parietal, and occipital areas to identify specific features for the various prefrontal subdivisions. Altogether, this study corroborates previous parcellations of the prefrontal cortex, adds new cortical subdivisions, and provides a neurochemical description of the prefrontal areas useful for comparative considerations and for guiding functional and clinical studies.

Strengths:<br /> - This study provides a detailed cytoarchitectonic map of the prefrontal cortex enriched with receptor density and functional connectivity data.<br /> - The authors shared the data via repositories and applied their map to a macaque MRI atlas to further facilitate data sharing.

Weaknesses:<br /> - The temporal cortex should be included in the functional connectivity analysis as it is known from anatomical studies that most prefrontal areas display rich connectivity with temporal areas. The aim of creating a comprehensive view of the frontal cortex makes the manuscript data-rich but cursory in discussing the relevant anatomical and functional literature.

Reviewer #1 (Public Review):

The authors have approached the study of the mechanism of maturation of retroviruses lattice, where Gag polyprotein is the main component. The Gag polyprotein is common to all retroviruses and makes up most of the observed lattice underlying the virion membrane. Within the lattice, 95% of the monomers are Gag, and 5% are Gag-Pol, which has the 6 domains of Gag followed by protease, reverse transcriptase and integrase domains (coming from Pol) embedded within the same polyprotein. For the maturation and infectivity of HIV retrovirus, the Gag proteins within the immature lattice must be cleaved by the protease formed from a dimer of Gag-Pol. Importantly, the lattice covers only 1/3 to 2/3 of the available space on the membrane. The incompleteness of the lattice results in a periphery of Gag monomers with unfulfilled intermolecular contacts. Recently, the structure of the immature lattice has been partially resolved using sub-tomogram averaging cryotomography (cryET) and it has been shown that the incompleteness of the lattice provides more accessible targets for the protease (Tan A. et al. 2021). Based on these, the authors have wondered: does the incompleteness of the lattice allow for dynamic rearrangements that ensure that protease domains embedded within the lattice can find one another to dimerize and activate? To answer this, they started from experimental cryoET data and used reaction-diffusion simulations of assembled Gag lattices with varying energies and kinetic rates to test how lattice structure and stability can support the dimerization of the Gag-Pols. They found that although they represent only 5% of the monomers that assemble into the lattice, the stochastic assembly ensure that at least a pair of them are adjacent within the lattice. They next showed that if the molecules are distant from one another, they would need to detach, diffuse, and reattach stochastically at the site of another Gag-Pol molecule.

I consider the work very interesting, which could contribute to a very important aspect of retroviruses maturation such as their infectivity. However, the observations made by the authors do not necessarily answer their initial question which seemed to be focused on studying the possible role of the incompleteness of the lattice on the protease activation rather than the mechanism of Pol activation itself. Maybe this is only a nuance to be polished in the writing.

The weakness of the work comes from both the fact their entire study has been done by computational methods and the exclusion in their computational approaches of well-known cellular components with a role in retrovirus maturation, which might obey to the fact of keeping their models into the simplest possible since handling atomistic models is already a heavy task. Maybe complementary molecular or structural studies would strengthen their results.

Reviewer #1 (Public Review):

The paper by Mohebi, Collins, and Berke describes the interactions between cholinergic interneurons and dopamine (DA) release in the core of the nucleus accumbens (NAc) in rats. The cholinergic triggering of DA release has been a debated issue in recent years, and this study provides data supporting cholinergic-dependent DA release.

The authors first show that optogenetic activation of cholinergic interneurons (CINs) induces DA release in the NAc, increasing with pulse width, frequency, and train pulse duration. They next show using simultaneous imaging of CIN calcium activity and DA release using RdLight that both are correlated in their response to sensory stimuli and entry to reward port in freely moving rats. They show that while CIN activity and DA release show ramping activity before entry to the center and food ports, such ramping is not seen in the spiking activity of DA cells. lastly, the authors show that blocking nicotinic receptors in the NAc by injection of DHBE impairs task performance, with similar (albeit weaker) effects as the DA antagonist flupenthixol. The uncoupling between DA release and DA cell firing, under certain conditions, has been shown by the authors in a previous paper (Mohebi et al, 2019). Here, the authors add the CINs calcium activity during the same task, showing that the dynamics of CIN activity resemble that of DA release. The results presented show correlations between CIN activity and DA release during behavior, however, the role of CINs in controlling DA release is not tested directly. The data presented in the paper are clear and it is well written. However, there are a few issues that need to be addressed, including some key experiments that could directly test the functional role of CIN-induced DA release.