a violent or sudden change or disruption to something

without embarrassment or shame.

Reviewer #2 (Public Review):

It is recommended to use a blind sample test to determine the specimen's status using the AI they developed.<br /> Where these markers promote tumorigenesis or metastasis if tested in vivo?<br /> The article would be very valuable in the future to promote using AI to predict disease status and facilitate cancer screening.<br /> Much more improvement is required for data validation and presentation.

Reviewer #2 (Public Review):

Summary:<br /> The evolution of resistance to antimalarial drugs follows a seemingly counterintuitive pattern, in which resistant strains typically originate in regions where malaria prevalence is relatively low. Previous investigations have suggested that frequent exposures in high-prevalence regions produce high levels of partial immunity in the host population, leading to subclinical infections that go untreated. These subclinical infections serve as refuges for sensitive strains, maintaining them in the population. Prior investigations have supported this hypothesis; however, many of them excluded important dynamics, and the results cannot be generalized. The authors have taken a novel approach using a deterministic model that includes both general and adaptive immunity. They find that high levels of population immunity produce refuges, maintaining the sensitive strains and allowing them to outcompete resistant strains. While general population immunity contributed, adaptive immunity is key to reproducing empirical patterns. These results are robust across a range of fitness costs, treatment rates, and resistance efficacies. They demonstrate that future investigations cannot overlook adaptive immunity and antigenic diversity.

Strengths:<br /> Overall, this is a very nice paper that makes a significant contribution to the field. It is well-framed within the body of literature and achieves its goal of providing a generalizable, unifying explanation for otherwise disparate investigations. As such, this work will likely serve as a foundation for future investigations. The approach is elegant and rigorous, with results that are supported across a broad range of parameters.

Weaknesses:<br /> Although the title states that the authors describe resistance invasion, they do not support or even explore this claim. As they state in the discussion (line 351), this work predicts the equilibrium state and doesn't address temporal patterns. While refuges in partially immune hosts may maintain resistance in a population, they do not account for the patterns of resistance spread, such as the rapid spread of chloroquine resistance in Africa once it was introduced from Asia.

As the authors state in the discussion, the evolution of compensatory mutations that negate the cost of resistance is possible, and in vitro experiments have found evidence of such. It appears that their results are dependent on there being a cost, but the lower range of the cost parameter space was not explored.

The use of a deterministic, compartmental model may be a structural weakness. This means that selection alone guides the fixation of new mutations on a semi-homogenous adaptive landscape. In reality, there are two severe bottlenecks in the transmission cycle of Plasmodium spp., introducing a substantial force of stochasticity via genetic drift. The well-mixed nature of this type of model is also likely to have affected the results. In reality, within-host selection is highly heterogeneous, strains are not found with equal frequency either in the population or within hosts, and there will be some linkage between the strain and a resistance mutation, at least at first. Of course, there is no recourse for that at this stage, but it is something that should be considered in future investigations.

The authors mention the observation that patterns of resistance in high-prevalence Papua New Guinea seem to be more similar to Southeast Asia, perhaps because of the low strain diversity in Papua New Guinea. However, they do not investigate that parameter space here. If they did and were able to replicate that observation, not only would that strengthen this work, it could profoundly shape research to come.

Reviewer #2 (Public Review):

Summary:<br /> This study seeks to understand the connection between protein sequence and function in disordered regions enriched in polar amino acids (specifically Q, N, S and T). While the authors suggest that specific motifs facilitate protein-enhancing activities, their findings are correlative, and the evidence is incomplete. Similarly, the authors propose that the re-assignment of stop codons to glutamine-encoding codons underlies the greater user of glutamine in a subset of ciliates, but again, the conclusions here are, at best, correlative. The authors perform extensive bioinformatic analysis, with detailed (albeit somewhat ad hoc) discussion on a number of proteins. Overall, the results presented here are interesting, but are unable to exclude competing hypotheses.

Strengths:<br /> Following up on previous work, the authors wish to uncover a mechanism associated with poly-Q and SCD motifs explaining proposed protein expression-enhancing activities. They note that these motifs often occur IDRs and hypothesize that structural plasticity could be capitalized upon as a mechanism of diversification in evolution. To investigate this further, they employ bioinformatics to investigate the sequence features of proteomes of 27 eukaryotes. They deepen their sequence space exploration uncovering sub-phylum-specific features associated with species in which a stop-codon substitution has occurred. The authors propose this stop-codon substitution underlies an expansion of ploy-Q repeats and increased glutamine distribution.

Weaknesses:<br /> The preprint provides extensive, detailed, and entirely unnecessary background information throughout, hampering reading and making it difficult to understand the ideas being proposed.<br /> The introduction provides a large amount of detailed background that appears entirely irrelevant for the paper. Many places detailed discussions on specific proteins that are likely of interest to the authors occur, yet without context, this does not enhance the paper for the reader.

The paper uses many unnecessary, new, or redefined acronyms which makes reading difficult. As examples: (1) Prion forming domains (PFDs). Do the authors mean prion-like domains (PLDs), an established term with an empirical definition from the PLAAC algorithm? If yes, they should say this. If not, they must define what a prion-forming domain is formally. (2) SCD is already an acronym in the IDP field (meaning sequence charge decoration) - the authors should avoid this as their chosen acronym for Serine(S) / threonine (T)-glutamine (Q) cluster domains. Moreover, do we really need another acronym here (we do not). (3) Protein expression-enhancing (PEE) - just say expression-enhancing, there is no need for an acronym here.

The results suggest autonomous protein expression-enhancing activities of regions of multiple proteins containing Q-rich and SCD motifs. Their definition of expression-enhancing activities is vague and the evidence they provide to support the claim is weak. While their previous work may support their claim with more evidence, it should be explained in more detail. The assay they choose is a fusion reporter measuring beta-galactosidase activity and tracking expression levels. Given the presented data they have shown that they can drive the expression of their reporters and that beta gal remains active, in addition to the increase in expression of fusion reporter during the stress response. They have not detailed what their control and mock treatment is, which makes complete understanding of their experimental approach difficult. Furthermore, their nuclear localization signal on the tag could be influencing the degradation kinetics or sequestering the reporter, leading to its accumulation and the appearance of enhanced expression. Their evidence refuting ubiquitin-mediated degradation does not have a convincing control.

Based on the experimental results, the authors then go on to perform bioinformatic analysis of SCD proteins and polyX proteins. Unfortunately, there is no clear hypothesis for what is being tested; there is a vague sense of investigating polyX/SCD regions, but I did not find the connection between the first and section compelling (especially given polar-rich regions have been shown to engage in many different functions). As such, this bioinformatic analysis largely presents as many lists of percentages without any meaningful interpretation. The bioinformatics analysis lacks any kind of rigorous statistical tests, making it difficult to evaluate the conclusions drawn.

The methods section is severely lacking. Specifically, many of the methods require the reader to read many other papers. While referencing prior work is of course, important, the authors should ensure the methods in this paper provide the details needed to allow a reader to evaluate the work being presented. As it stands, this is not the case.

Overall, my major concern with this work is that the authors make two central claims in this paper (as per the Discussion).

The authors claim that Q-rich motifs enhance protein expression. The implication here is that Q-rich motif IDRs are special, but this is not tested. As such, they cannot exclude the competing hypothesis ("N-terminal disordered regions enhance expression"). The authors also do not explore the possibility that this effect is in part/entirely driven by mRNA-level effects (see Verma Na Comms 2019). As such, while these observations are interesting, they feel preliminary and, in my opinion, cannot be used to draw hard conclusions on how N-terminal IDR sequence features influence protein expression. This does not mean the authors are necessarily wrong, but from the data presented here, I do not believe strong conclusions can be drawn.

That re-assignment of stop codons to Q increases proteome-wide Q usage. I was unable to understand what result led the authors to this conclusion. My reading of the results is that a subset of ciliates has re-assigned UAA and UAG from the stop codon to Q. Those ciliates have more polyQ-containing proteins. However, they also have more polyN-containing proteins and proteins enriched in S/T-Q clusters. Surely if this were a stop-codon-dependent effect, we'd ONLY see an enhancement in Q-richness, not a corresponding enhancement in all polar-rich IDR frequencies? It seems the better working hypothesis is that free-floating climate proteomes are enriched in polar amino acids compared to sessile ciliates. Regardless, the absence of any kind of statistical analysis makes it hard to draw strong conclusions here.

Reviewer #2 (Public Review):

In this study, multiple biophysical techniques were employed to investigate the activation mechanism of BTK, a multi-domain non-receptor protein kinase. Previous studies have elucidated the inhibitory effects of the SH3 and SH2 domains on the kinase and the potential activation mechanism involving the membrane-bound PIP3 inducing transient dimerization of the PH-TH domain, which binds to lipids.

The primary focus of the present study was on three new constructs: a full-length BTK construct, a construct where the PH-TH domain is connected to the kinase domain, and a construct featuring a kinase domain with a phosphomimetic at the autophosphorylation site Y551. The authors aimed to provide new insights into the autoinhibition and allosteric control of BTK.

The study reports that SAXS analysis of the full-length BTK protein construct, along with cryoEM visualization of the PH-TH domain, supports a model in which the N-terminal PH-TH domain exists in a conformational ensemble surrounding a compact/autoinhibited SH3-SH2-kinase core. This finding is interesting because it contradicts previous models proposing that each globular domain is tightly packed within the core.

Furthermore, the authors present a model for an inhibitory interaction between the N-lobe of the kinase and the PH-TH domain. This model is based on a study using a tethered complex with a longer tether than a previously reported construct where the PH-TH domain was tightly attached to the kinase domain (ref 5). The authors argue that the new structure is relevant. However, this assertion requires further explanation and discussion, particularly considering that the functional assays used to assess the impact of mutating residues within the PH-TH/kinase domain contradict the results of the previous study (ref 5).

Additionally, the study presents the structure of the kinase domain with swapped activation loops in a dimeric form, representing a previously unseen structure along the trans-phosphorylation pathway. This structure holds potential relevance. To better understand its significance, employing a structure/function approach like the one described for the PH-TH/kinase domain interface would be beneficial.

Overall, this study contributes to our understanding of the activation mechanism of BTK and sheds light on the autoinhibition and allosteric control of this protein kinase. It presents new structural insights and proposes novel models that challenge previous understandings.

Reviewer #2 (Public Review):

Summary:<br /> This paper consists of mostly descriptive data, judged from alpha-mannosidase-treated samples, in which they found an increase in core fucose, a product of Fut 8.

Strengths:<br /> This paper is interesting in the clinical field, but unfortunately, the data is mostly descriptive and does not have a significant impact on the scientific community in general.

Weaknesses:<br /> If core fucose is increased, at least the target glycan molecules of core fucose should be evaluated. They also found an increase in NO, suggesting that inflammatory processes also play an important role in OA in addition to glycan changes.<br /> It has already been reported that core fucose is decreased by administration of alpha-mannosidase inhibitors. Therefore, it is expected that alpha-mannosidase administration increases core fucose.

Reviewer #2 (Public Review):

In this manuscript, the authors aim to study the PML-nucleoli association (PNAs) by different genotoxic stress and to determine the underlying molecular mechanisms.

First, from a diverse set of genotoxic stress conditions (topoisomerases, RNA Pol I, rRNA processing, and DNA replication stress), the authors have found that the inhibition of topoisomerases and RNA Polymerase I has the highest PNA formation associated with p53 stabilization, gamma-H2AX, and PAF49 segregation. It was further demonstrated that Rad51-mediated HR pathway but not NHEJ pathway is associated with the PNA formation. Immuno-FISH assays show that doxorubicin induces DSBs (53BP1 foci) in rDNA and PNA interactions with rDNA/DJ regions. Furthermore, endonuclease I-Ppol induced DSB at a defined location in rDNA and led to PNAs.

Most claims by the authors are supported by the data provided. However, below weaknesses/concerns may need to be addressed to improve the quality of the study.

1) Top2B toxin doxorubicin had the highest degree of elevating PNAs; however, Top2B-knockdown had almost no noticeable effects on PNAs. How to reconcile the different phenotypes targeting Top2B?

2) To test the role of Rad51 and DNA-PKcs in the PNA formation, Rad51 inhibitor B02 and DNA-PKcs inhibitor NU-7441 were chosen to use in the study. To further exclude the possible off-target of B02 and NU-7441, siRNA-mediated knockdown of Rad51 and DNA-PKcs would be an appropriate complementary approach to the pharmaceutical inhibitor approach.

3) Several previous studies have shown the activation of the nucleolar ATM-mediated DNA damage response pathway by I-Ppol-induced DSBs in rDNA. What is the role of nucleolar ATM in the regulation of PNAs?

Reviewer #2 (Public Review):

Summary:<br /> This manuscript by Latini et al describes a methodology to develop Boolean-based predictive logic models that can be applied to uncover altered protein/signalling networks in cancer cells and discover potential new therapeutic targets. As a proof-of-concept, they have implemented their strategy on a hematopoietic cell line engineered to express one of two types of FLT3 internal tandem mutations (FLT3-ITD) found in patients, FLT3-ITD-TKD (which are less sensitive to tyrosine kinase inhibitors/TKIs) and FLT3-ITD-JMD (which are more sensitive to TKIs).

Strengths:<br /> This useful work could potentially represent a step forward towards personalised targeted therapy, by describing a methodology using Boolean-based predictive logic models to uncover altered protein/signalling networks within cancer cells. However, the weaknesses highlighted below severely limit the extent of any conclusions that can be drawn from the results.

Weaknesses:<br /> While the highly theoretical approach proposed by the authors is interesting, the potential relevance of their overall conclusions is severely undermined by a lack of validation of their predicted results in real-world data. Their predictive logic models are built upon a set of poorly-explained initial conditions, drawn from data generated in vitro from an engineered cell line, and no attempt was made to validate the predictions in independent settings. This is compounded by a lack of sufficient experimental detail or clear explanations at different steps. These concerns considerably temper one's enthusiasm about the conclusions that could be drawn from the manuscript. Some specific concerns include:

1. It remains unclear how robust the logic models are, or conversely, how affected they might be by specific initial conditions or priors that are chosen. The authors fail to explain the rationale underlying their input conditions at various points. For example:<br /> - at the start of the manuscript, they assert that they begin with a pre-PKN that contains "76 nodes and 193 edges", though this is then ostensibly refined with additional new edges (as outlined in Fig 2A). However, why these edges were added, nor model performance comparisons against the basal model are presented, precluding an evaluation of whether this model is better.

- At a later step (relevant to Fig S4 and Fig 3), they develop separate PKNs, for each of the mutation models, that contain "206 [or] 208 nodes" and "756 [or] 782 edges", without explaining how these seemingly arbitrary initial conditions were arrived at. Their relation to the original parameters in the previous model is also not investigated, raising concerns about model over-fitting and calling into question the general applicability of their proposed approach. The authors need to provide a clearer explanation of the logic underlying some of these initial parameter selections, and also investigate the biological/functional overlap between these sets of genes (nodes).

2. There is concern about the underlying experimental data underpinning the models that were generated, further compounded by the lack of a clear explanation of the logic. For example, data concerning the status of signalling changes as a result of perturbation appears to be generated from multiplex LUMINEX assays using phosphorylation-specific antibodies against just 14 "sentinel" proteins. However, very little detail is provided about the rationale underlying how these 14 were chosen to be "sentinels" (and why not just 13, or 15, or any other number, for that effect?). How reliable are the antibodies used to query the phosphorylation status? What are the signal thresholds and linear ranges for these assays, and how would these impact the performance/reliability of the logic models that are generated from them?

In addition, there are publicly available quantitative proteomics datasets from FLT3-mutant cell lines and primary samples treated with TKIs. At the very least, these should have been used by the authors to independently validate their models, selection of initial parameters, and signal performance of their antibody-based assays, to name a few unvalidated, yet critical, parameters.

3. There is an overwhelming reliance on theoretical predictions without taking advantage of real-world validation of their findings. For example, the authors identified a set of primary AML samples with relevant mutations (Fig 5) that could potentially have provided a valuable experimental validation platform for their predictions of effective drug combination. Yet, they have performed Boolean simulations of the predicted effects, a perplexing instance of adding theoretical predictions on top of a theoretical prediction!

Additionally, there are datasets of drug sensitivity on primary AML samples where mutational data is also known (for example, from the BEAT-AML consortia), that could be queried for independent validation of the authors' models.

4. There are additional examples of insufficient experimental detail that preclude a fuller appreciation of the relevance of the work. For example, it is alluded that RNA-sequencing was performed on a subset of patients, but the entire methodological section detailing the RNA-seq amounts to just 3 lines! It is unclear which samples were selected for sequencing nor where the data has been deposited (or might be available for the community - there are resources for restricted/controlled access to deidentified genomics/transcriptomics data).

Similarly, in the "combinatory treatment inference" methods, it states "...we computed the steady state of each cell line best model....." and "Then we inferred the activity of "apoptosis" and "proliferation" phenotypes", without explaining the details of how these were done. The outcomes of these methods are directly relevant to Fig 4, but with such sparse methodological detail, it is difficult to independently assess the validity of the presented data.

Overall, the theoretical nature of the work is hampered by real-world validation, and insufficient methodological details limit a fuller appreciation of the overall relevance of this work.

Reviewer #2 (Public Review):

Summary:<br /> Chen Chen et al. investigated the interaction between GHR and PRLR at the cell surface using STORM-type super-resolution microscopy, proximity ligation assay, and mutagenesis. They found that GH and PRL change the surface expression of GHR and PRLR. Upon stimulation, the hGHR cluster size significantly increases in a transient manner, whereas changes in hPRLR occur more slowly. In their previous publication, the authors found that hGHR and hPRLR co-immunoprecipitate in the absence of ligands. Based on that finding and the observations here, the authors examined colocalization of hGHR and hPRLR in clusters with proximity ligation assays and found that the receptors form complexes on the surface of T47D cells, and that these complexes respond differently to the ligands. Remarkably, the experiments in cells lacking either hGHR or hPRLR showed that PRLR is necessary for the reduction of surface hGHR induced by PRL. Studies with truncation or deletion of hPRLR mutants, suggest the box 1 region in hPRLR plays a critical role in stabilizing the hGHR-hPRLR complexes. This region contains the JAK2 binding site, and the authors show that binding of JAK2 to hGHR is also required for hPRLR-mediated regulation of hGHR surface expression. Cytokine receptors have very important broad-ranging roles in regulating cells and physiological roles. Therefore, the new findings described here will significantly expand our understanding of the structure-function relationship that drives a core signalling mechanism in cell biology.

Strengths:<br /> I particularly appreciate that the authors used different angles to examine the mechanism of GHR-PRLR interaction and that they also checked the conclusions with CRISPR/Cas9 technology and with a cellular reconstitution system.

Weaknesses:<br /> I could not fully evaluate some of the data, mainly because several details on acquisition and analysis are lacking. It would be useful to know what the background signal was in dSTORM and how the authors distinguished the specific signal from unspecific background fluorescence, which can be quite prominent in these experiments. Typically, one would evaluate the signal coming from antibodies randomly bound to a substrate around the cells to determine the switching properties of the dyes in their buffer and the average number of localisations representing one antibody. This would help evaluate if GHR or PRLR appeared as monomers or multimers in the plasma membrane before stimulation, which is currently a matter of debate. It would also provide better support for the model proposed in Figure 8. Since many of the findings in this work come from the evaluation of localisation clusters, an image showing actual localisations would help support the main conclusions. I believe that the dSTORM images in Figures 1 and 2 are density maps, although this was not explicitly stated. Alexa 568 and Alexa 647 typically give a very different number of localisations, and this is also dependent on the concentration of BME. Did the authors take that into account when interpreting the results and creating the model in Figures 2 and 8? I believe that including this information is important as findings in this paper heavily rely on the number of localisations detected under different conditions. Including information on proximity labelling and CRISPR/Cas9 in the methods section would help with the reproducibility of these findings by other groups.

Reviewer #2 (Public Review):

Summary:<br /> In their manuscript titled "Stimulation-induced cytokine polyfunctionality as a dynamic concept," the authors investigate the dynamic nature of polyfunctional cytokine responses to established stimulants. The authors use their previously published single-cell encapsulation droplet-microfluidic platform to analyse the response of peripheral blood mononuclear cells (PBMCs) to different stimulants and measure the secretion dynamics of individual cytokines. This assay shows that polyfunctionality in cytokine responses is a complex but short-lived phenomenon that decreases with prolonged stimulation times. The study finds that polyfunctional cells predominantly display elevated cytokine concentrations with similar secretion patterns but higher secretion levels compared to their monocytokine-secreting counterparts. The method is promising to analyse the correlation between the secretion dynamics of different cytokines in primary samples and heterogeneous cell populations.

Strengths:<br /> This method provides single-cell-resolved and dynamic cytokine concentration information, which might be used to identify "fingerprints" of secretion patterns for selected cytokines. When extending the available data to more than one donor, this might be the basis for a diagnostic tool. The combination of established droplet microfluidics with an epi-fluorescence microscope-based readout makes it convincing that the method is transferable to other labs. Specifically, the dynamic analysis of cytokine concentrations is interesting, and the differences or similarities in secretion timepoints might be missed with end-point methods. The authors convincingly show that they detect up to three different cytokines in single cells.

Weaknesses:<br /> The conclusions of the study are based on samples from a single donor, which makes the conclusions on secretion patterns difficult to interpret. The choice of cytokines is explained, but the justification of the groupings of the antibodies into the two panels is missing. It would further be helpful to discuss how the single cell incubation might affect the sectration dynamics vs. the influence of co-culture of all cell types during the 24 h activation. The authors compare average secretion rates and levels. However, the right panel in Fig. 6 looks like there might be two different populations of mono- or polyfuntional cells that have two secretion rates. As the authors have single-cell data, I would find the separation into these populations more meaningful than comparing the mean values. In line with this comment, comparing the mean values for these cytokines instead of the mean of the populations with distinct seretion properties might actually show stronger differences than the authors report here. Is the plateau of the cytokine concentration caused by the fluorescence signal saturating the camera, saturation of the magnetic beads, exhaustion of the fluorescent antibodies, or constant cytokine concentrations? The high number of non-CSCs and the limited number of droplets decrease the statistical power of the method. The authors discuss their choice to use PBMCs and not solely T cells, but this aspect is missing in the discussion.

Reviewer #2 (Public Review):

Summary<br /> In this work, the authors seek to test a version of an old idea, which is that our perception of the world and our understanding of the objects in it are deeply influenced by the nature of our bodies and the kinds of behaviours and actions that those objects afford. The studies presented here muster three kinds of evidence for a discontinuity in the encoding of objects, with a mental "border" between objects roughly of human body scale or smaller, which tend to relate to similar kinds of actions that are yet distinct from the kinds of actions implied by human-or-larger scale objects. This is demonstrated through observers' judgments of the kinds of actions different objects afford; through similar questioning of AI large-language models (LLMs); and through a neuroimaging study examining how brain regions implicated in object understanding make distinctions between kinds of objects at human and larger-than-human scales.

Strengths <br /> The authors address questions of longstanding interest in the cognitive neurosciences -- namely how we encode and interact with the many diverse kinds of objects we see and use in daily life. A key strength of the work lies in the application of multiple approaches, as noted in the summary. Examining the correlations among kinds of objects, with respect to their suitability for different action kinds, is novel, as are the complementary tests of judgments made by LLMs.

Weaknesses <br /> A limitation of the tests of LLMs may be that it is not always known what kinds of training material was used to build these models, leading to a possible "black box" problem. Further, presuming that those models are largely trained on previous human-written material, it may not necessarily be theoretically telling that the "judgments" of these models about action-object pairs show human-like discontinuities. Indeed, verbal descriptions of actions are very likely to mainly refer to typical human behaviour, and so the finding that these models demonstrate an affordance discontinuity may simply reflect those statistics, rather than evidence that affordance boundaries can arise independently even without "organism-environment interactions" as the authors claim here.

The authors include a clever manipulation in which participants are asked to judge action-object pairs, having first adopted the imagined size of either a cat or an elephant, showing that the discontinuity in similarity judgments effectively moved to a new boundary closer to the imagined scale than the veridical human scale. The dynamic nature of the discontinuity suggests a different interpretation of the authors' main findings. It may be that action affordance is not a dimension that stably characterises the long-term representation of object kinds, as suggested by the authors' interpretation of their brain findings, for example. Rather these may be computed more dynamically, "on the fly" in response to direct questions (as here) or perhaps during actual action behaviours with objects in the real world.

Reviewer #2 (Public Review):

Summary:

The present work addresses the mechanisms linking the sex-dependent temporal GH secretion patterns to the robust sex differences in chromatin accessibility and transcription factor binding that ultimately regulate sexually dimorphic liver gene expression. Using DNAseq analysis genomic sites hypersensitive to cleavage by DNase I, DNase hypersensitive sites [DHS] were studied in hepatocytes from male and female mice. DHS in the genome corresponds to accessible chromatin regions and encompasses key regulatory elements, including enhancers, promoters, insulators, and silencers, often flanked by specific histone modifications, and all of these players were described in different settings of GH action. Importantly, the dynamics of sex-dependent and independent chromatin accessibility linked to STAT5 binding were evaluated. For that purpose, hepatic samples from mice were divided into STAT high and STAT low binding by EMSA screening. With this information changes in DHS related to STAT binding were calculated in both sexes, giving an approximation of chromatin opening in response to STAT5, or alternatively to hypophsectomy, or a single GH pulse. More the 800 male-biased DHS (from a total of more than 70000 DHS) regions were identified in the STAT5 high groups, implying that the binding of a plasma GH pulse activates STAT5, and evokes a dynamic cycle of male liver chromatin opening and closing at sites that comprised 31% of all male-biased DHS. This proves that the pulsatility of plasma GH stimulation confers significant male bias in chromatin accessibility, and STAT5 binding at a fraction of the genomic sites linked to sex-biased liver gene expression and liver disease. As a proof of concept, authors show that a single physiological replacement dose or pulse of GH given to hypophysectomized mice recapitulate, within 30 min, the pulsatile re-opening of chromatin seen in pituitary-intact male mouse liver.

In another male-biased DHS set (69% of male-biased DHS), chromatin accessibility was static, that is unchanged across the peaks and valleys of GH-induced liver STAT5 activity and mapped to a set of target genes and processes distinct though sometimes overlapping those of the dynamic male-biased DHS.

In view of these distinct dynamic and static DHS in males, authors evaluated key epigenetic features distinguishing the dynamic STAT5-driven mechanism of chromatin opening from that of static male-biased DHS, which are constitutively open in the male liver but closed in the female liver. The analysis of histone marks enriched at each class of sex-biased DHS indicated exquisite differences in the epigenetic mechanisms that mediate sex-specific gene repression in each sex. For example, H3K27me3 and H3K9me3, two widely used repressive histone marks, are used in a unique way in each sex to enforce sex differences in chromatin states at sex-biased DHS.

Finally, the work recapitulates and explains the classifications of sex dimorphic genes made in previous works. Sex-biased and pituitary hormone-dependent DHS act as regulatory elements with a positive enhancer potential, to induce or maintain gene expression in the intact liver by sustaining an open chromatin in the case of class I male-biased DHS and class I male-biased genes in the male liver. Contrariwise DHS may participate in the inhibition of gene expression by maintaining a closed chromatin state, as in the case of class II male-biased DHS and class II female-biased genes in male liver.<br /> These results as a whole present a complex mechanism by which GH regulates the sexual dimorphism of liver genes in order to cope with the metabolic needs of each sex. In a complete story, the information on chromatin accessibility, histone modification, and transcription factor binding was integrated to elucidate the complex patterns of transcriptional regulation, which is sexually dimorphic in the liver.

Strengths:

The work presents a novel insight into the fundamental underlying epigenetic mechanisms of sex-biased gene regulation.

Results are supported by numerous Tables, and Supplementary Tables with the raw data, which present the advantage that they may be reanalyzed in the future to prove new hypotheses.

Weaknesses:

It is a complicated work to analyze, even though the main messages are clearly conveyed.

Reviewer #2 (Public Review):

Summary:<br /> The study by Cullen et al presents intriguing data regarding the contribution of mTOR complex 1 (mTORC1) versus mTORC2 or both in Pten-null-induced macrocephaly and epileptiform activity. The role of mTORC2 in mTORopathies, and in particular Pten loss-off-function (LOF)-induced pathology and seizures, is understudied and controversial. In addition, recent data provided evidence against the role of mTORC1 in PtenLOF-induced seizures. To address these controversies and the contribution of these mTOR complexes in PtenLOF-induced pathology and seizures, the authors injected a AAV9-Cre into the cortex of conditional single, double, and triple transgenic mice at postnatal day 0 to remove Pten, Pten+Raptor or Rictor, and Pten+raptor+rictor. Raptor and Rictor are essentially binding partners of mTORC1 and mTORC2, respectively. One major finding is that despite preventing mild macrocephaly and increased cell size, Raptor knockout (KO, decreased mTORC1 activity) did not prevent the occurrence of seizures and the rate of SWD event, and aggravated seizure duration. Similarly, Rictor KO (decreased mTORC2 activity) partially prevented mild macrocephaly and increased cell size but did not prevent the occurrence of seizures and did not affect seizure duration. However, Rictor KO reduced the rate of SWD events. Finally, the pathology and seizure/SWD activity were fully prevented in the double KO. These data suggest the contribution of both increased mTORC1 and mTORC2 in the pathology and epileptic activity of Pten LOF mice, emphasizing the importance of blocking both complexes for seizure treatment. Whether these data apply to other mTORopathies due to Tsc1, Tsc2, mTOR, AKT or other gene variants remains to be examined.

Strengths:<br /> The strengths are as follows: 1) they address an important and controversial question that has clinical application, 2) the study uses a reliable and relatively easy method to KO specific genes in cortical neurons, based on AAV9 injections in pups. 2) they perform careful video-EEG analyses correlated with some aspects of cellular pathology.

Weaknesses:<br /> The study has nevertheless a few weaknesses: 1) the conclusions are perhaps a bit overstated. The data do not show that increased mTORC1 or mTORC2 are sufficient to cause epilepsy. However the data clearly show that both increased mTORC1 and mTORC2 activity contribute to the pathology and seizure activity and as such are necessary for seizures to occur. 2) the data related to the EEG would benefit from having more mice. Adding more mice would have helped determine whether there was a decrease in seizure activity with the Rictor or Raptor KO. 3) it would have been interesting to examine the impact of mTORC2 and mTORC1 overexpression related to point #1 above.

Reviewer #2 (Public Review):

The aim of this study was to relate functional alterations in patients with bvFTD to neurotransmitter maps provided by the JuSpace toolbox in order to better understand the underlying pathological mechanisms of this disease.

A strength of the study is the novelty of this aim. Some weaknesses are the different fMRI parameters of patients belonging to each centre and a better explanation of some methodological choices as well a better description of the JuSpace toolbox.

The authors have achieved their aims and the results seem to support some conclusions, although the results should be interpreted in light of a potential lack of proper control for multiple comparisons.

This work will increase the use of approaches that relate brain abnormalities to neurotransmitters and transcriptomics.

There is an increasing trend to assess the correspondence between neuroimaging alterations and detailed information of neurotransmitters across the brain. This work represents this trend and adds to an increasing body of work doing the same with transcriptomics.

Reviewer #2 (Public Review):

The goal of this study is to show that the superficial superior colliculus (sSC) of mouse signals figure-ground differences defined by contrast, orientation, and phase, and that these signals are necessary for the animal to detect such figure-ground differences. By inhibiting sSC while the animals perform a figure-ground detection task, the study shows that detection performance decreases when sSC activity is suppressed during the onset of the visual stimulus. The study then intends to show that sSC neurons exhibit surround suppression based on orientation differences, and that surround suppression is stronger when the animal detects the correct location of the figure on the background.

The major strength of this study is the use of a behavioural paradigm to test detection performance of figure-ground stimuli while manipulating neural activity in the sSC during different times after stimulus onset. This paradigm would show whether activity in the sSC is relevant for performing the task. Secondly, the study collected data to confirm previous findings: sSC neurons exhibit orientation specific surround suppression. Additionally, it is impressive that the authors were able to train mice to generalize their task performance across different stimulus categories (figure-ground differences in orientation and phase). This should be highlighted as it may inform future studies.

The study has, however, methodological and analytical weaknesses so that the stated conclusions are not supported by the presented results.

1) Optogenetic inhibition is not limited to sSC (even expression may not be limited)<br /> About 30% of inhibitory neurons in the sSC project to other areas, e.g. ventral LGN, parabigeminal nucleus and pretectum (Whyland et al, 2019, see ref in manuscript). This means that these areas receive direct inhibition when inhibitory sSC neurons are optogenetically stimulated. This fact is mentioned in the discussion but the consequences and implications for the results are ignored. This is a major flaw of the optogenetic experiments of this study. Additionally, no evidence is given that opsin expression was limited to the superficial layers (except for one histological slice), which the authors acknowledge in line 285. Deeper layers may have other inhibitory neurons with long-range projections.<br /> The finding that sSC neurons show no figure-ground modulation for phase while the optogenetic manipulation has behavioural effects may be an indication for other areas being affected by the optogenetic manipulation.

2) Could other behavioural variables explain the results?<br /> a) Are there any task events other than the visual stimuli that the mice could use to make their decisions? The authors state the use of a custom made lick spout but it is not clear how this spout works, i.e. how do mechanics of the spout deliver water to the right versus the left output and could the mouse perceive these mechanics?<br /> b) Could the different neural responses to figure versus ground shown in Fig 2I-J and Fig 3B be explained by behaviours varying between the trial types, e.g. by early lick movements (which are conceivable even if the spout is not present), eye movements or changes in pupil-linked arousal? A behavioural difference seems even more likely to occur between hit and error/miss trials (Fig 4). If these behaviours were not measured, the possibility of behavioural modulation should be discussed.

3) What is the behavioural strategy of the animals?<br /> Only licks beyond 200 ms after stimulus onset determine the choice of the animal because "mice made early random licks" from 0 to 200 ms. To better understand the behavioural strategies of the animals we need to see their behavioural data, i.e. left and right licks aligned to stimulus onset. It would be particularly interesting to see how number and latency of licks changes during optogenetic manipulation.

4) Data relating to misses should be included in analyses to provide a complete picture of behaviour and neural responses<br /> a) In the optogenetic manipulations, an increase in misses seems to dominate the decreased accuracy (please, explain when a response was counted as a miss). A separate analysis of miss trials may be more robust than of error trials and also offers a different interpretation of the data, namely that the mouse did not see the stimulus rather than perceiving the figure on the opposite side. However, if the mice reduced their lick rate in general during optogenetic stimulation, this begs the question whether their motor performance was affected by optogenetic manipulation. Can this possibility be excluded?<br /> b) Related to Fig 4, it would be equally interesting to see how FGM changes during misses. Do the changes support the observations for error trials?

5) Statistical tests do not support the conclusions, are missing or inadequate<br /> a) In Fig 1E, accuracy is significantly affected at only 1-2 time points in each task, specifically either the 1st and 3rd or the 2nd time point. How do the authors interpret these results? If inhibition starting at the 2nd time point has no significant effects, why would it be significant when inhibition starts later (at the 3rd time)? Furthermore, given that all other starting points of laser stimulation have no significant effects, there is no reason to trust the latency of inhibition effects based on mostly insignificant data points. This analysis in its current form should be removed, including a comparison of latencies between tasks, which was not tested for significance. It may be more meaningful to analyse accuracy for each animal separately. This may reduce variability.<br /> b) Analyses regarding the difference in neural response to figure and ground (Fig 2I-J, Fig 3B, Fig 4B, Fig 5C) would be more convincing and informative if the differences were analysed on the level of single neurons in response to the same orientation within their RF (or at the location where the figure is presented, for edge-RF neurons). A histogram of these differences would show how many neurons are affected and how large the effect is in single neurons.<br /> c) All statistical tests performed across neurons should account for dependencies due to simultaneous recordings (dependency on session) and due to recordings in the same animal (dependency on animal). This can be done in most cases by using linear mixed-effects models.<br /> d) There was no significant difference between model weights (Fig 3D), so the statement in line 210 (RF-edge neurons had higher weights) should be removed.<br /> e) Fig 4B compares FGM during correct and error trials. This comparison has to be performed with the same set of neurons in correct and error trials (not the case for orientation). Again, the most compelling and informative comparison would be on the level of single neurons: response difference between figure and ground (same visual features at figure position) during hits versus errors.<br /> f) There is no evidence that FGM for phase was different between hit and error trials as stated in line 234.<br /> g) It is not clear why and how the mixed linear effects model was used pooling data across tasks (Fig 4C and Fig 5D). Different neurons were recorded for each task, so the sample points (neurons) are not affected by both task effects (orientation and phase). Each task should be analysed separately.<br /> h) Bonferroni correction in Fig 1E should correct multiple comparisons across time points, not across tasks (see Table 1).<br /> i) What is the reason to perform some tests one-tailed, others two-tailed?

6) The results relating to "multisensory neurons" are ambiguous regarding their interpretation (if significant at all) and seem unrelated to the goal of the study. It is particularly likely that behaviours like licking or other movements cause the response differences between figure and ground.

7) What depth were neurons recorded from (Fig 3 and 4)?

DOI: 10.3390/ijms242015225

Resource: ZFIN_ZDB-ALT-040107-2

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-ALT-040107-2

What is this?

Reviewer #2 (Public Review):

Prior results established that Lepr, Calcr, and Cck neurons are non-overlapping neuronal populations in the NTS that individually suppress food intake when activated. This paper examines the consequences of activating or inhibiting two or three of these populations simultaneously. Activating two or three populations inhibits food intake a body weight more than each individually. Activation of Lepr and/or Calcr neurons is not aversive based on the conditioned taste aversion test, whereas activating all three is aversive by this test, indicating that aversion due to Cck neurons activation is dominant. Vertical sleeve gastrectomy (VSG) causes weight loss, but inhibiting each of these neurons individual or all three of them does not prevent weight loss. Overall, this paper provides a solid set of results but does not provide mechanistic insight into any of the phenomena examined.

Reviewer #2 (Public Review):

Summary:<br /> In this study, Wilmot et al., ran a series of experiments to describe a dopaminergic projection from LC to dHPC, and its functional role in trace fear conditioning (TFC). Using fiber photometry in LC, they show convincingly that the activity of LC TH neurons is increased to both cues and footshock, and that this increases with acquisition or TFC, and decreases during extinction of this association. Projections from LC to dHPC show a similar pattern of activity, and dopamine release (measured by the fluorescent sensor GRAB-DA) is also comparable to calcium activity from LC. While the authors do show that activity at the dopamine D1R/D5R is necessary for TFC, a direct test of the necessity of dopamine release from LC during TFC is not shown.

Strengths:<br /> • The authors clearly and effectively show that the LC-dHPC projection is activated by an aversive outcome (i.e. shock), and that activity in this pathway changes in response to learning about a neutral cue that predicts this shock (i.e. TFC). Furthermore, they show that increased dopamine release in dHPC can be observed if LC is chemogenetically activated. A critical role for dopamine receptors (but not β- and ⍺-adrenergic receptors) in TFC was demonstrated, and intra-dHPC injection of a D1R/D5R antagonist blocks this learning. Finally, dopamine release (measured by GRAB-DA) in dHPC was shown to also occur during trace fear conditioning.

• The authors have conclusively shown that activity at the dopamine receptors in the dPHC during trace fear conditioning is of the same pattern as calcium activity recorded both in LC cell bodies, but more importantly in the axonal projections from LC to dHPC. This is very good evidence that this pathway is recruited during TFC.

Weaknesses:<br /> • The claim that dopamine release in dHPC is caused by LC neurons is not directly tested. Unfortunately, the most critical experiment for the claims that dopamine release comes from LC during conditioning is not tested. A lack of dopamine signal in dHPC caused by inhibition of LC during TFC would show this. It is indeed an interesting observation that chemoegenetic activation of LC causes dopamine release in the dHPC. However, in the absence of concurrent VTA inhibition or lesion, it remains a possibility that the dopamine release is mediated through indirect actions on other dopamine-expressing neurons. The authors do a good job of arguing against this interpretation in the discussion, and the literature seems appropriate for this. However, the title is still an overstatement of the data presented in this study.

• The primary alternative interpretations of the phasic activation experiment are whether only stimulation to the cue events (both on and off), or whether only stimulation to the shock. Thus this experiment would benefit from additional data showing either a no shock control, to show that enhanced activity of the LC to the tone is not inherently aversive, or manipulations to the tone but not to the shock.

• Specificity of the GRAB-NE and GRAB-DA sensors should be either justified through additional experiments testing the alternative antagonist (i.e. GRAB-NE CNO+eticloprode / GRAB-DA CNO+yohimbine) or additional citations that have tested this already. It is critical for the claims of the paper to show that these sensors are specific to dopamine or norepinephrine.

• The role of dopamine in prediction error was tested through a series of conditions whereby the shock was presented either signaled (i.e. predicted), or not. However, another way that prediction error is signaled is through the absence of an expected outcome. Admittedly it might not be possible to observe a decrease in dopamine signaling with this methodology.

• The difference between Fig. 6E and 6H needs to be clarified. What is shown in Fig. 6E is that the response to the shock decreases through experience (i.e. by the 10th trial). However in Fig 6H, there is no difference between signaled and signaled shock, but this is during conditioning, and not after learning (based on my understanding of the methods, line 482).

• Unless I missed it, at no point in the manuscript is the number of subjects described. Please add the n per experiment within each section describing each experiment in the methods (Behavioral procedures). Some more details in the photometry statistical analysis would be helpful. For example, what is the n per group for every data set that is presented? How many trials per analysis?

Reviewer #2 (Public Review):

In the revised manuscript, the authors aim to investigate brain-wide activation patterns following administration of the anesthetics ketamine and isoflurane, and conduct comparative analysis of these patterns to understand shared and distinct mechanisms of these two anesthetics. To this end, they perform Fos immunohistochemistry in perfused brain sections to label active nuclei, use a custom pipeline to register images to the ABA framework and quantify Fos+ nuclei, and perform multiple complementary analyses to compare activation patterns across groups. This is an interesting line of research and a tour de force in brain-wide Fos quantification.

I appreciate many of the changes that were made in the revised manuscript, including FDR correction and transparency in showing their results with and without transformation. However, several key issues described in our first review have not been addressed.

1-Aside from issues with their data transformation (see below), (a) I think they have some interesting Fos counts data in Figures 4B and 5B that indicate shared and distinct activation patterns after KET vs. ISO based anesthesia. These data are far closer to the raw data than PC analyses and need to be described and analyzed in the first figures long before figures with the more abstracted PC analyses. In other words, you need to show the concrete raw data before describing the highly transformed and abstracted PC analyses. (b) This gets to the main point that when selecting brain areas for follow up analyses, these should be chosen based on the concrete Fos counts data, not the highly transformed and abstracted PC analyses.

2-Now, the choice of data transformation for Fos counts is the most significant problem. First, the authors show in the response letter that not using this transformation (region density/brain density) leads to no clustering. However, they also showed the region-densities without transformation (which we appreciate) and it looks like overall Fos levels in the control group Home (ISO) are a magnitude (~10-fold) higher than those in the control group Saline (KET) across all regions shown. This large difference seems unlikely to be due to a biologically driven effect and seems more likely to be due to a technical issue, such as differences in staining or imaging between experiments. Was the Homecage-ISO experiment or at least the Fos labeling and imaging performed at the same time as for the Saline-Ketamine experiment? Please state the answer to this question in the Results section one way or the other.

3-Second, they need to deal with this large difference in overall staining or imaging for these two (Home/ISO and Saline/KET) experiments more directly; their current normalization choice does not really account for the large overall differences in mean values and variability in Fos counts (e.g. due to labeling and imaging differences).

3a-I think one option (not perfect but I think better than the current normalization choice) could be z-scoring each treatment to its respective control. They can analyze these z-scored data first, and then in later figures show PC analyses of these data and assess whether the two treatments separate on PC1/2. And if they don't separate, then they don't separate, and you have to go with these results.

3b-Alternatively, they need to figure out the overall intensity distributions from the different runs (if that the main reason of markedly different counts) and adjust their thresholds for Fos-positive cell detection based on this. I would expect that the saline and HC groups should have similar levels of activation, so they could use these as the 'control' group to determine a Fos-positive intensity threshold that gets applied to the corresponding 'treatment' group.

3c- If neither 3a nor 3b is an option then they need to show the outcomes of their analysis when using the untransformed data in the main figures (the untransformed data plots in their responses to reviewer are currently not in the main or supplementary figs) and discuss these as well.

This suggests to me that there is a lot of proprietary layer 2-3 in IOT. Is this the case?

Reviewer #2 (Public Review):

Summary:<br /> This work tests the hypothesis that water coordination in WNK kinases is linked to allosteric control of activity. It is proposed that dimeric WNK is inactive and bound to some conserved water molecules, and that monomerization/activation involves departure of these waters. New data here include a crystal structure of monomeric WNK1 which shows missing waters compared to the dimeric structure, in support of the hypothesis. Mutant proteins of a different isozyme (WNK3) designed to disrupt water coordination were produced, and activity and quaternary structure were measured. The results with WNK3 do not clearly support or refute the hypothesis as there is no systematic correlation between mutations designed to disrupt water coordination and activity or quaternary structure.

Strengths:<br /> The most interesting result presented here is that P1 crystals of WNK1 convert to P21 in the presence of PEG400 and still diffract (rather than being destroyed as the crystal contacts change, as one would expect). All of the assays for activity and osmolyte sensing are carried out well.

Weaknesses:<br /> The rationale for using WNK3 for the mutagenesis study is that it is more sensitive to osmotic pressure than WNK1. I think that WNK1 would have been a better platform because of the direct correlation to the structural work leading to the hypothesis being tested. All of the crystallographic work is WNK1; it is not logical to jump to WNK3 without other practical considerations.

Osmolyte sensing was tested by measuring ATP consumption as a function of PEG400 (Figure 6). Data for the subset of mutants analyzed by this assay showed increasing activity. It is not clear why the same collection of mutant proteins analyzed in the experiments of Figure 5 was not also measured for osmolyte sensing in Figure 6.

The last set of data presented uses light scattering to test whether the WNK3 mutant proteins exhibit quaternary structural changes consistent with the monomer/dimer hypothesis. If they did, one would expect a higher degree of monomer for those that are activated by mutation, and a lower amount of monomer (like wt) for those that are not. Instead, one of the mutant proteins that showed the most chloride inhibition (Y346F) had a quaternary structure similar to the wt protein, and others have similar monomer/dimer mixtures but distinct chloride inhibition profiles (K307A and M301A). I don't see how the light scattering data contribute to this story other than to refute the hypothesis by showing a lack of correlation between quaternary structure, water binding, and activity. This is another reason why the disconnect between WNK1 and WNK3 could be a problem. All of the detailed structural work with WNK1 must be assumed with WNK3; perhaps the light scattering data are contradicting this assumption?

Reviewer #2 (Public Review):

Summary:

The manuscript's main claim is that the absence of H2-O, a component of the MHC II presentation pathway, promotes regulatory T cell development and function.

Unfortunately, the submitted material is not sufficient for proper evaluation of the manuscript, both in terms of the significance of the findings and the strength of the supporting evidence.

Major issues include:

- the scRNAseq (shown in Fig. 5) is too rudimentary to allow any conclusion. Statements in the text (eg "Principle Component Analysis (PCA) of the normalized scRNA-seq data identified 11 distinct CD4 T cell clusters", line 166) suggest that additional expertise should be leveraged for these analyses.

- Most flow cytometry data (Figs. 1 and 2) shows marginal (at best) differences on y-axis truncated bar graphs, with no original data plot, gating strategies, etc., severely challenging conclusions drawn from this data.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Glaser et al. describe a new selective plane illumination microscope designed to image a large field of view that is optimized for expanded and cleared tissue samples. For the most part, the microscope design follows a standard formula that is common among many systems (e.g. Keller PJ et al Science 2008, Pitrone PG et al. Nature Methods 2013, Dean KM et al. Biophys J 2015, and Voigt FF et al. Nature Methods 2019). The primary conceptual and technical novelty is to use a detection objective from the metrology industry that has a large field of view and a large area camera. The authors characterize the system resolution, field curvature, and chromatic focal shift by measuring fluorescent beads in a hydrogel and then show example images of expanded samples from mouse, macaque, and human brain tissue.

Strengths:<br /> I commend the authors for making all of the documentation, models, and acquisition software openly accessible and believe that this will help assist others who would like to replicate the instrument. I anticipate that the protocols for imaging large expanded tissues (such as an entire mouse brain) will also be useful to the community.

Weaknesses:<br /> The characterization of the instrument needs to be improved to validate the claims. If the manuscript claims that the instrument allows for robust automated neuronal tracing, then this should be included in the data.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Zhang and Speer examine changes in the spatial organization of synaptic proteins during eye-specific segregation, a developmental period when axons from the two eyes initially mingle and gradually segregate into eye-specific regions of the dorsal lateral geniculate. The authors use STORM microscopy and immunostain presynaptic (VGluT2, Bassoon) and postsynaptic (Homer) proteins to identify synaptic release sites. Activity-dependent changes in this spatial organization are identified by comparing the β2KO mice to WT mice. They describe two types of presynaptic organization based on Bassoon clustering, the complex and the simple synapse. By analyzing the relative densities and distances between these proteins over age, the authors conclude that the complex synapses promote the clustering of simple synapses nearby to form the future mature glomerular synaptic structure.

Strengths:<br /> The data presented is of good quality and provides an unprecedented view at high resolution of the presynaptic components of the retinogeniculate synapse during active developmental remodeling. This approach offers an advance to the previous mouse EM studies of this synapse because of the CTB label allows identification of the eye from which the presynaptic terminal arises. Using this approach, the authors find that simple synapses cluster close to complex synapses over age, that complex synapse density increases with age.

Weaknesses:<br /> From these data, the authors conclude that the complex synapse serves to "promote clustering of like-eye synapses and prohibit synapse clustering from the opposite eye". However, the authors show no causal data to support these ideas. There are a number of issues that the authors should consider:

1. Clustering of retinal synapses is in part due to the fact that retinal inputs synapse on the proximal dendrites. With increased synaptogenesis, there will be increased density of retinal terminals that are closely localized. And with development, perhaps simple synapses mature into complex synapses. Simple synapses may also represent ones that are in the process of being eliminated as previously described by Campbell and Shatz, JNeurosci 1992 (consider citing). Can the authors distinguish these scenarios from the ones that they conclude?

2. The argument that "complex" synapses are the aggregate of "simple" synapses (Fig 2, S2) is not convincing.

3. The authors use of the β2KO mice to assess changes in the organization of synaptic proteins in retinal terminals that have disrupted retinal waves. However, β2-nAChRs are also expressed in the dLGN and other areas of the brain and glutamatergic synapse development has been reported in the CNS independent of the disruption in retinal waves. This issue should be considered when interpreting the total reduced retinal synapse density in the dLGN of the mutant.

4. Outside of a total synapse density difference between WT and β2KO mice, the changes in the spatial organization of synaptic proteins over development do not seem that different. In fact % simple synapses near complex synapses from the non-dominant eye in the mutant is not that different from WT at P8 (Fig 3C), an age when eye-specific segregation is very different between the genotypes. Can the authors explain this discrepancy?

5. The authors use nomenclature that has been previously used and associated with other aspects of retinogeniculate properties. For example, the phrases "simple" and "complex" synapses have been used to describe single boutons or aggregates of boutons from numerous retinal axons, whereas in this manuscript the phrases are used to describe vesicle clusters/release sites with no knowledge of whether they are from single or multiple boutons. Likewise, the use of the word "glomerulus" has been used in the context of the retinogeniculate synapse to refer to a specific pattern of bouton aggregates that involves inhibitory and neuromodulatory inputs. It is not clear how the release sites described by the authors fit in this picture. Finally the use of the word "punishment" is associated with a body of literature regarding the immune system and retinogeniculate refinement-which is not addressed in this study. This double use of the phrases can lead to confusion in the field and should be clarified by clear definitions of how they are used in the current study.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript examines how humans walk over uneven terrain using vision to decide where to step. There is a huge lack of evidence about this because the vast majority of locomotion studies have focused on steady, well-controlled conditions, and not on decisions made in the real world. The author team has already made great advances in this topic, but there has been no practical way to map 3D terrain features in naturalistic environments. They have now developed a way to integrate such measurements along with gaze and step tracking, which allows quantitative evaluation of the proposed trade-offs between stepping vertically onto vs. stepping around obstacles, along with how far people look to decide where to step.

Strengths:<br /> 1. I am impressed by the overarching outlook of the researchers. They seek to understand human decision-making in real-world locomotion tasks, a topic of obvious relevance to the human condition but not often examined in research. The field has been biased toward well-controlled studies, which have scientific advantages but also serious limitations. A well-controlled study may eliminate human decisions and favor steady or periodic motions in laboratory conditions that facilitate reliable and repeatable data collection. The present study discards all of these usually-favorable factors for rather uncontrolled conditions, yet still finds a way to explore real-world behaviors in a quantitative manner. It is an ambitious and forward-thinking approach, used to tackle an ecologically relevant question.

2. There are serious technical challenges to a study of this kind. It is true that there are existing solutions for motion tracking, eye tracking, and most recently, 3D terrain mapping. However most of the solutions do not have turn-key simplicity and require significant technical expertise. To integrate multiple such solutions together is even more challenging. The authors are to be commended on the technical integration here.

3. In the absence of prior studies on this issue, it was necessary to invent new analysis methods to go with the new experimental measures. This is non-trivial and places an added burden on the authors to communicate the new methods. It's harder to be at the forefront in the choice of topic, technical experimental techniques, and analysis methods all at once.

Weaknesses:<br /> 1. I am predisposed to agree with all of the major conclusions, which seem reasonable and likely to be correct. Ignoring that bias, I was confused by much of the analysis. There is an argument that the chosen paths were not random, based on a comparison of probability distributions that I could not understand. There are plots described as "turn probability vs. X" where the axes are unlabeled and the data range above 1. I hope the authors can provide a clearer description to support the findings. This manuscript stands to be cited well as THE evidence for looking ahead to plan steps, but that is only meaningful if others can understand (and ultimately replicate) the evidence.

2. I wish a bit more and simpler data could be provided. It is great that step parameter distributions are shown, but I am left wondering how this compares to level walking. The distributions also seem to use absolute values for slope and direction, for understandable reasons, but that also probably skews the actual distribution. Presumably, there should be (and is) a peak at zero slope and zero direction, but absolute values mean that non-zero steps may appear approximately doubled in frequency, compared to separate positive and negative. I would hope to see actual distributions, which moreover are likely not independent and probably have a covariance structure. The covariance might help with the argument that steps are not random, and might even be an easy way to suggest the trade-off between turning and stepping vertically. This is not to disregard the present use of absolute values but to suggest some basic summary of the data before taking that step.

3. Along these same lines, the manuscript could do more to enable others to digest and go further with the approach, and to facilitate interpretability of results. I like the use of a neural network to demonstrate the predictiveness of stepping, but aside from above-chance probability, what else can inform us about what visual data drives that? Similarly, the step distributions and height-turn trade-off curves are somewhat opaque and do not make it easy to envision further efforts by others, for example, people who want to model locomotion. For that, clearer (and perhaps) simpler measures would be helpful.

I am absolutely in support of this manuscript and expect it to have a high impact. I do feel that it could benefit from clarification of the analysis and how it supports the conclusions.

Reviewer #2 (Public Review):

Summary:<br /> Seignette et al. investigated the potential roles of axo-axonic (chandelier) cells (ChCs) in a sensory system, namely visual processing. As introduced by the authors, the axo-axonic cell type has remained (and still is) somehow mysterious in its function. Seignette and colleagues leveraged the development of a transgenic mouse line selective for ChC, and applied a very wide range of techniques: transsynaptic rabies tracing, optogenetic input activation, in vitro electrophysiology, 2-photon recording in vivo, behavior and chemogenetic manipulations, to precisely determine the contribution of ChCs to the primary visual cortex network.

The main findings are 1) the identification of synaptic inputs to ChC, with a majority of local, deep layer principal neurons (PN), 2) the demonstration that ChC is strongly and synchronously activated by visual stimuli with low specificity in naive animals, 3) the recruitment of ChC by arousal/visuomotor mismatch, 4) the induction of functional and structural plasticity at the ChC-PN module, and, 5) the weak disinhibition of PNs induced by ChCs silencing. All these findings are strongly supported by experimental data and thoroughly compared to available evidence.

Strengths:<br /> This article reports an impressive range of very demanding experiments, which were well executed and analyzed, and are presented in a very clear and balanced manner. Moreover, the manuscript is well-written throughout, making it appealing to future readers. It has also been a pleasure to review this article.

In sum, this is an impressive study and an excellent manuscript, that presents no major flaws.

Notably, this study is one of the first studies to report on the activities and potential roles of axo-axonic cells in an active, integrated brain process, beyond locomotion as reported and published in V1. This type of research was much awaited in the fields of interneuron and vision research.

Weaknesses:<br /> There are no fundamental weaknesses; the latter mainly concern the presentation of the main results.

The main weakness may be that the different sections appear somehow disconnected conceptually.

Additionally, some parts deserve a more in-depth clarification/simplification of concepts and analytic methods for scientists outside the subfield of V1 research. Indeed, this paper will be of key interest to researchers of various backgrounds.

Reviewer #2 (Public Review):

Summary:<br /> The work by Klaassen & Rasch investigates the influence of word learning difficulty on sleep-associated consolidation and reactivation. They elicited reactivation during sleep by applying targeted memory reactivation (TMR) and manipulated word learning difficulty by creating words more similar (easy) or more dissimilar (difficult) to our language. In one group of participants, they applied TMR of easy words and in another group of participants, they applied TMR of difficult words (between-subjects design). They showed that TMR leads to higher memory benefits in the easy compared to the difficult word group. On a neural level, they showed an increase in spindle power (in the up-state of an evoked response) when easy words were presented during sleep.

Strengths:<br /> The authors investigate a research question relevant to the field, that is, which experiences are actually consolidated during sleep. To address this question, they developed an innovative task and manipulated difficulty in an elegant way.

Overall, the paper is clearly structured, and results and methods are described in an understandable way. The analysis approach is solid.

Weaknesses:<br /> 1.Sample size<br /> For a between-subjects design, the sample size is too small (N = 22). The main finding (also found in the title "Difficulty in artificial word learning impacts targeted memory reactivation") is based on an independent samples t-test with 11 participants/group.

The authors explicitly mention the small sample size and the between-subjects design as a limitation in their discussion. Nevertheless, making meaningful inferences based on studies with such a small sample size is difficult, if not impossible.

2.Choice of task<br /> Even though the task itself is innovative, there would have been tasks better suited to address the research question. The main disadvantage the task and the operationalisation of memory performance (d') have is that single-trial performance cannot be calculated. Consequently, choosing individual items for TMR is not possible.

Additionally, TMR of low vs. high difficulty is conducted between subjects (and independently of pre-sleep memory performance) which is a consequence of the task design.

The motivation for why this task has been used is missing in the paper.

Reviewer #2 (Public Review):

Summary:<br /> The authors describe the structure-functional relationship of domains in S. pombe CAF-1, which promotes DNA replication-coupled deposition of histone H3-H4 dimer. The authors nicely showed that the ED domain with an intrinsically disordered structure binds to histone H3-H4, that the KER domain binds to DNA, and that, in addition to a PIP box, the KER domain also contributes to the PCNA binding. The ED and KER domains as well as the WHD domain are essential for nucleosome assembly in vitro. The ED, KER domains, and the PIP box are important for the maintenance of heterochromatin.

Strengths:<br /> The combination of structural analysis using NMR and Alphafold2 modeling with biophysical and biochemical analysis provided strong evidence on the role of the different domain structures of the large subunit of SpCAF-1, spPCF-1 in the binding to histone H3-H4, DNA as well as PCNA. The conclusion was further supported by genetic analysis of the various pcf1 mutants. The large amounts of data provided in the paper support the authors' conclusion very well.

Reviewer #2 (Public Review):

The manuscript deals with an interesting topic in metabolism: the so-called underground metabolism enabled by enzymes with broad substrate specificity. This is mainly relevant in secondary metabolisms. The authors deal, in particular, with the conversion of flavonoids, which have health-promoting effects. They present an algorithm for predicting the moonlight activities of enzymes, which must be given as inputs. Moreover, the authors performed experiments on the antioxidant activities of the flavonoids under study.

My focus was on the bioinformatics part. Overall, the bioinformatics part is not a major scientific achievement in my eyes, or it is too poorly described to see its merits. There may be difficulties understanding the presented algorithm.

Comments:

The prediction algorithm should be explained much better. Although the manuscript is quite long, it does not describe the approaches sufficiently well. It is quite hard to read.

As far as I can see, the method was only tested with a small sample of different flavonoid substances.

Major comments<br /> (1) I see the following contradiction. Line 18/19: "As flavonoids are not natural substrates of gut bacterial enzymes" and lines 76/77: "commensal gut microorganisms do not have specialized enzymes that utilize flavonoids as their native substrates" versus lines 72-74: "flavonoids ..., which makes them available to be metabolized". How can they be metabolized given what is said in the first two phrases?<br /> (2) It should be explained better what is meant by "reaction class" (e.g. in lines 97 and 99). Is this the same as the EC number (in the Enzyme Catalogue)? The term "reaction class" is indeed used in the KEGG database. On the webpage<br /> https://www.genome.jp/brite/br08204<br /> it seems indeed as if the terms "reaction class" and EC number are somehow equivalent. However, the term "RClass RC00392" in line 557 of the manuscript points to a difference in meaning.<br /> (3) The prediction algorithm should be explained much better. For example, in the Figure showing the workflow, it is shown that an EC number should be given as input. However, if we search for enzymes which could potentially degrade a given flavonoid, we may not know any suitable EC number. Line 122: "To match a given enzyme with its non-native polyphenolic substrates..." However, where can we take the enzyme name/EC number from? Moreover, given that it is assumed that the reaction is performed by underground metabolism, should the enzyme given as input come from another organism, for example, a plant?<br /> (4) Lines 521-523: Our prediction tool can take either a single enzyme in the form of Enzyme Commission (EC) number (e.g. "ec:2.1.1.75"), or a KEGG organism-identifier (e.g. "cpv") or a consortium, a list of different organism-identifiers, as input." I do not understand the wording "or a consortium". According to the Figure showing the workflow, it should read "and a consortium".<br /> (5) In the Materials and Methods section, the KEGG PATHWAY database is mentioned. This comes somewhat out of the blue. What is the connection to the "reaction class" concept in KEGG? Or is the PATHWAY database only used for extracting the negative controls?<br /> (6) Line 142,143. "Our analysis shows that RClass-based similarity can predict the correct reactions for known flavonoid-metabolizing enzymes". How do the authors know that the results are correct? If it is easy to check, then I assume the test whether a given enzyme is able to catalyze reactions with flavonoids can be done manually in KEGG, so that a computer algorithm is unnecessary.<br /> (7) Elaborating on the previous point - I have the impression that the algorithm is a rather simple search routine for finding reactions in the KEGG database that match certain criteria. This might be a helpful tool to save time in comparison to doing the search manually. However, at least the bioinformatics part of the paper is not a major scientific achievement as far as I can see.<br /> (8) It is not sufficiently clear whether the prediction algorithm only works for the example shown in the top figure (tilianin, acacetin etc), which would be quite a restricted application, or for many or even all flavonoids. In line 565, the authors say: "our tabulated 312 unique flavonoids", while in the upper part of the MS, e.g. in lines 26 and 109, only the pathway starting from tilianin is mentioned.<br /> (9) In which programming language was the algorithm implemented?<br /> (10) The connection between the theoretical and experimental parts of the paper is not fully clear. Some of the experiments serve to test the predictions, which is fine. The experiments on free radicals, however, seem to be somewhat unrelated.

Reviewer #2 (Public Review):

Summary:<br /> The study is titled "Leading an urban invasion: risk-sensitive learning is a winning strategy", and consists of three different parts. First, the authors analyse data on initial and reversal learning in Grackles confronted with a foraging task, derived from three populations labeled as "core", "middle" and "edge" in relation to the invasion front. The suggested difference between study populations does not surface, but the authors do find moderate support for a difference between male and female individuals. Secondly, the authors confirm that the proposed mechanism can actually generate patterns such as those observed in the Grackle data. In the third part, the authors present an evolutionary model, in which they show that learning strategies as observed in male Grackles do evolve in what they regard as conditions present in urban environments.

Strengths:<br /> The manuscript's strength is that it combines real learning data collected across different populations of the Great-tailed grackle (Quiscalus mexicanus) with theoretical approaches to better understand the processes with which grackles learn and how such learning processes might be advantageous during range expansion. Furthermore, the authors also take sex into account revealing that males, the dispersing sex, show moderately better reversal learning through higher reward-payoff sensitivity. I also find it refreshing to see that the authors took the time to preregister their study to improve transparency, especially regarding data analysis.

Weaknesses:<br /> One major weakness of this manuscript is the fact that the authors are working with quite low sample sizes when we look at the different populations of edge (11 males & 8 females), middle (4 males & 4 females), and core (17 males & 5 females) expansion range. Although I think that when all populations are pooled together, the sample size is sufficient to answer the questions regarding sex differences in learning performance and which learning processes might be used by grackles but insufficient when taking the different populations into account.

Another weakness of this manuscript is that it does not set up the background well in the introduction. Firstly, are grackles urban dwellers in their natural range and expand by colonising urban habitats because they are adapted to it? The introduction also fails to mention why urban habitats are special and why we expect them to be more challenging for animals to inhabit. If we consider that one of their main questions is related to how learning processes might help individuals deal with a challenging urban habitat, then this should be properly introduced.

Also, the authors provide a single example of how learning can differ between populations from more urban and more natural habitats. The authors also label the urban dwellers as the invaders, which might be the case for grackles but is not necessarily true for other species, such as the Indian rock agama in the example which are native to the area of study. Also, the authors need to be aware that only male lizards were tested in this study. I suggest being a bit more clear about what has been found across different studies looking at: (1) differences across individuals from invasive and native populations of invasive species and (2) differences across individuals from natural and urban populations.

Finally, the introduction is very much written with regard to the interaction between learning and dispersal, i.e. the 'invasion front' theme. The authors lay out four predictions, the most important of which is No. 4: "Such sex-mediated differences in learning to be more pronounced in grackles living at the edge, rather than the intermediate and/or core region of their range." The authors, however, never return to this prediction, at least not in a transparent way that clearly pronounces this pattern not being found. The model looking at the evolution of risk-sensitive learning in urban environments is based on the assumption that urban and natural environments "differ along two key ecological axes: environmental stability 𝑢 (How often does optimal behaviour change?) and environmental stochasticity 𝑠 (How often does optimal behaviour fail to pay off?). Urban environments are generally characterised as both stable (lower 𝑢) and stochastic (higher 𝑠)". Even though it is generally assumed that urban environments differ from natural environments the authors' assumption is just one way of looking at the differences which have generally not been confirmed and are highly debated. Additionally, it is not clear how this result relates to the rest of the paper: The three populations are distinguished according to their relation to the invasion front, not with respect to a gradient of urbanization, and further do not show a meaningful difference in learning behaviour possibly due to low sample sizes as mentioned above.

In conclusion, the manuscript was well written and for the most part easy to follow. The format of having the results before the methods makes it a bit harder to follow because the reader is not fully aware of the methods at the time the results are presented. It would, therefore, be important to more clearly delineate the different parts and purposes. Is this article about the interaction between urban invasion, dispersal, and learning? Or about the correct identification of learning mechanisms? Or about how learning mechanisms evolve in urban and natural environments? Maybe this article can harbor all three, but the borders need to be clear. The authors need to be transparent about what has and especially what has not been found, and be careful to not overstate their case.

Reviewer #2 (Public Review):

In their manuscript, Kato et al investigate a key aspect of membrane protein quality control in plant photosynthesis. They study the turnover of plant photosystem II (PSII), a hetero-oligomeric membrane protein complex that undertakes the crucial light-driven water oxidation reaction in photosynthesis. The formidable water oxidation reaction makes PSII prone to photooxidative damage. PSII repair cycle is a protein repair pathway that replaces the photodamaged reaction center protein D1 with a new copy. The manuscript addresses an important question in PSII repair cycle - how is the damaged D1 protein recognized and selectively degraded by the membrane-bound ATP-dependent zinc metalloprotease FtsH in a processive manner? The authors show that oxidative post-translational modification (OPTM) of the D1 N-terminus is likely critical for the proper recognition and degradation of the damaged D1 by FtsH. Authors use a wide range of approaches and techniques to test their hypothesis that the singlet oxygen (1O2)-mediated oxidation of tryptophan 14 (W14) residue of D1 to N-formylkynurenine (NFK) facilitates the selective degradation of damaged D1. Overall, the authors propose an interesting new hypothesis for D1 degradation and their hypothesis is supported by most of the experimental data provided. The study certainly addresses an elusive aspect of PSII turnover and the data provided go some way in explaining the light-induced D1 turnover. However, some of the data are correlative and do not provide mechanistic insight. A rigorous demonstration of OPTM as a marker for D1 degradation is yet to be made in my opinion. Some strengths and weaknesses of the study are summarized below:

Strengths:

1. In support of their hypothesis, the authors find that FtsH mutants of Arabidopsis have increased OPTM, especially the formation of NFK at multiple Trp residues of D1 including the W14; a site-directed mutation of W14 to phenylalanine (W14F), mimicking NFK, results in accelerated D1 degradation in Chlamydomonas; accelerated D1 degradation of W14F mutant is mitigated in an ftsH1 mutant background of Chlamydomonas; and that the W14F mutation augmented the interaction between FtsH and the D1 substrate.

2. Authors raise an intriguing possibility that the OPTM disrupts the hydrogen bonding between W14 residue of D1 and the serine 25 (S25) of PsbI. According to the authors, this leads to an increased fluctuation of the D1 N-terminal tail, and as a consequence, recognition and binding of the photodamaged D1 by the protease. This is an interesting hypothesis and the authors provide some molecular dynamics simulation data in support of this. If this hypothesis is further supported, it represents a significant advancement.

3. The interdisciplinary experimental approach is certainly a strength of the study. The authors have successfully combined mass spectrometric analysis with several biochemical assays and molecular dynamics simulation. These, together with the generation of transplastomic algal cell lines, have enabled a clear test of the role of Trp oxidation in selective D1 degradation.

4. Trp oxidative modification as a degradation signal has precedent in chloroplasts. The authors cite the case of 1O2 sensor protein EXECUTER 1 (EX1), whose degradation by FtsH2, the same protease that degrades D1, requires prior oxidation of a Trp residue. The earlier observation of an attenuated degradation of a truncated D1 protein lacking the N-terminal tail is also consistent with authors' suggestion of the importance of the D1 N-terminus recognition by FtsH. It is also noteworthy that in light of the current study, D1 phosphorylation is unlikely to be a marker for degradation as posited by earlier studies.

Weaknesses:

1. The study lacks some data that would have made the conclusions more rigorous and convincing. It is unclear why the level of Trp oxidation was not analyzed in the Chlamydomonas ftsH 1-1 mutant as done for the var 2 mutant. Increased oxidation of W14 OPTM in Chlamydomonas ftsH 1-1 is a key prediction of the hypothesis. It is also unclear to me what is the rationale for showing D1-FtsH interaction data only for the double mutant but not for the single mutant (W14F). Why is the FtsH pulldown of D2 not statistically significant (p value = {less than or equal to}0.1). Wouldn't one expect FtsH pulls down the RC47 complex containing D1, D2, and RC47. Probing the RC47 level would have been useful in settling this. A key proposition of the authors' is that the hydrogen bonding between D1 W14 and S25 of PsbI is disrupted by the oxidative modification of W14. Can this hypothesis be further tested by replacing the S25 of PsbI with Ala, for example?

2. Although most of the work described is in vivo analysis, which is desirable, some in vitro degradation assays would have strengthened the conclusions. An in vitro degradation assay using the recombinant FtsH and a synthetic peptide encompassing D1 N-terminus with and without OPTM will test the enhanced D1 degradation that the authors predict. This will also help to discern the possibility that whether CP43 detachment alone is sufficient for D1 degradation as suggested for cyanobacteria.

3. The rationale for analyzing a single oxidative modification (W14) as a D1 degradation signal is unclear. D1 N-terminus is modified at multiple sites. Please see Mckenzie and Puthiyaveetil, bioRxiv May 04 2023. Also, why is modification by only 1O2 considered while superoxide and hydroxide radicals can equally damage D1?

4. The D1 degradation assay seems not repeatable for the W14F mutant. High light minus CAM results in Fig. 3 shows a statistically significant decrease in D1 levels for W14F at multiple time points but the same assay in Fig. 4a does not produce a statistically significant decrease at 90 min of incubation. Why is this? Accelerated D1 degradation in the Phe mutant under high light is key evidence that the authors cite in support of their hypothesis.

5. The description of results at times is not nuanced enough, for e.g. lines 116-117 state "The oxidation levels in Trp-14 and Trp-314 increased 1.8-fold and 1.4-fold in var2 compared to the wild type, respectively (Fig. 1c)" while an inspection of the figure reveals that modification at W314 is significant only for NFK and not for KYN and OIA. Likewise, the authors write that CP43 mutant W353F has no growth phenotype under high light but Figure S6 reveals otherwise. The slow growth of this mutant is in line with the earlier observation made by Anderson et al., 2002. In lines 162-163, the authors talk about unchanged electron transport in some site-directed mutants and cite Fig. 2c but this figure only shows chl fluorescence trace and nothing else.

6. The authors rightly discuss an alternate hypothesis that the simple disassembly of the monomeric core into RC47 and CP43 alone may be sufficient for selective D1 degradation as in cyanobacteria. This hypothesis cannot yet be ruled out completely given the lack of some in vitro degradation data as mentioned in point 2. Oxidative protein modification indeed drives the disassembly of the monomeric core (Mckenzie and Puthiyaveetil, bioRxiv May 04 2023).

Reviewer #2 (Public Review):

The ATPase protein machine cohesin shapes the genome by loop extrusion and holds sister chromatids together by topological entrapment. When executing these functions, cohesin is tightly regulated by multiple cofactors, such as Scc2/Nipbl, Pds5, Wapl, and Eco1/Esco1/2, and it undergoes dynamic conformational changes with ATP binding and hydrolysis. The mechanisms by which cohesin extrudes DNA loops and medicates siter-chromatid cohesion are still not understood. A major reason for the lack of understanding of cohesin dynamics and regulation is the failure to capture the structures of intact cohesin in different nucleotide-bound states and in complex with various regulators. So far only the ATP state cohesin bound to NIPBL and DNA have been experimentally determined.

In this manuscript, Nasmyth et al. made use of the powerful protein structure prediction tool, AlphaFold2 (AF), to predict the models of tens of cohesin subcomplexes from different species. The results provide important insight into how the Smc3-Scc1 DNA exiting gate is opened, how Pds5 and Wapl maintain the opened gate, how Pds5 and Scc3/SA recruit different cofactors, how Eco1 and Sororin antagonize Wapl, and how Scc2/Nipbl interacts with Scc3/SA. The models are for the most part consistent with published mutations in these proteins that affect cohesin's functions in vitro and in vivo and raise testable hypotheses of cohesin dynamics and regulation. This study also serves as an example of how to use AF to build models of protein complexes that involve the docking of flexible regions to globular domains.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript focuses on a persistent question of why germline mutations in BRCA1 which impair homology-directed repair of DNA double-strand breaks predispose to primarily breast and ovarian cancers but not other tissues. The authors propose that replication stress is elevated in the luminal progenitor (LP) cells and apply the gene signature from Dreyer et al as a measure of replication stress in populations of cells selected by FACS previously (published by Lim et al.) and suggest an enrichment of replication stress among the LP cells. This is followed by single-cell RNA seq data from a small number of breast tissues from a small number of BRCA1 mutation carriers but the pathogenic variants are not listed. The authors perform an elegant analysis of the effects of BRCA1 knockdown in MCF10A cells, but these cells are not considered a model of LP cells.

Overall, the manuscript suffers from significant gaps and leaps in logic among the datasets used. The connection to luminal progenitor cells is not adequately established because the models used are not representative of this population of cells. Therefore, the central hypothesis is not sufficiently justified.

Strengths:<br /> The inducible knockdown of BRCA1 provided compelling data pointing to an upregulation of ELF3 in this setting as well as a small number of other genes. It would be useful to discuss the other genes for completeness and explain the logic for focusing on ELF3. Nonetheless, the connection with ELF 3 is reasonable. The authors provide significant data showing a role for ELF3 in breast epithelial cells and its role in cell survival.

Weaknesses:<br /> The initial observations in primary breast cells have small sample sizes. The mutations in BRCA1 seem to be presumed to be all the same, but we know that pathogenic variants differ among individuals and range from missense mutations affecting interactions with one critical partner to large-scale truncations of the protein.

The figure legends are missing critical details that make it difficult for the reader to evaluate the data. The data support the notion that ELF3 may participate in relieving replication stress, but does not appear to be limited to LP cells as proposed in the hypothesis.

Reviewer #2 (Public Review):

Summary:<br /> This study utilizes a large series of neurulation human embryos to address several questions about the similarities and differences between human neurulation and model systems such as the chicken and rodent.

Strengths:<br /> The number of specimens utilized for the analysis provides robustness to the findings.

Weaknesses:<br /> It is not clear how the gestational age of the specimens was determined or how that can be known with certainty. There is no information given in the methods on this. With this in mind, bunching the samples at 2-day intervals in Figure 1J will lead to inaccuracies in assessing the rate of somite formation. This is pointed out as a major difference between specimens and organoids in the abstract but a similar result in the results section. The data supporting either of these statements is not convincing.

Whenever possible, give the numbers of specimens that had the described findings. For example, in Figure 2C - how many embryos were examined with the massive rounded end at CS13? Apoptosis in Figures 3 and 4?

For Figure 2I-K, it would be informative to superimpose the individual data points on the box plots distinguishing males from females, as in Figure 1I.

Is it possible to quantitate apoptosis and proliferation data?

The Tunel staining in Figure 3 is difficult to make out.

Additional improvements to the presentation of figures, writing, and quantization of results are suggested.

Reviewer #2 (Public Review):

Summary:<br /> This work performs structural analysis on isolated or purified rootlets.

Strengths:<br /> To date, most studies of this cellular assembly have been from fluorescence microscopy, conventional TEM methods, or through biochemical analysis of constituents. It is clearly a challenging target for structural analysis due to its complexity and heterogeneity. The authors combine observations from cryo-electron tomograms, automated segmentations, subtomogram averaging, and previous data from the literature to present an overall model of how the rootlet is organised.

Their model will serve as a jumping-off point for future studies, and as such it is something of considerable value and interest.

Weaknesses:<br /> It is speculative but is presented as such, and is well-reasoned, plausible, and thorough.

https://en.wikipedia.org/wiki/Shmita

During shmita, the land is left to lie fallow and all agricultural activity, including plowing, planting, pruning and harvesting, is forbidden by halakha (Jewish law).

The sabbath year (shmita; Hebrew: שמיטה, literally "release"), also called the sabbatical year or shǝvi'it (שביעית‎, literally "seventh"), or "Sabbath of The Land", is the seventh year of the seven-year agricultural cycle mandated by the Torah in the Land of Israel and is observed in Judaism.

Reviewer #2 (Public Review):

Joshi et al. investigated the use of dantrolene, an RyR stabilizing drug, in improving contractile function and slowing pathological progression of pressure-overload heart failure. In a guinea pig model, they found that dantrolene treatment reduced cytosolic Ca2+ levels, improved contractility, reduced the incidence of arrhythmias, reduced fibrosis, and slowed the progression of heart failure. Importantly, delaying treatment until 3 weeks after aortic banding (when heart failure was already established) also resulted in improvements in function and decreased arrhythmogenesis. While some of the mechanistic details remain to be worked out, the data suggest that improving intracellular Ca2+ handling can break the vicious cycle of sympathetic activation, ROS production, and further deterioration of cardiac function.

The functional ECG and echo data are convincing, and very clearly demonstrate the positive effects of dantrolene in heart failure. This is important because dantrolene is already FDA-approved to treat malignant hyperthermia and muscle spasms, so repurposing this drug as a heart failure therapeutic might have a straightforward path to clinical implementation. This also highlights the non-specific nature of dantrolene to interact with RyR1, and therefore, potential side effects. However, this does not detract from the main proof-of-concept demonstrated here.

The guinea pig model employed here is also a strength, as the guinea pig has intracellular Ca2+ handling and ionic currents that are much more similar to human (vs. a murine model, for example).

One weakness is the exclusion of female animals from the study. The authors report more heterogeneity in the progression of HF in the female guinea pig model, however it will be very important to determine effects of dantrolene in the female heart, as there are considerable known sex differences in intracellular Ca2+ handling and contractility. Therefore, it is possible that dantrolene could have sex-dependent effects.

Reviewer #2 (Public Review):

Summary:<br /> This study reports the levels of expression of selected genes implicated in Wnt signaling in trabecular bone from femur heads obtained after surgery from post-menopausal women with (15 women) or without (21 women) type 2 diabetes. They found higher expression levels of SOST and WNT5A, and lower expression levels of LEF-1 and WNT10B in tissues from subjects with T2D, correlating with glycemia and advanced glycation products. No significant differences in bone density were observed. Overall, this is a cross-sectional, observational study measuring a limited set of genes found to vary with glycemia in postmenopausal women undergoing hip surgery.

Strengths:<br /> The study demonstrates the feasibility of measuring gene expression in post-surgical trabecular bone samples, and finds differences associated with glycemia despite a relatively small number of subjects. It can form the basis for further research on the causes and consequences of changes in elements of the WNT signaling pathway in bone biology and disease.

Weaknesses:<br /> The small number of targeted genes does not provide a comprehensive view of the transcriptional landscape within which the effects are observed. The gene expression changes are not associated with cellular or physiological properties of the tissue, raising questions about the biological significance of the observations.

Reviewer #2 (Public Review):

In the manuscript by Yang et al titled "Mecp2 fine-tunes quiescent exit by targeting nuclear receptors", the authors found that Mecp, a well-known protein because of its crucial role in neurological disorders, has a cell cycle-dependent ability to negatively regulate quiescent exit by transcriptional activation of metabolic genes while repressing proliferation-related genes. Conceptually, this is an interesting study with very well-executed experiments and controls.

Since the mutation of MeCP2 was identified as the cause of Rett syndrome, the previous reports have been focused on the exhaustive biochemical and functional characterization of this protein. In this study, the authors show that MeCP2 expression is cell-cycle related, and acute reduction of Mecp2 is essential for efficient quiescence exit in cells. They also identified a novel E3 ligase Nedd4 contributes to Mecp2 degradation during G0 exit. These findings are the first description of MeCP2 protein expression during the cell cycle. The variation in MeCP2 levels at different stages of the cell cycle phases should be taken into consideration when examining MeCP2-related disordered disease.

Reviewer #2 (Public Review):

In this study the authors sought to investigate how the metabolic state of iNKT cells impacts their potential pathological role in allergic asthma. The authors used two mouse models, OVA and HDM-induced asthma, and assessed genes in glycolysis, TCA, B-oxidation and FAS. They found that acetyl-coA-carboxylase 1 (ACC1) was highly expressed by lung iNKT cells and that ACC1 deficient mice failed to develop OVA-induced and HDM-induced asthma. Importantly, when they performed bone marrow chimera studies, when mice that lacked iNKT cells were given ACC1 deficient iNKT cells, the mice did not develop asthma, in contrast to mice given wildtype NKT cells. In addition, these observed effects were specific to NKT cells, not classic CD4 T cells. Mechanistically, iNKT cell that lack AAC1 had decreased expression of fatty acid-binding proteins (FABPs) and peroxisome proliferator-activated receptor (PPAR)γ, but increased glycolytic capacity and increased cell death. Moreover, the authors were able to reverse the phenotype with the addition of a PPARg agonist. When the authors examined iNKT cells in patient samples, they observed higher levels of ACC1 and PPARG levels, compared to healthy donors and non-allergic-asthma patients.

Reviewer #2 (Public Review):

This work provides a new tool (H3-Opt) for the prediction of antibody and nanobody structures, based on the combination of AlphaFold2 and a pre-trained protein language model, with a focus on predicting the challenging CDR-H3 loops with enhanced accuracy than previously developed approaches. This task is of high value for the development of new therapeutic antibodies. The paper provides an external validation consisting of 131 sequences, with further analysis of the results by segregating the test sets into three subsets of varying difficulty and comparison with other available methods. Furthermore, the approach was validated by comparing three experimentally solved 3D structures of anti-VEGF nanobodies with the H3-Opt predictions

Strengths:<br /> The experimental design to train and validate the new approach has been clearly described, including the dataset compilation and its representative sampling into training, validation and test sets, and structure preparation. The results of the in silico validation are quite convincing and support the authors' conclusions.

The datasets used to train and validate the tool and the code are made available by the authors, which ensures transparency and reproducibility, and allows future benchmarking exercises with incoming new tools.

Compared to AlphaFold2, the authors' optimization seems to produce better results for the most challenging subsets of the test set.

Weaknesses:<br /> The scope of the binding affinity prediction using molecular dynamics is not that clearly justified in the paper.

Some parts of the manuscript should be clarified, particularly the ones that relate to the experimental validation of the predictions made by the reported method. It is not absolutely clear whether the experimental validation is truly a prospective validation. Since the methodological aspects of the experimental determination are not provided here, it seems that this may not be the case. This is a key aspect of the manuscript that should be described more clearly.

Some Figures would benefit from a more clear presentation.

Reviewer #2 (Public Review):

The work presented in this manuscript focuses on the role of Cylicins in spermiogenesis and the consequences of their absence on infertility. The manuscript is presented in two parts: the first part studies the absence of Cylicins from KO mouse models and shows in mice that both isoforms of Cylicins are necessary for normal spermiogenesis. The evaluation of double heterozygotes is particularly useful for the second part which looks at the presence of mutations in these genes in a cohort of infertile men. A patient with two hemizygous/heterozygous mutations in the CYLC1 and 2 genes, respectively, was identified for the first time and the results obtained with the KO models support the hypothesis of the pathogenicity of the mutations.

In general, the experiments are perfectly performed and the results are clear. Numerous techniques in the state of the art in male reproduction are used to obtain high-quality phenotyping of the mouse models.

The discovery of two concomitant mutations in an infertile patient is very interesting and the work carried out on mice allows supporting that an absence of CYLC1 and a heterozygous mutation of CYLC2 could lead to a phenotype of complete infertility. However, as the mutation on CYLC2 is not identified as pathogenic, the pathogenicity of this mutation remains in question (the authors note this point in the discussion). It would be interesting to see if the mutated amino acid is conserved between different species. In mice, the authors have shown the importance of these proteins on the morphology of the acrosome. What about in humans?

Reviewer #2 (Public Review):

Summary:<br /> In this study, Olivieri & Wang et.al. probe the role of the conserved alphaC-beta4 loop in the allosteric regulation of the PKA catalytic subunit. The authors employ a combination of NMR-restrained molecular dynamics simulations and mutational analysis to uncover the conformational transitions between distinct excited states and identify a pivotal role for the alphaC-beta4 loop in facilitating these conformational transitions. These studies support previous models proposing the alphaC-beta4 loop as a critical element in kinase conformational regulation. Overall, this is a timely and fitting study.

Strengths:<br /> 1. Exciting application of NMR and MD to explore hidden conformation states of kinases.<br /> 2. Novel mechanistic insights into the role of the alphaC-beta4 loop in PKA.

Weaknesses:<br /> 1. While the alphaC-beta4 loop is a conserved feature of protein kinases, the residues within this loop vary across various kinase families and groups, enabling group and family-specific control of activity through cis and trans acting elements. F102 in PKA interacts with co-conserved residues in the C-tail, which has been proposed to function as a cis regulatory element. The authors should elaborate on the conformational changes in the C-tail, particularly in the arginine that packs against F102, in the results and discussion. This would further extend the impact and scope of the manuscript, which is currently confined to PKA.<br /> 2. The MD data and conformational states would be a valuable resource for the community and should be shared via some open-source repositories.<br /> 3. The authors state that ES1 and ES2 states are novel and not observed in previous crystal structures. The authors should quantify this through comparisons with PKA inactive states and with other AGC kinases.<br /> 4. Based on the results, can the authors speculate on the impact of oncogenic mutations in the alphaC-beta4 loop mutations in PKA?

Massebah for pagan worship: - Exodus 23:24 (https://hypothes.is/a/r3m5QmyDEe6SC8eLYcJE1Q) - Hosea 10:1 (https://hypothes.is/a/4PK2GGyDEe6wZg_r2YpVCA ) - 2 Kings 18:4 - 2 Kings 23:14

Massebah for worship of Yahweh: - Genesis 28:18 Jacob's pillow (https://hypothes.is/a/NF5p8Gx6Ee65Rg_J4tfaMQ)<br /> - Genesis 31:44-45 Jacob and Laban's covenant - Exodus 24:4 - Joshua 24:25-27

Link to https://hypothes.is/a/r3m5QmyDEe6SC8eLYcJE1Q

In neighboring cultures in which both have oral practices relating to massebah, one is not just destroying "sacred stones" to stamp out their religion, but it's also destroying their culture and cultural memory as well as likely their laws and other valuable memories for the function of their society.

View this in light also of the people of Israel keeping their own sacred stones (Hosea 10:1) as well as the destruction of pillars dedicated to Baal in 2 Kings 18:4 and 2 Kings 23:14.

(Link and) Compare this to the British fencing off the land in Australia and thereby destroying Songlines and access to them and the impact this had on Indigenous Australians.

It's also somewhat similar to the colonialization activity of stamping out of Indigenous Americans and First Nations' language in North America, though the decimation of their language wasn't viewed in as reciprocal way as it might be viewed now. (Did colonizers of the time know about the tremendous damage of language destruction, or was it just a power over function?)

Use of massebah for remembrance of a name...

Potentially used for other factors? translation? context?

See also: https://hypothes.is/a/oqgH4mx9Ee68_dMgihgD0A (Rachel's massebah in Genesis 35:19-20)

Reviewer #2 (Public Review):

The authors sought to define the molecular structure of autoinhibited Kinesin-1, which is the major kinesin providing plus-end directed transport on microtubules. The paper reports a structural model of full-length kinesin-1 which builds on the known folded conformation of kinesin-1 and describes its autoinhibitory mechanism using cryo-EM, alphafold structural predictions, cross-linking, and mass spectrometry. The authors study the conformation of dimeric Kinesin Heavy Chain (KHC) and tetrameric KHC bound to the Kinesin Light Chains (KLCs), where KLC stabilizes the autoinhibited conformation. The combination of these various approaches leads to an integrated molecular model of autoinhibited Kinesin-1. Until now, there was some debate over the role of the small coiled-coil 3 (a and b) and where the hinge region of Kinesin-1. The authors resolve this question and present data indicating the hinge is between cc3a and cc3b.

In some places the absence of crosslinks is reported as a lack of interaction, however, it could also be that there are no residues that can be crosslinked in this region. Some crosslinks also are too long to satisfy the model, so it is possible, while most crosslinks occur when Kinesin-1 is inhibited, that a small number of crosslinks arise from when Kinesin-1 adopts another conformation. The structural data are supported by single-molecule motility assays with various mutants of Kinesin-1, which greatly help characterising the domains functionally.

Overall there are some interesting novel data on the autoinhibitory mechanism of Kinesin-1, with well performed and analyzed data with KLC and TRAP. The topic and paper will be of interest to many.

Reviewer #2 (Public Review):

Summary:<br /> In eukaryotes, sterols are crucial for signaling and regulating membrane fluidity, however, the mechanism governing cholesterol production and transport across the cell membrane in bacteria remains enigmatic. The manuscript by Zhai et al. sheds light on this topic by uncovering three potential cholesterol transport proteins. Through comprehensive bioinformatics analysis, the authors identified three genes bstA, bstB, and bstC encoding proteins which share homology with transporters, periplasmic binding proteins, and periplasmic components superfamily, respectively. Furthermore, the authors confirmed the specific interaction between these three proteins and C-4 methylated sterols and determined the structures of BstB and BstC. Combining these structural insights with molecular dynamics simulation, they postulated several plausible substrate binding sites within each protein.

Strengths:<br /> The authors have identified 3 proteins that seem likely to be involved in sterol transport between the inner and outer membrane. The structures are of high quality, and the sterol binding experiments support a role for these proteins in sterol transport.

Weaknesses:<br /> While the author's model is very plausible, direct evidence for a role of BstABC in transport, or that the 3 proteins function together in a single pathway, is limited.

Reviewer #2 (Public Review):

Summary:

Recent advances in single-cell profiling of gene expression (RNA) permit the analysis of specialized cell types, an approach that has great value in the nervous system which is characterized by prodigious neuronal diversity. The novel data in this study focus primarily on genetic profiling to compare autonomic neurons from ganglia associated with the cranial parasympathetic outflow (sphenopalatine (also known as pteropalatine), the thoraco-lumbar sympathetic outflow (stellate, coeliac) and the sacral parasympathetic outflow (pelvic). Using statistical methods to reduce the dimensionality of the data and map gene expression, the authors provide interesting evidence that cranial parasympathetic and sacral sympathetic ganglia differ from each other and from sympathetic ganglia (Figures 1, S1 - S4). The authors interpret the mapping analysis as evidence that the cranial and sacral outflows differ so calling them both parasympathetic is unjustified. Based on anatomical localization of markers (Figure 2 ) (mainly transcription factors) the authors show a similarity between the sympathetic and pelvic ganglion. In Figure 3 they present evidence that some pelvic ganglionic neurons are dually innervated by sympathetic preganglionic neurons and sacral preganglionic neurons. These observations are interpreted to mean that the pelvic ganglion is not parasympathetic, but rather a modified sympathetic ganglion - hence the title of the manuscript.

Strengths:<br /> The extensive use of single-cell profiling in this work is both interesting and exciting. Although still in its early stages, it holds promise for a deepened understanding of autonomic development and function. As noted in the introduction, this study extends previous work by Professor Brunet and his associates.

Weaknesses:<br /> This work further documents differences between the cranial and sacral parasympathetic outflows that have been known since the time of Langley - 100 years ago. The approach taken by Brunet et al. has focused on late neonatal and early postnatal development, a time when autonomic function is still maturing. In addition, the sphenopalatine and other cranial ganglia develop from placodes and the neural crest, while sympathetic and sacral ganglia develop from the neural crest alone. How then do genetic programs specifying brainstem and spinal development differ and how can this account for kinship that Brunet documents between spinal and sacral ganglia? One feature that seems to set the pelvic ganglion apart is the mixture of 'sympathetic' and 'parasympthetic' ganglion cells and the convergence of preganglionic sympathetic and parasympathetic synapses on individual ganglion cells (Figure 3). This unusual organization has been reported before using microelectrode recordings (see Crowcroft and Szurszewski, J Physiol (1971) and Janig and McLachlan, Physiol Rev (1987)). Anatomical evidence of convergence in the pelvic ganglion has been reported by Keast, Neuroscience (1995). It should also be noted that the anatomy of the pelvic ganglion in male rodents is unique. Unlike other species where the ganglion forms a distributed plexus of mini-ganglia, in male rodents the ganglion coalesces into one structure that is easier to find and study. Interestingly the image in Figure 3A appears to show a clustering of Chat-positive and Th-positive neurons. Does this result from the developmental fusion of mini ganglia having distinct sympathetic and parasympathetic origins? In addition, Brunet et al dismiss the cholinergic and noradrenergic phenotypes as a basis for defining parasympathetic and parasympathetic neurons. However, see the bottom of Figure S4 and further counterarguments in Horn (Clin Auton Res (2018)). What then about neuropeptides, whose expression pattern is incompatible with the revised nomenclature proposed by Brunet et al.? Figure 1B indicates that VIP is expressed by sacral and cranial ganglion cells, but not thoracolumbar ganglion cells. The authors do not mention neuropeptide Y (NPY). The immunocytochemistry literature indicates that NPY is expressed by a large subpopulation of sympathetic neurons but never by sacral or cranial parasympathetic neurons.

Reviewer #2 (Public Review):

In this study, the authors aimed to evaluate the contribution of brain-age indices in capturing variance in cognitive decline and proposed an alternative index, brain-cognition, for consideration.

The study employs suitable methods and data to address the research questions, and the methods and results sections are generally clear and easy to follow.

Comments on revised submission:

I appreciate the authors' efforts in significantly improving the paper, including some considerable changes, from the original submission. While not all reviewer points were tackled, the majority of them were adequately addressed. These include additional analyses, more clarity in the methods and a much richer and nuanced discussion. While recognising the merits of the revised paper, I have a few additional comments.

Perhaps it would help the reader to note that it might be expected for brain-cognition to account for a significantly larger variance (11%) in fluid cognition, in contrast to brain-age. This stems from the fact that the authors specifically trained brain-cognition to predict fluid cognition, the very variable under consideration. In line with this, the authors later recommend that researchers considering the use of brain-age should evaluate its utility using a regression approach. The latter involves including a brain index (e.g. brain-cognition) previously trained to predict the regression's target variable (e.g. fluid cognition) alongside a brain-age index (e.g., corrected brain-age gap). If the target-trained brain index outperforms the brain-age metric, it suggests that relying solely on brain-age might not be the optimal choice. Although not necessarily the case, is it surprising for the target-trained brain index to demonstrate better performance than brain-age? This harks back to the broader point raised in the initial review: while brain-age may prove useful (though sometimes with modest effect sizes) across diverse outcomes as a generally applicable metric, a brain index tailored for predicting a specific outcome, such as brain-cognition in this case, might capture a considerably larger share of variance in that specific context but could lack broader applicability. The latter aspect needs to be empirically assessed.

Furthermore, the discussion pertaining to training brain-age models on healthy populations for subsequent testing on individuals with neurological or psychological disorders seems somewhat one-sided within the broader debate. This one-sidedness might potentially confuse readers. It is worth noting that the choice to employ healthy participants in the training model is likely deliberate, serving as a norm against which atypical populations are compared. To provide a more comprehensive understanding, referencing Tim Hans's counterargument to Bashyam's perspective could offer a more complete view (https://academic.oup.com/brain/article/144/3/e31/6214475?login=false).

Overall, this paper makes a significant contribution to the field of brain-age and related brain indices and their utility.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Howard et al reports the development of high-affinity WDR5-interaction site inhibitors (WINi) that engage the protein to block the arginine-dependent engagement with its partners. Treatment of MLL-rearranged leukemia cells with high-affinity WINi (C16) decreases the expression of genes encoding most ribosomal proteins and other proteins required for translation. Notably, although these targets are enriched for WDR5-ChIP-seq peaks, such peaks are not universally present in the target genes. High concordance was found between the alterations in gene expression due to C16 treatment and the changes resulting from treatment with an earlier, lower affinity WINi (C6). Besides protein synthesis, genes involved in DNA replication or MYC responses are downregulated, while p53 targets and apoptosis genes are upregulated. Ribosome profiling reveals a global decrease in translational efficiency due to WINi with overall ribosome occupancies of mRNAs ~50% of control samples. The magnitude of the decrements of translation for most individual mRNAs exceeds the respective changes in mRNA levels genome-wide. From these results and other considerations, the authors hypothesize that WINi results in ribosome depletion. Quantitative mass spec documents the decrement in ribosomal proteins following WINi treatment along with increases in p53 targets and proteins involved in apoptosis occurring over 3 days. Notably, RPL22L1 is essentially completely lost upon WINi treatment. The investigators next conduct a CRISPR screen to find moderators and cooperators with WINi. They identify components of p53 and DNA repair pathways as mediators of WINi-inflicted cell death (so gRNAs against these genes permit cell survival). Next, WINi are tested in combination with a variety of other agents to explore synergistic killing to improve their expected therapeutic efficacy. The authors document the loss of the p53 antagonist MDM4 (in combination with splicing alterations of RPL22L1), an observation that supports the notion that WINi killing is p53-mediated.

Strengths:<br /> This is a scientifically very strong and well-written manuscript that applies a variety of state-of-the art molecular approaches to interrogate the role of the WDR5 interaction site and WINi. They reveal that the effects of WINi seem to be focused on the overall synthesis of protein components of the translation apparatus, especially ribosomal proteins-even those that do not bind WDR5 by ChIP (a question left unanswered is how much the WDR5-less genes are nevertheless WINi targeted). They convincingly show that disruption of the synthesis of these proteins is accompanied by DNA damage inferred by H2AX-activation, activation of the p53-pathway, and apoptosis. Pathways of possible WINi resistance and synergies with other anti-neoplastic approaches are explored. These experiments are all well-executed and strongly invite more extensive pre-clinical and translational studies of WINi in animal studies. The studies also may anticipate the use of WINi as probes of nucleolar function and ribosome synthesis though this was not really explored in the current manuscript.

Weaknesses:<br /> A mild deficiency in the current manuscript is the absence of cell biological methods to complement the molecular biological and biochemical approaches so ably employed. Some microscopic observations and confirmation of nucleolar dysfunction and DNA damage would be reassuring.

Reviewer #2 (Public Review):

Understanding disease conditions often yields valuable insights into the physiological regulation of biological functions, as well as potential therapeutic approaches. In previous investigations, the author's research group identified abnormal expression of brain-derived neurotrophic factor (BDNF) in the hypothalamus of a mouse model exhibiting Smith-Magenis syndrome (SMS), which is caused by heterozygous mutations of the Rai1 gene. Human SMS is associated with distinct facial characteristics, sleep disturbances, behavioral issues, and intellectual disabilities, often accompanied by obesity. Conditional knockout (cKO) of the Bdnf gene from the paraventricular hypothalamus (PVH) in mice led to hyperphagic obesity, while overexpression of the Bdnf gene in the PVH of Rai1 heterozygous mice restored the SMS-like obese phenotype. Based on these preceding findings, the authors of the present study discovered that homozygous Rai1 cKO restricted to Bdnf-expressing cells, or Rai1 gene knockdown solely in Bdnf-positive neurons in the PVH, induced obesity along with intricate alterations in adipose tissue composition, energy expenditure, locomotion, feeding patterns, and glucose tolerance, some of which varied between sexes. Additionally, the authors demonstrated that a brain-penetrating drug capable of activating the AKT cascades, a downstream signaling pathway of BDNF, partially alleviated the SMS-like obesity phenotype in female mice with Rai1 heterozygous mutations. Although the specific (neural) cell type responsible for this signaling remains an open question, the present study unequivocally highlights the importance of Rai1 gene function in PVH Bdnf neurons for the obesity phenotype, providing valuable insights into potential therapeutic strategies for managing obesity associated with SMS.

In the proteomic analysis (Fig. 1), the authors elucidated that multiple phospho-protein signaling pathways, including Akt and mTOR pathways, exhibited significant attenuation in the SMS model mice. Of significance, the manifestation of haploinsufficiency of the Rai1 gene exclusively within the BDNF+ cells demonstrated negligible impact on body weight (Fig. 2-supple 3D), despite observing a reduction in BDNF levels in the heterozygous Rai1 mutant (Fig. 1A). Conversely, the homozygous Rai1 cKO in the BDNF+ cells prominently displayed an obesity phenotype, suggesting substantial dissimilarities in the gene expression profiles between Rai1 heterozygous and homozygous conditions within the BDNF+ cell population. It would be advantageous to precisely identify the responsible differentially expressed genes, possibly including Bdnf itself, in the homozygous cKO model. The observed reduction in the excitability of PVH BDNF+ cells (Fig. 3) is presumably attributed to aberrant gene expression other than Bdnf itself, which may serve as a prospective target for gene expression analysis. Notably, the Rai1 homozygous cKO mice in BDNF+ cells exhibited some sexual dimorphisms in feeding and energy expenditures, as evidenced by Fig. 2 and related figures. Exploring the potential relevance of these sexual differences to human SMS cases and investigating the underlying cellular/molecular mechanisms in the future would provide valuable insights.

The CRISPR-mediated knockdown of the Rai1 gene appears to be highly effective, and the majority of Rai1 cKO effects in Bdnf+ cells are primarily attributed to PVH-Bdnf+ cells based on the similarity of phenotypes observed. With regards to the apparent rescue of the body weight phenotype in Rai1 heterozygous mutants using an AKT pathway activator, the specific biological processes, and neurons responsible for this effect remain unclear. Elucidating these aspects in future studies would be significant when considering potential applications to human SMS cases.

Overall, the present study represents a valuable addition to the authors' series of high-quality molecular genetic investigations into the in vivo functions of the Rai1 gene. This reviewer particularly commends their diligent efforts to enhance our comprehension of SMS and contribute to the future development of more effective therapies for this syndrome.

Reviewer #2 (Public Review):

Summary:<br /> Chong Chen and colleagues reported that mutations were identified in the ZC3H11A gene in four adolescents from 1015 high myopia subjects in their myopia cohort. They further generated Zc3h11a knockout mice utilizing the CRISPR/Cas9 technology. They analyzed the heterozygotes knockout mice compared to control littermates and found refractive error changes, electrophysiological differences, and retinal inflammation-related gene expression differences. They concluded that ZC3H11A may play a role in the early onset of myopia by regulating inflammatory responses.

Strengths:<br /> Data were shown from both clinical cohort and animal models.

Weaknesses:<br /> Their findings are interesting and important, however; they need to resolve several points to make the current conclusion.

1. They described the ZC3H11A gene as a pathogenic variant for high myopia. It should be classified as pathogenic according to the guidelines of the American College of Medical Genetics and Genomics (Richards et al., Genet Med 17(5):405-24, 2015). The modes of inheritance for the families need to be shown. They also described identifying the gene as a "new" candidate. It should be checked in databases such as gnomAD and ClinVar, and any previous publications and be declared as a novel variant.

2. The phenotypes of the heterozygote mice are weak overall. The het mice showed mild to moderate myopic refractive shifts from 4 to 10 weeks of age. However, this cannot be explained by other ocular biometrics such as anterior chamber depth or lens thickness. Some differences are found between het and WT littermates in axial length and vitreous chamber depth but disappear after 8 weeks old. Furthermore, the early differences are not enough to explain the refractive error changes. They mentioned that they did not use homozygotes because of the embryonic lethality. I would strongly suggest employing conditional knockout systems to analyze homozygotes. This will also be able to identify the causative tissues/cells because they assume bipolar cells are functional. The cells in the retinal pigment epithelium and choroid are also important to contribute to myopia development.

3. Their hypothesis regarding inflammatory gene changes and myopic development is not logical. Are the inflammatory responses evoked from bipolar cells? Did the mice show an accumulation of inflammatory cells in the inner retina? Visible retinal inflammation is not generally seen in either early-onset or high-myopia human subjects. Can this be seen in the actual subjects in the cohort? To me, this is difficult to adapt the retina-to-sclera signaling they mentioned in the discussion so far. Egr-1 may be examined as described.

Reviewer #2 (Public Review):

Summary:<br /> Syntaxin17 (STX17) is a SNARE protein that is recruited to mature (i.e., closed) autophagosomes, but not to immature (i.e., unclosed) ones, and mediates the autophagosome-lysosome fusion. How STX17 recognizes the mature autophagosome is an unresolved interesting question in the autophagy field. Shinoda and colleagues set out to answer this question by focusing on the C-terminal domain of STX17 and found that PI4P is a strong candidate that causes the STX17 recruitment to the autophasome.

Strengths:<br /> The main findings are: 1) Rich positive charges in the C-terminal domain of STX17 are sufficient for the recruitment to the mature autophagosome; 2) Fluorescence charge sensors of different strengths suggest that autophagic membranes have negative charges and the charge increases as they mature; 3) Among a battery of fluorescence biosensors, only PI4P-binding biosensors distribute to the mature autophagosome; 4) STX17 bound to isolated autophagosomes is released by treatment with Sac1 phosphatase; 5) By dynamic molecular simulation, STX17 TM is shown to be inserted to a membrane containing PI4P but not to a membrane without it. These results indicate that PI4P is a strong candidate that STX17 binds to in the autophagosome.

Weaknesses:<br /> • It was not answered whether PI4P is crucial for the STX17 recruitment in cells because manipulation of the PI4P content in autophagic membranes was not successful for unknown reasons.<br /> • The molecular simulation study did not show whether PI4P is necessary for the STX17 TM insertion or whether other negatively charged lipids can play a similar role.<br /> • The question that the authors posed in the beginning, i.e., why is STX17 recruited to the mature (closed) autophagosome but not to immature autophagic membranes, was not answered. The authors speculate that the seemingly gradual increase of negative charges in autophagic membranes is caused by an increase in PI4P. However, this was not supported by the PI4P fluorescence biosensor experiment that showed their distribution to the mature autophagosome only. Here, there are at least two possibilities: 1) The increase of negative charges in immature autophagic membranes is derived from PI4P. However the fluorescence biosensors do not bind there for some reason; for example, they are not sensitive enough to recognize PI4P until it reaches a certain level, or simply, their binding does not occur in a quantitative manner. 2) The negative charge in immature membranes is not derived from PI4P, and PI4P is generated abundantly only after autophagosomes are closed. In either case, it is not easy to explain why STX17 is recruited to the mature autophagosome only. For the first scenario, it is not clear how the PI4P synthesis is regulated so that it reaches a sufficient level only after the membrane closure. In the second case, the mechanism that produces PI4P only after the autophagosome closure needs to be elucidated (so, in this case, the question of the temporal regulation issue remains the same).

Reviewer #2 (Public Review):

Summary:<br /> The research conducted by Yaning Cui and colleagues delves into understanding FLS2-mediated immunity. This is achieved by comparing the spatiotemporal dynamics of an FLS2-S938A mutant and FLS2-WT, especially in relation to their association with the remorin protein. To delineate the differences between the FLS2-S938A mutant and FLS2-WT, they utilized a plethora of advanced fluorescent imaging techniques. By analyzing surface dynamics and interactions involving the receptor signal co-receptor BAK1 and remorin proteins, the authors propose a model of how FLS2 and BAK1 are assembled and positioned within a remorin-specific nano-environment during FLS2 ligand-induced immune responses.

Strengths:<br /> These techniques offer direct visualizations of molecular dynamics and interactions, helping us understand their spatial relationships and interactions during innate immune responses.

Advanced cell biology imaging techniques are crucial for obtaining high-resolution insights into the intracellular dynamics of biomolecules. The demonstrated imaging systems are excellent examples to be used in studying plant immunity by integrating other functional assays.

Weaknesses:<br /> It's essential to acknowledge that every fluorescence-based method, just like biochemical assays, comes with its unique limitations. These often pertain to spatial and temporal resolutions, as well as the sensitivity of the cameras employed in each setup. Meticulous interpretation is pivotal to guarantee an accurate depiction and to steer clear of potential misunderstandings when employing specific imaging systems to analyze molecular attributes. Moreover, a discerning interpretation and accurate image analysis can offer invaluable guidance for future studies on plant signaling molecules using these nice cell imaging techniques.

For instance, although single-particle analysis couldn't conclusively link FLS2 and remorin, FLIM-FRET effectively highlighted their ligand-triggered association and the disengagement brought on by mutations. While these methodologies seemed to present differing outcomes, they were described in the manuscript as harmonious. In reality, these differences could highlight distinct protein populations active in immune responses, each accentuated differently by the respective imaging techniques due to their individual spatial and temporal limitations. Addressing these variations is imperative, especially when designing future imaging explorations of immune complexes.

Reviewer #2 (Public Review):

Summary:<br /> The mechanisms by which seizures induce neurogenesis has remained unclear. Prior work from the authors demonstrated Mossy cell expressed Shh, that altered Shh expression follows epileptic seizures, and that Shh is a neural mitogen. Here authors show that Shh from mossy cells, which are well positioned between the pyramidal and granule cell layers, are a major source of signaling after seizures, contributing to seizure-induced neurogenesis. Moreover, they find that Mossy cell-sourced Shh is required for self-renewal of NSCs even outside of the context of seizures.

SVZ Gli1 expression was detected in NSCs and Gli1 reporter activity follows kainate-induced seizures. Heterozygous Shh mice show reduced seizure induced Shh signaling and reduced neurogenesis. After localizing Shh production to Mossy cells, authors removed Shh from Mossy cells and found reduced neurogenesis. By activating mossy cells through chemogenetic DREADD, they found that the effect of mossy cells on SVZ neurogenesis is activity-dependent, that Shh signaling activity is upregulated in NSCs by mossy cell neuronal activity, and that the induction of neurogenesis by mossy cell neuronal activity is compromised in the absence of Shh from mossy cells. In a series of experiments incorporating AAV DREADD, they find that mossy cell activity can contribute to neurogenesis in contralateral DG, and that seizure induced Shh may be transported along mossy axons. To examine long-term effects, they study mice several weeks after seizure, and find that suggesting that NSCs are less likely to return to their stem cell state after seizure-induced proliferation in the absence of Shh from mossy cells, and that Shh from mossy cells contributes to persistence of the NSC state during aging.

Strengths:<br /> The results are compelling and impactful, and the study is extremely well done. The various genetic lines in the study ensure robust results. Adequate consideration of statistics, methods of quantification, and avoidance of artifact is given.

Weaknesses:<br /> None identified.

Reviewer #2 (Public Review):

Summary:<br /> The author's main goal is to understand the mechanism by which pyroptosis (through the formation of Gasdermin D (GSDMD) pores in the plasma membrane) contributes to increased release of procoagulant Tissue Factor-containing microvesicles (MV). Their previous data demonstrate that GSDMD is critical for the release of MV that contains Tissue Factor (TF), thus making a link between pyroptosis and hypercoagulation. Given the recent identification of NINJ1 being responsible for plasma membrane rupture (Kayagaki et al. Nature 2011), the authors wanted to determine if NINJ1 is responsible for TF-containing MV release. Given the constitutive ninj1 KO mouse leads to partial embryonic lethality, the authors decided to use a heterozygous ninj1 KO mouse (ninj1+/-). While the data are well controlled, there is limited understanding of the mechanism of action. Also, given that the GSDMD pores have an ~18 nm inner diameter enough to release IL-1β, while larger molecules like LDH (140 kDa) and other DAMPs require plasma membrane rupture (likely mediated by NINJ1), it s not unexpected that large MVs require NINJ1-mediated plasma cell rupture.

Strengths:<br /> The authors convincingly demonstrate that ninj1 haploinsufficiency leads to decreased prothrombin time, plasma TAT and plasma cytokines 90 minutes post-treatment in mice, which leads to partial protection from lethality.

Weaknesses:<br /> - In the abstract, the authors say "...cytokines and protected against blood coagulation and lethality triggered by bacterial flagellin". This conclusion is not substantiated by the data, as you still see 70% mortality at 24 hours in the ninj1+/- mice.

- The previous publication by the authors (Wu et al. Immunity 2019) clearly shows that GSDMD-dependent pyroptosis is required for inflammasome-induced coagulation and mouse lethality. However, as it is not possible for the authors to use the homozygous ninj1 KO mouse due to partial embryonic lethality, it becomes challenging to compare these two studies and the contributions of GSDMD vs. NINJ1. Comparing the contributions of GSDMD and NINJ1 in human blood-derived monocytes/macrophages where you can delete both genes and assess their relevant contributions to TF-containing MV release within the same background would be crucial in comparing how much contribution NINJ1 has versus what has been published for GSDMD? This would help support the in vivo findings and further corroborate the proposed conclusions made in this manuscript.

- What are the levels of plasma TAT, PT, and inflammatory cytokines if you collect plasma after 90 minutes? Given the majority (~70%) of the ninj+/- mice are dead by 24 hours, it is imperative to determine whether the 90-minute timeframe data (in Fig 1A-G) is also representative of later time points. The question is whether ninj1+/- just delays the increases in prothrombin time, plasma TAT, and plasma cytokines.

Reviewer #2 (Public Review):

Summary: In this manuscript, Trautman et al. set out to test the hypothesis that increased intake of dietary protein is deleterious to health when uncoupled from resistance training.

Strengths: The experimental design is well crafted and the experiments provide useful information supporting the hypothesis. The authors take into account the limitations of their study in the discussion, and guide the reader through their results and the interpretation in a fair and measured way, without overstating claims.

Weaknesses: As acknowledged by the authors in the discussion section, this study only features a small sample of male mice from a single strain. Thus the results may not hold when female mice and diverse genetic backgrounds are analyzed. The lack of repeated measures of physiological parameters is also a limitation of the study. Measurements of body weight, body composition, food (calorie) consumption, and locomotor/strength assays could have been provided throughout the study and compared to a baseline value for each animal.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Kandoi et al. describes a new 3D retinal organoid model of a mono-allelic copy number variant of the rhodopsin gene that was previously shown to induce autosomal dominant retinitis pigmentosa via a dominant negative mechanism in patients. With advancements in the low-cost genomics application to detect copy number variations, this is a timely article that highlights a potential disease mechanism that goes beyond the retina field. The evidence is relatively strong that the rod photoreceptor phenotype observed in an adult patient with RP in vivo is similar to that phenotype observed in human stem cell-derived retinal organoids. Increases in RHO expression detected by qPCR, RNA-seq, and IHC support this phenotype. Importantly, the amelioration of photoreceptor rhodopsin mislocalization and related defects using the small molecule drug photoregulin demonstrates an important potential clinical application.

Overall, the authors succeeded in providing solid evidence that copy number variation via a genomic RHO duplication leads to abnormalities in rod photoreceptors that can be partially blocked by photoregulin. However, there are several points that should be addressed that will enhance this paper.

Strengths:<br /> - The use of patient-derived organoids from patients that have visual defects is a major strength of this work and adds relevance to the disease phenotype.<br /> - The rod phenotype assessed by qPCR, RNA-seq, and IHC supports a phenotype that shares similarities with the patient.<br /> - The use of a small molecule drug that selectively targets rod photoreceptors, as opposed to cones, is a noteworthy strength.

Weaknesses:<br /> 1. The chromosomal segment that was duplicated had 3 copies of RHO in addition to three copies of each of the flanking genes (IFT122, HIF100, PLXND1). Discussion of the involvement of these genes would be helpful. Would duplication of any of these genes alone cause or contribute to adRP? As an example, a missense mutation in IFT122 was previously implicated in photoreceptor loss (PMID: 33606121 PMCID: PMC8519925).

2. Related to #1, have the authors considered inserting extra copies of RHO (and/or the flanking genes) of these at a genomic safe harbor site? Although not required, this would allow one to study cells with isogenic-matched genetic backgrounds and would partially address the technical challenge of repairing a 188kb duplication, which as the authors note would be difficult to do. Demonstrating that excess copy numbers in different genetic backgrounds would be a huge contribution to the field. At a minimum, a discussion of the role of the nearby genes should be included. 


3. In the patient, the central foveal region was spared suggesting that cones were normal. Was there a similar assessment that cones are unaffected in retinal organoids? 


4. Pathway analysis indicated that glycosylation was perturbed and this was proposed as an explanation as to why rhodopsin was mislocalized. Have the authors verified that there is an actual decrease in glycosylation? 


5. Line 182: by what criteria are the authors able to state that " there were no clear visible anatomical changes in apical-basal retinal cell type distribution during the early differentiation timeframe (data not shown)." Was this based on histological staining with antibodies, nuclear counter-staining, or some other evaluation?


6. Figure 2C - the appearance of the inner segments in RC and RM looks very different from one another. Have the authors ruled out the possibility that the RC organoid cell isn't a cone? In addition, the RM structure has what appears to be a well-defined OLM which would suggest well-formed Muller glia. Do these structures also exist in RC organoids? Typically the OLM does form in older organoids. In addition, was this representative in numerous EM preparations?


7. What criteria were used to assess cell loss? Has any TUNEL labeling been performed to confirm cell loss? From the existing data, it seems that rod outer segments appear to be affected in organoids. However, it's not clear if the photoreceptors themselves actually die in this model.

8. Figure 5B. The RHO staining in the vehicle-treated sample is perturbed relative to the PR3 treatments as indicated in the text. In the vehicle-treated sample, the number of DAPI-positive cells that are completely negative proximal to the inner segments suggests that there might be non-rod cells there. Have the authors confirmed whether these are cones? Labels would be helpful in the left vehicle panel as the morphology looks very different than the treated samples.
<br /> <br /> 9. It is interesting that in addition to increases in RHO, and photo-transduction, there are also increases in PTPRT which is related to synaptic adhesion. Is there evidence of ectopic neurites that result from PTPRT over-expression?

Reviewer #2 (Public Review):

Summary:<br /> In this study, Rana and colleagues present interesting findings demonstrating the potential beneficial effects of AMPA receptor modulators with ampakines in the context of the neurogenic bladder following acute spinal cord injury. Neurogenic bladder dysfunction is characterized by urinary retention and/or incontinence, with limited treatments available. Based on recent observations showing that ampakines improved respiratory function in rats with SCI, the authors explored the use of ampakine CX1739 on bladder and external urethral sphincter (EUS) function and coordination early after mid-thoracic contusion injury. Using continuous flow cystometry and EUS myography the authors showed that ampakine treatment led to decreased peak pressures, threshold pressure, intercontraction interval, and voided volume in SCI rats versus vehicle-treated controls. Although CX1739 did not alter EUS EMG burst duration, treatment did lead to EUS EMG bursting at lower bladder pressure compared to baseline. In a subset of rats that did not show regular cystometric voiding, CX1739 treatment diminished non-voiding contractions and improved coordinated EUS EMG bursting. Based on these findings the authors conclude that ampakines may have utility in recovery of bladder function following SCI.

Strengths:<br /> The experimental design is thoughtful and rigorous, providing an evaluation of both the bladder and external urethral sphincter function in the absence and presence of ampakine treatment. The data in support of a role for CX1789 treatment in the context of the neurogenic bladder are presented clearly, and the conclusions are adequately supported by the findings.

Weaknesses:<br /> Since CX1789 was administered in the context of cystometry and urethral sphincter EMG, a brief discussion of how ampakines could be used in a therapeutic context in humans would help to understand the translational significance of the work. The study lacks information on the half-life of CX1789 and how might this impact the implementation of CX1789 for clinical use. In addition, the study was limited to female rats. Lastly, given the male bias of traumatic SCI in humans, a brief discussion of this limitation is warranted.

Reviewer #2 (Public Review):

The current manuscript by Ban et al describes that cells undergoing EMT have increased rRNA synthesis, as analyzed by RNA seq-based gene expression analysis, and that the increased rRNA synthesis provides a therapeutic opportunity to target chemoresistance. The cells utilized in this manuscript were isolated from the authors' Tri-PyMT EMT lineage tracing model published a few years ago which demonstrated that cells undergoing EMT are not the cells that are contributing to metastasis but rather to tumor chemoresistance (Fischer, Nature 2015). This in vivo model has since then been criticized for not capturing all relevant EMT events which the authors also acknowledge in the introduction. The authors therefore reason that they use this lineage tracing model to better understand the role of EMT in chemoresistance.

A major problem with the current manuscript is that the authors present many of their findings as a novel without the proper acknowledgment of previously published literature in particular, Prakash et al., Nature Communications, 2019 and Dermitt, Dev Cell, 2020. In the studies by Prakash, the authors demonstrate that maintaining ongoing rRNA biogenesis is essential for the execution of the EMT program, and thus the ability of cancer cells to become migratory and invasive. Further, Prakash et al showed that blocking rRNA biogenesis with a small molecule inhibitor, CX-5461 (which is also used in the study by Ban et al) specifically inhibits breast cancer growth, invasion, EMT, and metastasis in animal models without significant toxicity to normal tissues. As such a significant revision that is necessary at this time is a rewrite of the manuscript especially the introduction and the discussion to more accurately describe and cite previously published findings and then highlight the current work by Ban et al which nicely builds on the previously published literature as it highlights the contribution of EMT to chemoresistance rather than metastasis. The suggestion for the authors is that they therefore should focus on highlighting the chemotherapy resistance angle as their Tri-PyMT EMT lineage tracing was chosen to test this angle and as such focus on both primary tumor growth and metastasis.

Additional major revisions:<br /> The authors use the FSP1-Cre Model which in the field has been questioned as to not capture all the relevant EMT events and therefore their findings should be corroborated by another EMT model system.

In the current version of the manuscript, there are no measurements of rRNA synthesis, but the gene expression profiles are used as a proxy for rRNA synthesis. The authors therefore need to include measurements of rRNA synthesis corroborating the RNA sequencing data to support their scientific findings and claims. This can be accomplished by qPCR, Northern blot, or EU staining of the respective sorted cell population. Quantification of rRNA synthesis is also needed for the CX-5461/BMH-21 and silencing studies.

Currently, there is no mechanistic insight as to how rRNA synthesis is increased during EMT, which would also strengthen the manuscript. This could be done through targeted ChIP analysis.

rRNA synthesis has canonically been linked to the cell cycle therefore it will be necessary for the authors to determine the cell cycle state of their respective cell populations throughout the manuscript.

Statistics and quantifications are currently missing in several figures and need to be better explained throughout the manuscript to strengthen the scientific rigor of the studies.

Only metastasis studies are shown in the current version of the manuscript. These studies should be complemented with primary tumor studies as the main focus of the paper is the contribution of EMT to chemoresistance.

Reviewer #2 (Public Review):

Overview:

As a report of the first structure of VMAT2, indeed the first structure of any vesicular monoamine transporter, this manuscript represents an important milestone in the field of neurotransmitter transport. VMAT2 belongs to a large family (the major facilitator superfamily, MFS) containing transporters from all living species. There is a wealth of information relating to the way that MFS transporters bind substrates, undergo conformational changes to transport them across the membrane, and couple these events to the transmembrane movement of ions. VMAT2 couples the movement of protons out of synaptic vesicles to the vesicular uptake of biogenic amines (serotonin, dopamine, and norepinephrine) from the cytoplasm. The new structure presented in this manuscript can be expected to contribute to an understanding of this proton/amine antiport process.

The structure contains a molecule of the inhibitor TBZ bound in a central cavity, with no access to either luminal or cytoplasmic compartments. The authors carefully analyze which residues interact with bound TBZ and measure TBZ binding to VMAT2 mutated at some of those residues. These measurements allow well-reasoned conclusions about the differences in inhibitor selectivity between VMAT1 and VMAT2 and differences in affinity between TBZ derivatives.

The structure also reveals polar networks within the protein and hydrophobic residues in positions that may allow them to open and close pathways between the central binding site and the cytoplasm or the vesicle lumen. The authors propose the involvement of these networks and hydrophobic residues in the coupling of transport to proton translocation and conformational changes. However, these proposals are quite speculative in the absence of supporting structures and experimentation that would test specific mechanistic details.

Critique:

Although the structure presented in this MS is clearly important, I feel that the authors have overstated several of the conclusions that can be drawn from it. I don't agree that the structure clearly indicates why TBZ is a non-competitive inhibitor; the proposal that specific hydrophobic residues function as gates will depend on lumen- and cytoplasm-facing structures for verification; the polar networks could have any number of functions - indeed it would be surprising if they were all involved in proton transport. Several of these issues could be resolved by a clearer illustration of the data, but I believe that a more rigorous description of the conclusions and where they fall between firm findings and speculation would help the reader put the results in perspective.

Non-competitive inhibition occurs when the action of an inhibitor can't be overcome by increasing substrate concentration. The structure shows TBZ sequestered in the central cavity with no access to either cytoplasm or lumen. The explanation of competitive vs non-competitive inhibition depends entirely on how TBZ got there. If it is bound from the cytoplasm, cytoplasmic substrate should have been able to compete with TBZ and overcome the inhibition. If it is bound from the lumen, or from within the bilayer, cytoplasmic substrate would not be able to compete, and inhibition would be non-competitive. The structure does not tell us how TBZ got there, only that it was eventually occluded from both aqueous compartments and the bilayer.

The issue of how VMAT2 opens access to the central binding site from luminal and cytoplasmic sides is an important and interesting one, and comparison with other MFS structures in cytoplasmic-open or extracellular/luminal-open is a very reasonable approach. However, any conclusions for VMAT2 should be clearly indicated as speculative in the absence of comparable open structures of VMAT2. As a matter of presentation, I found the illustrations in ED Fig. 6 to be less helpful than they could have been. Specifically, illustrations that focus on the proposed gates, comparing that region of the new structure with the corresponding region of either VGLUT or GLUT4 would better help the reader to compare the position of the proposed gate residues with the corresponding region of the open structure. I realize that is the intended purpose of ED Fig. 6b and 6c, but currently, those show the entire protein, and a focus on the gate regions might make the proposed gate movements clearer. I also appreciate the difference between the Alphafold prediction and the new structure, but I'm not convinced that ED Fig. 6a adds anything helpful.

The polar networks described in the manuscript provide interesting possibilities for interactions with substrates and protons whose binding to VMAT2 must control conformational change. Aside from the description of these networks, there is little evidence presented to assess the role of these networks in transport. Are the networks conserved in other closely related transporters? How could the interaction of the networks with substrate or protons affect conformational change? Of course, any potential role proposed for the networks would be highly speculative at this point, and any discussion of their role should point out their speculative nature and the need for experimental verification. Some speculation, however, can be useful for focusing the field's attention on future directions. However, statements in the abstract (three distinct polar networks... play a role in proton transduction.) and the discussion (...are likely also involved in mediating proton transduction.) should be clearly presented as speculation until they are validated experimentally.

The strongest aspect of this work (aside from the structure itself) is the analysis of TBZ binding. There is a problematic aspect to this analysis. The discussion on how TBZ stabilizes the occluded conformation of VMAT2 is premature without structures of apo-VMAT2 and possibly structures with other ligands bound. We don't really know at this point whether VMAT2 might be in the same occluded conformation in the absence of TBZ. Any statements regarding the effect of interactions between VMAT2 and TBZ depend on demonstrating that TBZ has a conformational effect. The same applies to the discussion of the role of W318 on conformation and to the loops proposed to "occlude the luminal side of the transporter" (line 131).

The description of VMAT2 mechanism makes many assumptions that are based on studies with other MFS transporters. Rather than stating these assumptions as fact (VMAT2 functions by alternating access...), it would be preferable to explain why a reader should believe these assumptions. In general, this discussion presents conclusions as established facts rather than proposals that need to be tested experimentally.

The MD simulations are not described well enough for a general reader. What is the significance of the different runs? ED Fig. 4d is not high enough resolution to see the details.

Reviewer #2 (Public Review):

Summary:<br /> The authors aimed to identify the neural sources of behavioral variation in a decision between odor and air, or between two odors.

Strengths:<br /> -The question is of fundamental importance.<br /> -The behavioral studies are automated, and high-throughput.<br /> -The data analyses are sophisticated and appropriate.<br /> -The paper is clear and well-written aside from some strong wording.<br /> -The figures beautifully illustrate their results.<br /> -The modeling efforts mechanistically ground observed data correlations.

Weaknesses:<br /> -The correlations between behavioral variations and neural activity/synapse morphology are (i) relatively weak, (ii) framed using the inappropriate words "predict", "link", and "explain", and (iii) sometimes non-intuitive (e.g., PC 1 of neural activity).<br /> -No attempts were made to perturb the relevant circuits to establish a causal relationship between behavioral variations and functional/morphological variations.

Reviewer #2 (Public Review):

Erbacher et al. have used new techniques to explore the neuro-cutaneous structures of human epidermis, which is a valuable goal given the lack of in-depth studies in human skin. Human skin is less studied than rodent skin because it presents challenges in obtaining samples and finding excellent immunohistological labels. They have employed expansion microscopy and super resolution array tomography for histological studies and have developed a human keratinocyte and human iPSC-derived sensory neuron co-culture. The authors have used these techniques to investigate the relation of intraepidermal nerve fibers (IENF) and keratinocytes, as well as to probe the localization of connexin 43. The data offer some anatomical insights, but as is does not add to our understanding of keratinocyte-neuron coupling.

Strengths:<br /> This paper is applying newer techniques to probe structure in human skin and establishes some useful immunohistochemical labels to do this, which sets up a foundation that will be valuable for future studies. The observation that IENF sometimes tunnel through keratinocytes is interesting, and the manuscript does show that Cx43 hemichannels are localized near IENF. Their data definitely represents a technical achievement, as these studies are challenging.

Weaknesses:<br /> Throughout the paper, the authors imply that they make discoveries that shed light on neuro-cutaneous interactions, but the data in this manuscript do not offer any functional insight into connections between IENF and keratinocytes. For example, the final figure legend indicates they have found evidence of "electrical and chemical synapse-like contacts to nerve fibers" (Figure 9), but no such evidence was shown. Only a single neuron vesicular marker (synaptophysin) was shown to localize to neurons in culture, as expected. They also "...propose a crucial role of nerve fiber ensheathment and Cx43-based keratinocyte-fiber contacts in neuropathic pain and small fiber pathology." but do not show any data regarding the contribution of their anatomical findings to sensory function.

Their data do show that IENF are anatomically closely apposed to keratinocytes, but this is inevitable given their location in the epidermis. The expression of Cx43 in human epidermis is also known (PMID: 7518858) and localizing Cx43 plaques near IENF does not add to current knowledge, as wide expression in keratinocytes naturally positions them near the embedded IENF. There is no indication whether IENF also expresses Cx43 to form gap junctions. Moreover, due to the lack of quantification, it is not clear whether Cx43 labeling is enriched at IENF sites as compared to other areas on the keratinocytes.

The authors' implication that their anatomical data offers insight into neuro-cutaneous functional coupling is a leap that is evident throughout the manuscript.

Reviewer #2 (Public Review):

Results from these experiments confirm the role of TRP channels but raise serious doubts that HCN channels contribute to the light response, refuting the findings of an influential paper that appeared in Cell (Jiang et al., 2018). Instead, a major role for T-type voltage-gated Ca2+channels is suggested. Together, these results further clarify our understanding of intrinsic photosensitivity in ganglion cells. However, there are several technical issues that need to be clarified before the major claims of this paper are justified.

Reviewer #2 (Public Review):

The manuscript by Chi, et al., mainly investigated the mutational characteristics of ctDNA, ctDNA-related markers in metastasis triple-negative breast cancer (mTNBC). They evaluated the translational value of ctDNA in predicting the prognosis and monitoring the treatment response of patients with mTNBC. Overall, this study is interesting and decent with great clinical significance.

Reviewer #2 (Public Review):

In this study authors study how OFP operates in control healthy humans and people that suffered of lesions of the OFP. Authors used a variation of the local vs. global oddball paradigm to study different levels of regularity violations. Overall the data is very interesting and having the study based on healthy and lesion humans make the results much more valuable than, other studies on healthy subject or even in animal studies.

However, the current version of the manuscript is overall very long and verbose, for example, the introduction is 5 pages long and includes up to 102 references. In my view this is way too much. I suppose authors wish to be very detailed, but somehow they get an opposite effect, the main message of the introduction and aims get diluted.

I wonder if the presentation rate used, SOA; 150 is too fast and the stimuli too short 50 ms. Please prove a rationale for this. Also, one of the conditions is 'omissions', but results are not reported, so either authors do not mention this at all, or they report these data, which would be probably interesting.<br /> The results are complex themselves and difficult to follow for a non-specialist in the field and there is not much to simplify here, but again, the Discussion is very long and in some aspect even too speculative. For example, in the conclusions authors claim that the OFC contributes to a top-down predictive process that modulates the deviance detection system in the primary auditory cortices and may be involved in connecting PEs at lower hierarchical areas with predictions at higher areas. I am not sure the current data support this. This would-be probably more appropriate if they could compare results from OFP and AC etc. so it is a more dynamic study.

At the beginning of Discussion, the authors mention that overall, these findings provide novel information about the role of the OFC in detecting violation of auditory prediction at two levels of stimuli abstraction/time scale. I think this needs to be detailed more specifically rather than mention they provide novel results

I am not sure I like to have a section as a general discussion within the discussion itself, probably this heading should be reformatted to be more specific to what is discussed.

In sum, while I find that this paper is potentially very interesting, it needs to be recast and shortened to make it more direct and appealing.

Reviewer #2 (Public Review):

In this manuscript, Lu et al. aimed to identify and characterize how cyclin D1 is ubiquitinated and degraded through Cullin-E3 ligases in addition to the well-documented CUL1/7-F-box proteins (Fbxw8, Fbx4, and Fbx031). The authors first confirmed that in addition to CUL1/7, all seven Cullin proteins (CUL1, 2,3, 4A, 4B, 5, and 7) are required for cyclin D1 degradation via overexpression or siRNA-mediated knockdown approach. Next, these seven Cullin proteins are validated as critical factors for cyclin D1 ubiquitination and proteosome-mediated degradation via a phosphorylation-dependent mechanism. A siRNA library of 154 e3 ligases was screened to identify 24 E3 ligases for cyclin D1 degradation, five of which (Fbxw8, Keap1, DDB2, WSB2, and Rbx1) were selected for further analysis. Functional validation and characterization analyses have shown that Keap1, DDB2, WSB2, and Rbx1 interact with cyclin D1, and that Wild Type but not mutant version of Keap1, DDB2, WSB2, and Rbx1 induces cyclin D1 ubiquitination and degradation. Finally, these cullin-E3 ligases-mediated cyclin D1 degradation is important for cell cycle progression, DNA replication, and cell viability.

Although the experimental design is overall sound and the presentation of the data is great, some major weaknesses (see details below) dampen the rigor of the study and overall claim.

Major weaknesses:<br /> 1) The biggest weakness of the manuscript is the lack of appropriate explanation and interpretation of these observed cyclin D1 ubiquitination and degradation by at least five different combinations of Cullin-E3 ligases. Are all the five cullin-E3 combinations exclusive and/or redundant to each other for cyclin D1 ubiquitination? What are the speculations in terms of the underlying mechanism? At least a working model should be included to better interpret the data.

2) Although a phosphorylation-mutant cyclin D1 (i.e., T286) was included in the manuscript, there is no Lysine residue mutant within cyclin D1 identified and characterized for the critical function of cyclin D1 ubiquitination.

3) The significance of these different Cullin 1-7 and associated E3 ligases (Keap1-CUL3, DDB2-CUL4A/4B, WSB2-CUL2/5, and RBX1-CUL1-7) in cyclin D1 ubiquitination is mainly determined by siRNA-mediated knockdown or overexpression of target cullin/E3 proteins. However, it is not clear whether the observed phenotypes of cyclin D1 are due to these cullin-E3 ligases directly or indirectly. In vitro ubiquitination assay with E1, E2, and E3 should be performed to demonstrate whether recombinant cyclin D1 is ubiquitinated.

Reviewer #2 (Public Review):

The preliminary cohort study has provided the efficacy and safety profile of immunotherapy combined with SBRT and cytotoxic chemotherapy, and the data are solid to support the findings, which could serve as evidence for future basic research and larger scale randomized control trials. While the major innovation of this study concentrates on immunotherapy, the description of specific issues regarding immunotherapy should be strengthened and more detailed.

Reviewer #2 (Public Review):

The analysis of bundle morphology using both confocal and SEM imaging is a strength of the paper and the authors have some nice images, especially with SEM. Still, the main weakness is that it is unclear how significant their findings are in terms of understanding bundle development; the mouse phenotypes are not distinct enough to make it clear that they serve different functions so the reader is left wondering what the main takeaway is.

In Figure 1 and 3, changes in bundle morphology clearly don't occur until after P5. Widening still occurs to some extent but lengthening does not and instead the stereocilia appear to shrink in length. EPS8 levels appear to be the most reduced of all the tip proteins (Srf mutants) so I wonder if these mutants are just similar to an EPS8 KO if the loss of EPS8 occurred postnatally (P0-P5).

A major shortcoming is that there are few details on how the image analyses were done. Were SEM images corrected for shrinkage? How was each of the immunocytochemistry quantitation (e.g., cuticular plates for phalloidin and tip staining for antibodies) done? There are multiple ways of doing this but there are few indications in the manuscript.

The tip protein analysis in Figs 2 and 4 is nice but it would be nice for the authors to show the protein staining separately from the phalloidin so you could see how restricted to the tips it is (each in grayscale). This is especially true for the CNN2 labeling in Fig 7 as it does not look particularly tip specific in the x-y panels. It would be especially important to see the antibody staining in the reslices separate from phalloidin.

In Fig 6, why was the transcriptome analysis at P2 given that the phenotype in these mice occurs much later? While redoing the transcriptome analysis is probably not an option, an alternative would be to show more examples of EPS8/GNAI/CNN2 staining in the KO, but at younger ages closer to the time of PCR analysis, such as at P5. Pinpointing when the tip protein intensities start to decrease in the KOs would be useful rather than just showing one age (P10).

While it is certainly interesting if it turns out CNN2 is indeed at tips in this phase, the experiments do not tell us that much about what role CNN2 may be playing. It is notable that in Fig 7E in the control+GFP panel, CNN2 does not appear to be at the tips. Those images are at P11 whereas the images in panel A are at P6 so perhaps CNN2 decreases after the widening phase. An important missing control is the Anc80L65-Cnn2 AAV in a wild-type cochlea.

Reviewer #2 (Public Review):

In this article Zhang et al. report that the Histone Deacetylase-3 (HDAC3) is highly expressed in mouse pDC and that pDC development is severely affected both in vivo and in vitro when using mice harbouring conditional deletion of HDAC3. However, pDC numbers are not affected in Hdac3fl/fl Itgax-Cre mice, indicating that HDCA3 is dispensable in CD11c+ late stages of pDC differentiation. Indeed, the authors provide wide experimental evidence for a role of HDAC3 in early precursors of pDC development, by combining adoptive transfer, gene expression profiling and in vitro differentiation experiments. Mechanistically, the authors have demonstrated that HDAC3 activity represses the expression of several transcription factors promoting cDC1 development, thus allowing the expression of genes involved in pDC development. In conclusion, these findings reveals HDAC3 as a key epigenetic regulator of the expression of the transcription factors required for pDC vs cDC1 developmental fate.

These results are novel and very promising. However, supplementary information and eventual further investigations are required to improve the clarity and the robustness of this article.

Major points<br /> 1) The gating strategy adopted to identify pDC in the BM and in the spleen should be entirely described and shown, at least as a Supplementary Figure. For the BM the authors indicate in the M & M section that they negatively selected cells for CD8a and B220, but both markers are actually expressed by differentiated pDC. However, in the Figures 1 and 2 pDC has been shown to be gated on CD19- CD11b- CD11c+. What is the precise protocol followed for pDC gating in the different organs and experiments?

2) pDC identified in the BM as SiglecH+ B220+ can actually contain DC precursors, that can express these markers, too. This could explain why the impact of HDAC3 deletion appears stronger in the spleen than in the BM (Figures 1A and 2A). Along the same line, I think that it would important to show the phenotype of pDC in control vs HDAC3-deleted mice for the different pDC markers used (SiglecH, B220, Bst2) and I would suggest to include also Ly6D, taking also in account the results obtained in Figures 4 and 7. Finally, as HDCA3 deletion induces downregulation of CD8a in cDC1 and pDC express CD8a, it would important to analyse the expression of this marker on control vs HDAC3-deleted pDC.

3) How do the authors explain that in the absence of HDAC3 cDC2 development increased in vivo in chimeric mice, but reduced in vitro (Figures 2B and 2E)? More generally, as reported also by authors (line 207), the reconstitution with HDAC3-deleted cells is poorly efficient. Although cDC seem not to be impacted, are other lymphoid or myeloid cells affected? This should be expected as HDAC3 regulates T and B development, as well as macrophage function. This should be important to know, although this does not call into question the results shown, as obtained in a competitive context.

4) What are the precise gating strategies used to identify the different hematopoietic precursors in the Figure 4 ? In particular, is there any lineage exclusion performed? Moreover, what is the SiglecH+ CD11c- population appearing in the spleen of mice reconstituted with HDAC3-deleted CDP? Data shown in Figure 4F should be expressed as log2 and not10. Finally, how do the authors explain that Hdac3fl/fl express Il7r, while they are supposed to be sorted CD127- cells?

5) What is known about the expression of HDAC3 in the different hematopoietic precursors analysed in this study? This information is available only for a few of them in Supplementary Figure 1. If not yet studied, they should be addressed.

6) It would be highly informative to extend CUT and Tag studies to Irf8 and Tcf4, if this is technically feasible.

Reviewer #2 (Public Review):

In this manuscript, Yu and colleagues profile the lysosome content in C. elegans. They implement lysosome immunoprecipitation (Lyso-IP) for C. elegans and they convincingly show that this method successfully isolates lysosomes from whole worms. The authors find that the lysosomes of worms overexpressing the lysosomal lipase lipl-4 are enriched for AMPK subunits and nucleoporins and that these proteins are required for the longevity of lipl-4 overexpressing worms. The authors also show that this is specific to this longevity pathway given that another long-lived worm strain (daf-2) does not exhibit enrichment for nucleoporins nor does it require them for longevity. The authors go on to express the Lyso-IP tag in different tissues of C. elegans (muscle, hypodermis, intestine, neurons) and identify the tissue-specific lysosome proteomes. Finally, the authors use this method to identify lysosome proteins in mature lysosomes and they find new proteins that regulate lysosomal acidification.

The authors present a powerful tool to unbiasedly identify lysosome-associated proteins in C. elegans, and they provide an in-depth assessment of how this method can be used to understand longevity pathways and identify novel proteins. Understanding lysosomal differences in specific tissues or in response to different longevity conditions are exciting as it provides new insight into how organelles could control specific homeostasis responses. This tool and proteomics datasets also represent a great resource for the C. elegans community and should pry open new studies on the regulation and role of the lysosome at the organismal level.

Addressing the following suggestions would help strengthen this already strong manuscript. First, it would be helpful to validate selected candidates from the tissue-specific Lyso-IP to verify that the protocol is still specific with lower sample amounts. Second, it would be helpful to provide more details on the methods, notably for sample preparation and analysis, so that it can serve as a guideline for the community. Third, the manuscript contains a lot of data and conditions, which is great, but they may also feel disconnected in some cases and it could be helpful to focus the study on the main key findings.

Reviewer #2 (Public Review):

The manuscript examined the behavioural and neural profile of weak and strong fear memories. The data provide strong evidence that weak but not strong fear memories are subject to extinction and reconsolidation disruption. Strong memories also show greater generalization. These differences were echoed in differential neural connectivity with weak fear memories showing greater connectivity between brains areas than strong fear memories.

The findings are of a great importance and offer insight into why resistance to extinction and reconsolidation may underlie fear-related psychopathology.<br /> The study uses key behavioural tests to study the durability of weak vs strong memories (extinction and reconsolidation) as well as studies the generalisation of those memories. These behavioural effects nicely dovetail with the neural connectivity analyses that were performed.<br /> The data presented in this paper will be the basis for future hypothesis driven examinations on the causal influence of specific pathways involved in contextual fear.<br /> Excellent use of the open field to control for motor effects.

This is a strong paper and the results support the conclusions. The findings are of broad interest and are important for future research.

Reviewer #2 (Public Review):

In Bolumar, Moncayo-Arlandi et al. the authors explore whether endometrium-derived extracellular vesicles contribute DNA to embryos and therefore influence embryo metabolism and respiration. The manuscript combines techniques for isolating different populations of extracellular vesicles, DNA sequencing, embryo culture, and respiration assays performed on human endometrial samples and mouse embryos.

Vesicle isolation is technically difficult and therefore collection from human samples is commendable. Also, the influence of maternally derived DNA on the bioenergetics of embryos is unknown and therefore novel. However, several experiments presented in the manuscript fail to reach statistical significance, likely due to the small sample sizes. This manuscript is a good but incomplete start as to the potential function of maternal DNA transfer via vesicles.

In my opinion the manuscript supports the following of the authors' claims:

1. Different amounts of nDNA and mtDNA are shed in human endometrial extracellular vesicles during different phases of the menstrual cycle.<br /> 2. Endometrial microvesicles are more enriched for mitochondrial DNA sequences compared to other types of vesicles present in the human samples.<br /> 3. Fluorescently labelled DNA from extracellular vesicles derived from an endometrial adenocarcinoma cell line can be incorporated into hatched mouse embryos.<br /> 4. Culture of mouse embryos with endometrial extracellular vesicles can influence embryo respiration and the effect is greater when cultured with isolated exosomes compared to other isolated microvesicles.

My main concerns with the manuscript:

1. Several experiments presented fail to reach statistical significance or are qualitative.<br /> 2. The definitive experiments presented in the manuscript are limited to the transfer of DNA in general not mtDNA. Therefore a strong connection with metabolism is missing, diminishing the significance of the findings.

Reviewer #2 (Public Review):

Pinpoint by Birman et al. serves not only as a probe trajectory planning tool but also offers a far richer suite of functionalities. It provides a simple and intuitive environment that users can learn within minutes and start planning trajectories for multiple probes based on the Allen mouse brain atlas. Pinpoint further includes two MRI-based transformations to better map the Allen atlas to live brains. It features a coefficient to adjust for different Bregma-Lambda distances and includes a mouse skull model to provide a better approximation of the craniotomy coordinates, rather than the coordinate of the point of insertion on the brain. It also offers tools to link the application to manipulator controllers to visualise the position of probes in the brain in real-time. Remarkably, most of these features are available right from the web browser, without the need to install anything or any coding knowledge.

The authors developed an open-source and well-documented software. Although I did not test it myself, it can communicate with the most common recording softwares (Open Ephys, SpikeGLX) and manipulators (New Scale, Sensapex) in the field. The current level of support by the developers on GitHub is reassuring, and I hope this continues as Pinpoint matures into a more stable and robust version.

Reviewer #2 (Public Review):

Summary:<br /> This is an interesting and well-written study assessing the role of dopaminergic inputs from the VTA on orexin cell responses in an opto-pavlovian conditioning task. These data are consistent with a possible role of this system in reward expectation and are surprisingly one of the first demonstrations of a role for dopamine in this phenomenon.

Strengths:

The study has used an interesting opto-Pavlovian approach combined with fibre photometry.

Weaknesses:

It is unclear what n size was used or analysed, particularly for AUC measures e.g. Figures 1 D/E and 3 G. The number of trials reflected and the animal numbers need clarification.

The study focussed on opto-stim omissions - this work would be significantly strengthened by a comparison to a real-world examination where animals are trained for a radiation reward (food pellet). Have the authors considered the role of orexin in the opposing situation i.e. a surprise addition of reward? Similarly, there remains some conjecture regarding the role of these systems in reward and aversion - have the authors considered aversive learning paradigms - fear, or fear extinction - to further explore the roles of this system? There are some (important) discussions about the possible role of orexin in negative reinforcement. Further studies to address this could be warranted.

I think some further discussion of the work by Lineman concerning the interesting bidirectional actions of d1/d2 r signalling on glutamatergic transmission onto orexin neurons is worthwhile. While this work is currently cited, the nuance and perhaps relevance to d1 and d2 signalling could be contextualised a little more (https://doi.org/10.1152/ajpregu.00150.2018).

Reviewer #2 (Public Review):

The question of whether the neural mechanisms for reward and punishment learning are similar has been a constant debate over the last two decades. Numerous studies have shown that the midbrain dopamine neurons respond to both negative and salient stimuli, some of which can't be well accounted for by the classic RL theory (Delgado et al., 2007). Other research even proposed that aversive learning can be viewed as reward learning, by treating the omission of aversive stimuli as a negative PE (Seymour et al., 2004).

Although the current study took an axiomatic approach to search for the PE encoding brain regions, which I like, I have major concerns regarding their experimental design and hence the results they obtained. My biggest concern comes from the false description of their task to the participants. To increase the number of "valid" trials for data analysis, the instructed and actual probabilities were different. Under such a circumstance, testing axiom 2 seems completely artificial. How does the experimenter know that the participants truly believe that the 75% is more probable than, say, the 25% stimulation? The potential confusion of the subjects may explain why the SCR and relief report were rather flat across the instructed probability range, and some of the canonical PE encoding regions showed a rather mixed activity pattern across different probabilities. Also for the post-hoc selection criteria, why pick the larger SCR in the 75% compared to the 25% instructions? How would the results change if other criteria were used?

To test axiom 3, which was to compare the 100% stimulation to the 0% stimulation conditions, how did the actual shock delivery affect the fMRI contrast result? It would be more reasonable if this analysis could control for the shock delivery, which itself could contaminate the fMRI signal, with extra confound that subjects may engage certain behavioral strategies to "prepare for" the aversive outcome in the 100% stimulation condition. Therefore, I agree with the authors that this contrast may not be a good way to test axiom 3, not only because of the arguments made in the discussion but also the technical complexities involved in the contrast.

Reviewer #2 (Public Review):

Summary<br /> The paper introduces a valuable method, tCFS, for measuring suppression depth in continuous flash suppression (CFS) experiments. tCFS uses a continuous-trial design instead of the discrete trials standard in the literature, resulting in faster, better controlled, and lower-variance estimates. The authors measured suppression depth during CFS for the first time and found similar suppression depths for different image categories. This finding provides an interesting contrast to previous results that breakthrough thresholds differ for different image categories and refine inferences of subconscious processing based solely on breakthrough thresholds. However, the paper overreaches by claiming breakthrough thresholds are insufficient for drawing certain conclusions about subconscious processing.

Strengths<br /> 1. The tCFS method, by using a continuous-trial design, quickly estimates breakthrough and re-suppression thresholds. Continuous trials better control for slowly varying factors such as adaptation and attention. Indeed, tCFS produces estimates with lower across-subject variance than the standard discrete-trial method (Fig. 2). The tCFS method is straightforward to adopt in future research on CFS and binocular rivalry.<br /> 2. The CFS literature has lacked re-suppression threshold measurements. By measuring both breakthrough and re-suppression thresholds, this work calculated suppression depth (i.e., the difference between the two thresholds), which warrants different interpretations from the breakthrough threshold alone.<br /> 3. The work found that different image categories show similar suppression depths, suggesting some aspects of CFS are not category-specific. This result enriches previous findings that breakthrough thresholds vary with image categories. Re-suppression thresholds vary symmetrically, such that their differences are constant.

Weaknesses<br /> 1. The results and arguments in the paper do not support the claim that 'variations in breakthrough thresholds alone are insufficient for inferring unconscious or preferential processing of given image categories,' to take one example phrasing from the abstract. The same leap in reasoning recurs on lines 28, 39, 125, 566, 666, 686, 759, etc.<br /> Take, for example, the arguments on lines 81-83. Grant that images are inequivalent, and this explains different breakthrough times. This is still no argument against differential subconscious processing. Why are images non-equivalent? Whatever the answer, does it qualify as 'residual processing outside of awareness'? Even detecting salience requires some processing. The authors appear to argue otherwise on lines 694-696, for example, by invoking the concept of effective contrasts, but why is effective contrast incompatible with partial processing? Again, does detecting (effective) contrast not involve some processing? The phrases 'residual processing outside of awareness' and 'unconscious processing' are broad enough to encompass bottom-up salience and effective contrast. Salience and (effective) contrast are arguably uninteresting, but that is a different discussion. The authors contrast 'image categories' or semantics with 'low-level factors.' In my opinion, this is a clearer contrast worth emphasizing more. However, semantic processing is not equal to subconscious processing writ large. The preceding does not detract from the interest in finding uniform suppression depth. Suppression depth and absolute bCFS can conceivably be due to orthogonal mechanisms warranting their own interpretations. In fact, the authors briefly take this position in the Discussion (lines 696-704, 'A hybrid model ...'). The involvement of different mechanisms would defeat the argument on lines 668-670.

2. These two hypotheses are confusing and should be more clearly distinguished: a) varying breakthrough times may be due to low-level factors (lines 76-79); b) uniform suppression depth may also arise from early visual mechanisms (e.g., lines 25-27).

Neutral remarks<br /> The depth between bCFS and reCFS depended on measurement details such as contrast change speed and continuous vs. discrete trials. With discrete trials, the two thresholds showed inverse relations (i.e., reCFS > bCFS) in some participants. The authors discuss possible reasons at some length (adaptation, attention, etc. ). Still, a variable measure does not clearly indicate a uniform mechanism.

Reviewer #2 (Public Review):

Summary:<br /> The authors analyze how individual amacrine cells in the salamander retina can affect the sensitivity of retinal ganglion cells to different visual features. They use simultaneous recordings of amacrine and ganglion cells and apply current injection into the amacrine cells to assess the evoked response modulation of ganglion cells. The resulting transmission filter is combined with the amacrine cell's temporal receptive field to determine a visual feature that stands for the visual signal processing from stimulus to a ganglion cell via the recorded amacrine cell. This sets the stage for analyzing how activation of this "amacrine pathway" affects the encoding of other (orthogonal) visual features by the ganglion cell.

Strengths:<br /> The direct measurements of amacrine cell signals and their signal transmission to ganglion cells in challenging dual recordings is certainly a strength of this paper. In addition, the authors use an original and intriguing computational framework to analyze interactions of different visual features encoded by a ganglion cell and ask important questions about how inhibitory interneurons modulate stimulus encoding. The concept of distinct types of amacrine cell function with feature-specific modulation of input sensitivity and global modulation of output strength is thought-provoking and an interesting concept for follow-up investigations.

Weaknesses:<br /> However, despite the emphasis on a causal approach and direct measurements of amacrine cell effects, the paper does not use actual amacrine cell signals for the main analyses, but rather a proxy given by visual signals that are consistent with the amacrine-to-ganglion signal transmission. In doing so, it is largely disregarded that visual filters of other pathways (including, e.g., fatigue or desensitization in the excitatory signals) may overlap with the deduced amacrine pathways. It thus remains unclear how much such alternative pathways may contribute to the signals assigned to the amacrine pathway and how this might influence the findings and their interpretations. In addition, the analysis and interpretation of the amacrine pathway are hard to follow and easy to misunderstand, because the paper often applies ambiguous language by referring to the visual stimulus dimension of the amacrine pathway as "amacrine output" and "amacrine effects" and by equating activation or deactivation of the amacrine pathway with hyperpolarization or depolarization of the amacrine cell.

Some other interpretations are also unclear, by taking the results a bit too far. For example, the emphasis on divisive normalization remains unclear, as divisive normalization seems more specific than the general suppressive effects described here. Similarly, the connection to the previously observed reversal of preferred contrast by ganglion cells is somewhat tenuous. Here, the potential reversal in the analyzed response nonlinearities only concerns specific features that nonlinearly interact with other features and therefore do not easily translate to the contrast sensitivity of the ganglion cell as a whole, as is suggested in the text. In addition, the two examples of reversals shown in the figures are not fully convincing.

Regarding the clustering analysis of the pairs of amacrine cells and ganglion cell features (Fig. 4), a specific concern is that it is unclear how well the analyzed parameters can actually be extracted from the firing rate response nonlinearities. From the examples in Fig. 4A, it looks like many nonlinearities do not show a clear saturation (but might still yield a good fit by the piece-wise linear model and thus be included in the analysis). It seems plausible that this could result in a bias towards lower gain (defined via the saturation level) when nonlinearities are shifted rightward (higher threshold). It is thus not entirely clear how strong the evidence is for the correlation between gain and threshold changes.

Further, minor caveats are that only 11 amacrine cells go into the analysis, and it remains uncertain to what degree they cover the diversity of amacrine cells in the retina or rather represent a specific subset of types. Also, the restriction to visual signals with no spatial structure, though understandable, limits the generality of the findings. The extracted temporal features remain rather abstract with unspecified significance, in particular since quite a large number of features are extracted per ganglion cell (a total of 321 features, which presumably come from 39 ganglion cells that had a significant amacrine transmission filter).

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Salazar-Lázaro et al. systematically dissects the different functional properties of the SNARE-domains of syntaxin-1 and syntaxin-2. By systematically substituting the SNARE-domain (or its C- or N-terminal half) into the non-cognate counterpart, the authors find that the C-terminal half of the SNARE-complex is especially important for maintaining RRP size and clamping spontaneous release. They also mutate single residues, to further nail down the effect. Overall, this is an interesting manuscript, which sheds light on the functionality of different co-expressed SNARES.

Strengths:<br /> The strength of the manuscript is the systematic dissection, using substitution of either SNARE-domain into the other syntaxin, together with the state-of-the art methods. The authors follow up with a substitution of single and paired residues. This is a large undertaking, which has been very well carried out.

Weaknesses:<br /> No major weaknesses. The large number of experiments paint a somewhat complicated picture. The writing could be improved in places to increase clarity.

Reviewer #2 (Public Review):

Summary:<br /> This study examined the possible effect of spike-wave discharges (SWDs) on the response to visual or somatosensory stimulation using fMRI and EEG. This is a significant topic because SWDs often are called seizures and because there is non-responsiveness at this time, it would be logical that responses to sensory stimulation are reduced. On the other hand, in rodents with SWDs, sensory stimulation (a noise, for example) often terminates the SWD/seizure.

In humans, these periods of SWDs are due to thalamocortical oscillations. A certain percentage of the normal population can have SWDs in response to photic stimulation at specific frequencies. Other individuals develop SWDs without stimulation. They disrupt consciousness. Individuals have an absent look, or "absence", which is called absence epilepsy.

The authors use a rat model to study the responses to stimulation of the visual or somatosensory systems during and in between SWDs. They report that the response to stimulation is reduced during the SWDs. While some data show this nicely, the authors also report on lines 396-8 "When comparing statistical responses between both states, significant changes (p<0.05, cluster-) were noticed in somatosensory auditory frontal..., with these regions being less activated in interictal state (see also Figure 4). That statement is at odds with their conclusion.

They also conclude that stimulation slows the pathways activated by the stimulus. I do not see any data proving this. It would require repeated assessments of the pathways in time.

The authors also study the hemodynamic response function (HRF) and it is not clear what conclusions can be made from the data.

Finally, the authors use a model to analyze the data. This model is novel and while that is a strength, its validation is unclear. The conclusion is that the modeling supports the conclusions of the study, which is useful.

Strengths:<br /> Use of fMRI and EEG to study SWDs in rats.

Weaknesses:<br /> Several aspects of the Methods and Results are unclear.

Reviewer #2 (Public Review):

This work investigates the possible association between language experience and morphology of the superior temporal cortex, a part of the brain responsible for the processing of auditory stimuli. Previous studies have found associations between language and music proficiency as well as language learning aptitude and cortical morphometric measures in regions in the primary and associated auditory cortex. These studies have most often, however, focused on finding neuroanatomical effects of difference between features in a few (often two) languages or from learning single phonetic/phonological features and have often been limited in terms of N. On this background, the authors use more sophisticated measures of language experience that take into account the age of onset and the differences in phonology between languages the subjects have been exposed to as well as a larger number of subjects (N = 146 + 69) to relate language experience to the shape and structure of the superior temporal cortex, measured from T1-weighted MRI data. It shows solid evidence for there being a negative relationship between language experience and the right 2nd transverse temporal gyrus as well as some evidence for the relationship representing phoneme-level cross-linguistic information.

Strengths<br /> The use of entropy measures to quantify language experience and include typological distance measures allows for a more general interpretation of the results and is an important step toward respecting and making use of linguistic diversity in neurolinguistic experiments.

A relatively large group of subjects with a range of linguistic backgrounds.

The full analysis of the structure of the superior temporal cortex including cortical volume, area, as well as the shape of the transverse gyrus/gyri. There is a growing literature on the meaning of the shape and number of the transverse gyri in relation to language proficiency and the authors explore all measures given the available data.

The authors chose to use a replication data set to verify their data, which is applaudable. However, see the relevant point under "Weaknesses".

Weaknesses<br /> The authors fail to explain how a thinner cortex could reflect the specialization of the auditory cortex in the processing of diverse speech input. The Dynamic Restructuring Model (Pliatsikas, 2020) which is referred to does not offer clear guidance to interpretation. A more detailed discussion of how a phonologically diverse environment could lead to a thinner cortex would be very helpful.

It is difficult to understand what measure of language experience is used when. Clearer and more explicit nomenclature would assist in the interpretation of the results.

There is a lack of description of the language backgrounds of the included subjects. How many came from each of the possible linguistic backgrounds? How did they differ in language exposure? This would be informative to evaluate the generalizability of the conclusions.

Only the result from the multiple transverse temporal gyri (2nd TTG) is analyzed in the replicated dataset. Only the association in the right hemisphere 2nd TTG is replicated but this is not reflected in the discussion or the conclusions. The positive correlation in the right TTG is thus not attempted to be replicated.

The replication dataset differed in more ways than the more frequent combination of English and German experience, as mentioned in the discussion. Specifically, the fraction of monolinguals was higher in the replication dataset and the samples came from different scanners. It would be better if the primary and replication datasets were more equally matched.

Even if the language experience and typological distance measures are a step in the right direction for correctly associating language exposure with cortical plasticity, it still is a measure that is insensitive to the intensity of the exposure. The consequences of this are not discussed.

Reviewer #2 (Public Review):

The strengths of this paper begin with the topic. Specifically, this approaches the question of how GPCR signals are directed to different outcomes under different conditions. There is rich complexity within this question; there are potentially billions of molecules that could interact with >800 human GPCRs and thousands of molecular effectors that may be activated. However, these outcomes are filtered through a small number of GPCR-interacting proteins that direct the signal.

Experimentally, strengths include the initial experimental controls employed in characterizing their ever-important antisera, on which their conclusions hinge. In showing strong agonist-dependent and phosphosite-dependent recognition, as well as the addition of GRK inhibitors and eventually an antagonist and phosphatase treatment, the authors substantiate the role of the antiserum in recognizing their intended motifs. When employed, those antisera overall give clear indications of differences across variables in immunoblots, and while the immunocytochemical studies are qualitative and at times not visually significantly different across all variables, they are in large part congruent with the results of the immunoblots and provide secondary supporting evidence for the author's major claims. One confounding aspect of the immunocytochemical images is the presence of background pThr306/pThr310, like in Figures 4C and 6A and B. In 4A and C, while the immunoblot shows a complete absence of pThr306/pThr310, Figure 4C's immuno image does not. In 6A and B, a similar presence of pThr306/pThr310 is seen in the vehicle image, which is not strikingly over-shown by the MOMBA-treated image. In addition, only Ser/Thr residues of the C-terminus were investigated, while residues of ICL3 have long been known to direct signaling in many GPCRs. Because of the presentations of sequences, it was not clear whether there were residues of ICL3 that have the possibility of being involved.

It may be possible and further testable to show whether the residues that maintain basal phosphorylation could also be tissue-specific, especially considering the presence of pThr306/pThr310 detection in both the Figure 6A immunoblot's vehicle lane (but not MOMBA lane). The aforementioned detection in the immunocytochemical vehicle image could support differential basal phosphorylation in the enteroendocrine cells. Should this be the case, it could have confounded the initial mass-spec screen wherein the Ser residues were basally active in that cell type, while in a distinct cell type that may not be the case. Lastly, should normalized quantification of these images be possible, it may help in clearing up these hard-to-compare visual images.

It is noted that aspects of the writing and presentation may lead to confusion for some readers, but this does not affect the overall significance of the work.

Nevertheless, in terms of the global goal of the authors, the indication of differences in phosphorylation states between tissues is still evident across the experiments. Accordingly, the paper is overall strongly well-researched, well-controlled, and the conclusions made by the authors are data-grounded and not overly extrapolated. Providing direct evidence for the tissue-based branch of the barcode hypothesis is both novel and significant for the field, and the paper leaves room for much more exciting research to be done in the area, opening the door for new questions and hypotheses.

Reviewer #2 (Public Review):

Summary:

This manuscript describes the study protocol, structure and logic of the PAVE strategy. The PAVE study is a multicentric study to evaluate a novel cervical screen-triage-treat strategy for resource-limited settings as part of a global strategy to reduce cervical cancer burden. The PAVE strategy involves: 1) screening with self-sampled HPV testing; 2) triage of HPV-positive participants with a combination of extended genotyping and visual evaluation of the cervix assisted by deep-learning-based automated visual evaluation (AVE); and 3) treatment with thermal ablation or excision (Large Loop Excision of the Transformation Zone). The PAVE study has two phases: efficacy (2023-2024) and effectiveness (planned to begin in 2024-2025). The efficacy phase aims to refine and validate the screen-triage portion of the protocol. The effectiveness phase will examine implementation of the PAVE strategy into clinical practice.

Strengths and weaknesses:

The Pave Study develops and evaluates a novel strategy that combines HPV self-collection, that has been proven effective to increase screening coverage in different settings, with genotyping and Automated Visual Evaluation as triage. The proposed strategy combined three key innovations to improve an important step in the cervical cancer care continuum. If the strategy is effective it will contribute to enhancing cervical cancer prevention in low resource settings.

As the authors mentioned, despite the existence of effective preventive technologies (e.g., HPV vaccine and HPV test) translation of the HPV prevention methods has not yet occurred in many Low-Middle-Income Countries. So, in this context, new screen-triage-treat strategies are needed and if PAVE strategy were effective, it could be a landmark for cervical cancer prevention.

The PAVE Study is a solid and important study that is aimed to be carried out in nine countries and recruit tens of thousands of women. It is a study with a large and diverse sample that can provide useful information for the development of this new screen-triage-treat strategy. Another strength is the fact that the PAVE project is integrated into the screening activities placed in the selected countries that will allow to evaluate efficacy and effectiveness in real-word context.

The manuscript does not present results because its aim is to describe the study protocol, structure and logic of the PAVE strategy.

Phase 1 aims to evaluate the efficacy of the strategy. Methods are well described and are consistent with the study aims.

Phase 2 aims to evaluate the implementation of the PAVE strategy in clinical practice. The inclusion of implementation evaluation in this type of studies is an important milestone in the field of cervical cancer prevention. It has been shown that many strategies that have proven to be effective in controlled studies face barriers when they are implemented in real life. In that sense, the results of phase 2 are key to ensure the future implementation of the strategy.

However, some aspects of Phase 2 need to be clarified and extended. Although authors mentioned that implementation outcomes, such as acceptability and feasibility will be evaluated, more information is needed about method (i.e. qualitative/quantitative), data collection tools (i.e., survey, semi-structure interviews, focus groups, etc.) and frameworks that will be used to evaluate these implementation outcomes.

Reviewer #2 (Public Review):

This study aims to characterize transcriptional and epigenetic activity-dependent striatal neuronal adaptations using rat primary cultures, a model still poorly characterized up to date. In addition, the authors aim to interrogate regulatory mechanisms that could modulate the expression of a highly-striatal enriched gene responding to neuronal activation in striatal neuronal cells, the Pdyn gene.

Among the major strengths of the article there is the generation of high quality neuronal RNA-seq and ATAC-seq data in rat striatal neuronal cells in basal level and upon neuronal activity, a experimental setup that has not been so characterized as other more common ones such as mouse hippocampal neuronal cells. In this model, the authors clearly demonstrate the need of protein translation to induce the transcriptional waves of late response genes. In addition, the functional characterization of an enhancer of the Pdyn gene might be of great interest for translational applications in which alterations of this gene might be occurring in neurological disorders.

On the other hand, the manuscript presents some limitations to be considered. One of the major points in this regard is that, at least in part, some of the conclusions reached by the study related to the induction of particular transcriptional programs upon neuronal activation, the changes in chromatin state, and the need of protein translation for proper induction of LRGs have been already previously described in the literature, affecting the novelty of the study. However, it is needed to be mentioned, that these previous studies were not conducted using the same model (rat striatal neurons), which can make some differences in the final outputs. The other major cautionary point in the study is the selection of the time point for distinguishing early versus late response genes, as the short difference in time and the overlap of part of the transcriptional signature between them suggest that the transcriptional waves are somehow partially overlapping (also probably in part because of the recurrent stimulation of the primary cultures with KCl), which could result in missing part of the late-response genes.

Despite this, the conclusions raised in the study are well supported by the data generated in it.

In summary, the study presents a useful set of transcriptomic and epigenomic data of activity-dependent striatal neuronal programs in rats, which will be of great use for the scientific community working in this not so well characterized model. In addition, the characterization of a Pdyn distal regulatory genomic region involved in its transcriptional regulation, both at basal levels and upon neuronal activation in this particular system, can present translational relevance for striatal disorders such as Huntington's disease or other neuropsychiatric disorders.

Reviewer #2 (Public Review):

The revised paper addresses most of my major comments and concerns. The authors have added more detail explaining their model, they have added more background information, citations, and discussion for termite humidity sensing capabilities. With these modifications, this paper now provides a convincing presentation of valuable results of the drivers of nest construction for one termite species, and they briefly discuss possible relevance to other termite species. However, the authors have not yet addressed how their results may be important outside the field of termite nest construction. I could imagine the significance of the paper being elevated to important if there is a broader discussion about the impact of this work, e.g., the relevance of the results, the approach, and/or next steps to related fields outside of termite nest construction. Similarly, on a related note, as someone not directly in the field of termite nest construction but wanting to understand the system (and the results) presented here in a broader context, I found the additional information about species and natural habitat very helpful and interesting, though I was rather disappointed to find it relegated to supplementary material where most readers will not see it.

Reviewer #2 (Public Review):

The work by Bærentsen et al., entitled "Structural basis for regulation of a tripartite toxin-antitoxin system by dual phosphorylation" deals with the structural aspects of the control of the hipBST TA operon, the role of auto-phosphorylation in the activation and neutralisation of the enzyme and the direct effects of HipS and HipB in neutralisation. This is a follow-up to the Vang Nielsen et al., and Gerdes et al., papers from the same authors on this very unique TA module, that brings forth a thorough and well written dissection of an unusually complex regulatory system.

Reviewer #2 (Public Review):

Summary:<br /> Dolcemascolo and colleagues describe the use of the mammalian RNA-binding protein Musashi-1 (MSI-1) to implement translational regulation systems in E. coli. They perform detailed in vitro studies of MSI-1 and its binding to different RNA sequences. They provide compelling evidence of the effectiveness of the regulatory system in multiple circuits using different mRNA sequence motifs. They harness allosteric inhibition of MSI-1 by omega-9 monounsaturated fatty acids to demonstrate a fatty-acid-responsive circuit in E. coli.

Strengths:<br /> The experimental results are compelling and the characterization of the binding between MSI-1 and different RNA sequences is thorough and performed via multiple complementary techniques. Several new useful circuit components are demonstrated.

Weaknesses:<br /> MSI-1 provides 8.6-fold downregulation of sfGFP with an optimized mRNA sequence. In some applications, a larger degree of repression may be required.

Reviewer #2 (Public Review):

This model of skeletal muscle includes springs and dampers which aim to capture the effect of crossbridge and titin stiffness during the stretch of active muscle. While both crossbridge and titin stiffness have previously been incorporated, in some form, into models, this model is the first to simultaneously include both. The authors suggest that this will allow for the prediction of muscle force in response to short-, mid- and long-range stretches. All these types of stretch are likely to be experienced by muscle during in vivo perturbations, and are known to elicit different muscle responses. Hence, it is valuable to have a single model which can predict muscle force under all these physiologically relevant conditions. In addition, this model dramatically simplifies sarcomere structure to enable this muscle model to be used in multi-muscle simulations of whole-body movement.

In order to test this model, its force predictions are compared to 3 sets of experimental data which focus on short-, mid- and long-range perturbations, and to the predictions of a Hill-type muscle model. The choice of data sets is excellent and provide a robust test of the model's ability to predict forces over a range of length perturbations. However, I find the comparison to a Hill-type muscle model to be somewhat limiting. It is well established that Hill-type models do not have any mechanism by which they can predict the effect of active muscle stretch. Hence, that the model proposed here represents an improvement over such a model is not a surprise. Many other models, some of which are also simple enough to be incorporated into whole-body simulations, have incorporated mechanistic elements which allow for the prediction of force responses to muscle stretch. It is not clear from the results presented here that this model would outperform such models.

The paper begins by outlining the phenomenological vs mechanistic approaches taken to muscle modelling, historically. It appears, although is not directly specified, that this model combines these approaches. A somewhat mechanistic model of the response of the crossbridges and titin to active stretch is combined with a phenomenological implementation of force-length and force-velocity relationships. This combination of approaches may be useful in improving the accuracy of predictions of muscle models and whole-body simulations, which is certainly a worthy goal. However, it also may limit the insight that can be gained. For example, it does not seem that this model could reflect any effect of active titin properties on muscle shortening. In addition, it is not clear to me, either physiologically or in the model, what drives the shift from the high stiffness in short-range perturbations to the somewhat lower stiffness in mid-range perturbations.

Reviewer #2 (Public Review):

Summary:

This study represents an ambitious endeavor to comprehensively analyze the role of miR-199a/b-5p and its networks in cartilage formation. By conducting experiments that go beyond in vitro MSC differentiation models, more robust conclusions can be achieved.

Strengths:

This research investigates the role of miR-199a/b-5p during chondrogenesis using bioinformatics and in vitro experimental systems. The significance of miRNAs in chondrogenesis and OA is crucial, warranting further research, and this study contributes novel insights.

Weaknesses:

While miR-140 and miR-455 are used as controls, these miRNAs have been demonstrated to be more relevant to Cartilage Homeostasis than chondrogenesis itself. Their deficiency has been genetically proven to induce Osteoarthritis in mice. Therefore, the results of this study should be considered in comparison with these existing findings.

Reviewer #2 (Public Review):

Summary:

Zhang et al investigated the biophysical mechanism of potassium-mediated chemotactic behavior in E coli. Previously, it was reported by Humphries et al that the potassium waves from oscillating B subtilis biofilm attract P aeruginosa through chemotactic behavior of motile P aeruginosa cells. It was proposed that K+ waves alter PMF of P aeruginosa. However, the mechanism was this behaviour was not elusive. In this study, Zhang et al demonstrated that motile E coli cells accumulate in regions of high potassium levels. They found that this behavior is likely resulting from the chemotaxis signalling pathway, mediated by an elevation of intracellular pH. Overall, a solid body of evidence is provided to support the claims. However, the impacts of pH on the fluorescence proteins need to be better evaluated. In its current form, the evidence is insufficient to say that the fluoresce intensity ratio results from FRET. It may well be an artefact of pH change. Nevertheless, this is an important piece of work. The text is well written, with a good balance of background information to help the reader follow the questions investigated in this research work.

In my view, the effect of pH on the FRET between CheY-eYFP and CheZ-eCFP is not fully examined. The authors demonstrated in Fig. S3 that CFP intensity itself changes by KCl, likely due to pH. They showed that CFP itself is affected by pH. This result raises a question of whether the FRET data in Fig3-5 could result from the intensity changes of FPs, but not FRET. The measured dynamics may have nothing to do with the interaction between CheY and CheZ. It should be noted that CFP and YFP have different sensitivities to pH. So, the measurement is likely confounded by the change in intracellular pH. Without further experiments to evaluate the effect of pH on CFP and YFP, the data using this FRET pair is inconclusive.

The data in Figure 1 is convincing. It would be helpful to include example videos. There is also ambiguity in the method section for this experiment. It states 100mM KCl was flown to the source channel. However, it is not clear if 100 mM KCl was prepared in water or in the potassium-depleted motility buffer. If KCl was prepared with water, there would be a gradient of other chemicals in the buffer, which confound the data.

The authors show that the FRET data with both KCl and K2SO4, and concluded that the chemotactic response mainly resulted from potassium ions. However, this was only measured by FRET. It would be more convincing if the motility assay in Fig1 is also performed with K2SO4.

Methods:

- Please clarify the promotes used for the constitutive expression of FliCsticky and LacI.<br /> - Fluorescence filters and imaging conditions (exposure time, light intensity) are missing.<br /> - Please clarify if the temperature was controlled in motility assays.<br /> - L513. It is not clear how theta was selected. Was theta set to be between 0 and pi? If not, P(theta) can be negative?<br /> - Typo in L442 (and) and L519 (Koff)

Reviewer #2 (Public Review):

Summary:

Guan and colleagues address the question of how a single neuroblast produces a defined number of progeny, and what influences its decommissioning. The focus of the experiments are two well-studied RNA-binding proteins: Imp and Syp. The Authors find that these factors play an important role in determining the number of neurons in their preferred model system of VNC motor neurons coming from a single lineage (LinA/15) by separate functions taking place at specific stages of development of this lineage: influencing the life-span of the LinA neuroblast to control its timely decommissioning and functioning in the Late-born post-mitotic neurons to influence cell death after the appropriate number of progeny is generated. The post-mitotic role of Imp/Syp in regulating programmed-cell death (PCD) is also correlated with a specific code of key transcription factors that are suspected to influence neuronal identity, linking the fate of neuronal survival with its specification. This paper addresses a wide scope of phenotypes related to the same factors, thus providing an intriguing demonstration of how the nervous system is constructed by context-specific changes in key developmental regulators.

The bulk of conclusions drawn by the authors are supported by careful experimental evidence, and the findings are a useful addition to an important topic in developmental neuroscience.

Strengths:

A major strength is the use of a genetic labeling tool that allows the authors to specifically analyze and manipulate one neuronal lineage. This allows for simultaneous study of both the progenitors and post-mitotic progeny. As a result the paper conveys a lot of useful information for this particular neuronal lineage. Furthermore addressing the association of cell fate specification, taking advantage of this lab's extensive prior work in the system, with developmentally-regulated programmed cell-death is an important contribution to the field.<br /> Beyond Imp/Syp, additional characterization of this model system is provided in characterizing a previously unrecognized death of a hemilineage in early-born neurons.

Weaknesses:

The main observations that distinguish this study from others that have investigated Imp/Syp in the fly nervous system is the role played in late-born post-mitotic neurons to regulate programmed cell-death. This is an important and plausible (based on the presented findings) newly discovered role for these proteins. However the precision of experiments is not particularly strong, which limits the authors claims. The genetic strategy used to manipulate Imp/Syp or the TF code appears to be done throughout the entire lineage, or all neuronal progeny, and not restricted to only the late born cells. Can the authors rule out survival of the early born hemi-lineage normally fated to die? Therefore statements such as this: To further investigate this possibility, we used the MARCM technique to change the TF code<br /> of last-born MNs without affecting the expression of Imp and Syp<br /> should be qualified to specify that the result is obtained by misexpressing these factors throughout the entire lineage.

The authors make an observation that differs from other systems in which Imp/Syp have been studied: that the expression of the two proteins appears to be independent and not influenced by cross-regulation. However there is a lack of investigation as to what effect this may have on how Imp/Syp regulate temporal identity. A key implication of the previously observed cross-regulation in the fly mushroom body is that the ratio of Imp/Syp could change over the life of the NB which would permit different neuronal identities. Without cross-regulation, do the authors still observe a gradient in the expression pattern of time? Because the data is presented with Imp and Syp stained in different brain samples, and without quantification across different stages, this is unclear. The authors use the term 'gradient' but changes in levels of these factors are not evident from the presented data.

Reviewer #2 (Public Review):

Summary: The authors of this manuscript are interested in discovering and functionally characterizing genes that might cause obesity. To find such genes, they conducted a forward genetic screen in mice, selecting strains which displayed increased body weight and adiposity. They found a strain, with germ-line deficiency in the gene Spag7, which displayed significantly increased body weight, fat mass, and adipose depot sizes manifesting after the onset of adulthood (20 weeks). The mice also display decreased organ sizes, leading to decreased lean body mass. The increased adiposity was traced to decreased energy expenditure at both room temperature and thermoneutrality, correlating with decreased locomotor activity and muscle atrophy. Major metabolic abnormalities such as impaired glucose tolerance and insulin sensitivity also accompanied the phenotype. Unexpectedly, when the authors generated an inducible, whole body knockout mouse using a globally expressed Cre-ERT2 along with a globally floxed Spag7, and induced Spag7 knockout before the onset of obesity, none of the phenotypes seen in the original strain were recapitulated. The authors trace this discrepancy to the major effect of Spag7 being on placental development.

Strengths: Strengths of the manuscript are its inherently unbiased approach, using a forward genetic screen to discover previously unknown genes linked to obesity phenotypes. Another strong aspect of the work was the generation of an independent, complementary, strain consisting of an inducible knockout model, in which the deficiency of the gene could be assessed in a more granular form. This approach enabled the discovery of Spag7 as a gene involved in the establishment of the mature placenta, which determines the metabolic fate of the offspring. Additional strengths include the extensive array of physiological parameters measured, which provided a deep understanding of the whole-body metabolic phenotype and pinpointed its likely origin to muscle energetic dysfunction.

Weaknesses: Weaknesses that can be raised are the lack of molecular mechanistic understanding of the numerous phenotypic observations. For example, the specific role of Spag7 to promote placental development remains unclear. Also, the reason why placental developmental abnormalities lead to muscle dysfunction, and whether indeed the entire metabolic phenotype of the offspring can be attributed solely to decreased muscle energetics is not fully explored.

Overall, the authors achieved a remarkable success in identifying genes associated with development of obesity and metabolic disease, discovering the role of Spag7 in placental development, and highlighting the fundamental role of in-utero development in setting future metabolic state of the offspring.

Reviewer #2 (Public Review):

Summary: There is increasing evidence in the literature that rodent models of stress can produce phenotypes that persist through multiple generations. Nevertheless, the mechanism(s) by which stress exposure produces phenotypes are unknown in the directly affected individual as well as in subsequent offspring that did not directly experience stress. Moreover, it has also been shown that glucocorticoid stress hormones can recapitulate the effects of programmed stress. In this manuscript, the authors test the compelling hypothesis that glucocorticoid receptor (GR)-signaling is responsible for the transmission of phenotypes across generations. As a first step, the investigators test for a role of GR in the male and female germline. Using knockouts and GR agonists, they show that although germ cells in male and female mice have GR that appears to localize to the nucleus when stimulated, oocytes are resistant to changes in GR levels. In contrast, the male germline exhibits changes in splicing but no overt changes in fertility.

Strengths: Although many of the results in this manuscript are negative, this is a careful and timely study that informs additional work to address mechanisms of transmission of stress phenotypes across generations and suggests a sexually dimorphic response to glucocorticoids in the germline. The work presented here is well-done and rigorous and the discussion of the data is thoughtful. Overall, this is an important contribution to the literature.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Yan et al. assess the effect of two facets of habitat fragmentation (i.e., habitat loss and habitat fragmentation per se) on biodiversity, ecosystem function, and the biodiversity-ecosystem function (BEF) relationship in grasslands of an agro-pastoral ecotone landscape in northern China. The authors use stratified random sampling to select 130 study sites located within 500m-radius landscapes varying along gradients of habitat loss and habitat fragmentation per se. In these study sites, the authors measure grassland specialist and generalist plant richness via field surveys, as well as above-ground biomass by harvesting and dry-weighting the grass communities in each 3 x 1m2 plots of the 130 study sites. The authors find that habitat loss and fragmentation per se have different effects on biodiversity, ecosystem function and the BEF relationship: whereas habitat loss was associated with a decrease in plant richness, fragmentation per se was not; and whereas fragmentation per se was associated with a decrease in above-ground biomass, habitat loss was not. Finally, habitat loss, but not fragmentation per se was linked to a decrease in the magnitude of the positive biodiversity-ecosystem functioning relationship, by reducing the percentage of grassland specialists in the community.

Strengths:<br /> This study by Yan et al. is an exceptionally well-designed, well-written, clear and concise study shedding light on a longstanding, important question in landscape ecology and biodiversity-ecosystem functioning research. Via a stratified random sampling approach (cf. also "quasi-experimental design" Butsic et al. 2017), Yan et al. create an ideal set of study sites, where habitat loss and habitat fragmentation per se (usually highly correlated) are decorrelated and hence, separate effects of each of these facets on biodiversity and ecosystem function can be assessed statistically in "real-world" (and not experimental, cf. Duffy et al. 2017) communities. The authors use adequate and well-described methods to investigate their questions. The findings of this study add important empirical evidence from real-world grassland ecosystems that help to advance our theoretical understanding of landscape-moderation of biodiversity effects and provide important guidelines for conservation management.

Weaknesses:<br /> I found only a few minor issues, mostly unclear descriptions in the study that could be revised for more clarity.

References:<br /> Butsic, V., Lewis, D. J., Radeloff, V. C., Baumann, M., & Kuemmerle, T. (2017). Quasi-experimental methods enable stronger inferences from observational data in ecology. Basic and Applied Ecology, 19, 1-10.

Duffy, J.E., Godwin, C.M. & Cardinale, B.J. (2017). Biodiversity effects in the wild are common and as strong as key drivers of productivity. Nature.

Reviewer #2 (Public Review):

Summary:

Although Trabid missense mutations are identified across a range of neurodevelopmental disorders, its role in neurodevelopment is not understood. Here the authors study two different patient mutations and implicate defects in its deubiquitylating activity and interactions with STRIPAK. Knockin mice for these mutations impaired trafficking of APC to microtubule plus ends, with consequent defects in neuronal growth cone and neurite outgrowth.

The authors focus on R438W and A451V, two missense mutations seen in patients. Recombinant fragments showed R438W is nearly completely DUB-dead whereas A451V showed normal activity but failed to efficiently precipitate STRIPAK. Knockin of these mutations showed a partially penetrant reduced cortical neuronal and glial cell numbers and reduced TH+ neurons and their neuronal processes. Cell culture demonstrated that both DUB and STRIPAK-binding activities of Trabid are required for efficient deubiquitylation of APC in cells, and alter APC transport along neurites. APC-tdTomato fluorescent reporter mice crossed with the Trabid mutants confirmed these results. The results suggest that Trabid's mechanism of action is to suppress APC ubiquitylation to regulate its intracellular trafficking and neurite formation.

Strengths:

Solid manuscript with in vivo and in vitro demonstration of mechanism of action

Weaknesses:

Much of the work relies on prior discoveries of Trabid's role in STRIPAK and APC related functions, so the novelty is somewhat reduced.

Reviewer #2 (Public Review):

Summary:

In this study, Winstel and colleagues test if the deoxycytidine kinase inhibitor, (R)-DI-87 provides therapeutic benefit during infection with Staphylococcus aureus. The premise behind the current work is a series of prior studies that found that S. aureus can disable functional immune clearance by generating NET-derived deoxyribonucleosides to induce macrophage apoptosis via purine salvage. Here, the authors use in vitro and in vivo experiments with (R)-DI-87 to demonstrate that inhibition of deoxycytidine kinase prevents S. aureus-induced deoxyribonucleoside-mediated macrophage cell death, to bolster immune cell function and promote more effective clearance during infection. The authors conclude that (R)-DI-87 represents and potentially important Host-Directed Therapy (HDT) with good potential to promote natural clearance of infection without targeting the bacterium. Overall, the study represents an important next step in the exploration of purine salvage and deoxyribonucleoside toxicity as a targetable pathway to bolster infection clearance and provides early-stage evidence of the therapeutic potential of (R)-DI-87 during S. aureus infection.

Strengths:

The study has several strengths that support its conclusions:<br /> 1. Well-controlled in vitro studies that firmly establish (R)-DI-87 is capable of blocking deoxyribonucleoside-mediated apoptosis of immune cell lines and primary cells.<br /> 2. Solid evidence to support that administration of (R)-DI-87 can have therapeutic benefits during infection (reduced number of abscesses and reduced CFU).<br /> 3. Controls included to ascertain the degree to which (R)-DI-87 might have secondary effects on immune cell distribution.<br /> 4. Controls included to ascertain whether or not (R)-DI-87 has intrinsic antibacterial properties.

Weaknesses:

However, there are several important weaknesses related to the rigor of the research and the conclusions drawn. The most relevant weaknesses noted by this reviewer are:

1. Drawing firm conclusions about the therapeutic potential of (R)-DI-87 using only S. aureus strain Newman, a methicillin-susceptible S. aureus, that while a clinical isolate is not clearly representative of the strains of S. aureus causing infection in hospitals and communities. Newman also harbors an unusual mutation in a regulator that dramatically changes virulence factor gene expression. While the data with Newman remains valuable, the absence of consideration of other strains, including MRSA, makes it more difficult to support the relatively broad conclusions about therapeutic potential made by the authors.

2. In vitro (R)-DI-87 efficacy studies with dAdo and dGuo are strong, however, the authors do not test the in vitro efficacy of (R)-DI-87 using S. aureus. They have done this type of work in prior studies (See doi: 10.1073/pnas.1805622115 - Figure 5). If included it would greatly strengthen their argument that (R)-DI-87 is directly affecting the S. aureus --> Nuclease --> AdsA macrophage-killing pathway. Without it, the evidence provided remains indirect, and several conclusions may be overstated.

3. Caspase-3 immunoblot experiments seem to suggest an alternative conclusion to what was made by the authors. They point out that Caspase-3 cleavage does not occur upon treatment with (R)-DI-87. However, the data seem to argue that there is almost no caspase-3 present in (R)-DI-87 treated cells (cleaved or uncleaved). Might this suggest that caspase-3 is not even produced when cells are not experiencing deoxyribonucleoside toxicity? Perhaps the authors could reconsider the interpretation of this data.

4. There are some concerns over experimental rigor and clarity of the experimental design in the methods. The most important points noted by this reviewer are included here. (a.) There is no description of the number of replicates or representation of the Western blots and no uncropped blots are provided. (b.) the methods describing the treatment conditions for in vivo studies are not sufficiently clear. For example, it is hard to tell when (R)-DI-87 is first administered to mice. Is it immediately before the infection, immediately after, or at the same time? This has important implications for interpreting the results in terms of therapeutic potential. (c.) There are several statements made that (R)-DI-87 does not have a negative impact on the mice however, it is not sufficiently clear that the studies conducted are sufficient to make this broader claim that (R)-DI-87 has no impact on the animal, except as it relates to the distribution of immune cells, which is directly tested. (d.) there are no quantitative measures of apoptosis or macrophage infiltration, which impacts the rigor of these imaging experiments. (d.) only female mice are used in the in vivo studies. There is no justification provided for this choice; however, the rigor of the study design and the ability to draw conclusions about therapeutic potential is impacted in the absence of consideration of both sexes.

5. Animal studies show significant disease burden (CFU) even after administration of (R)-DI-87. Given the absence of robust clearance of infection, the author's claims read as an overstatement of the data. The authors may wish to reframe their conclusions to better highlight the potential benefit of this therapy at reducing severe disease but also to point out relevant limitations, especially considering that it does not lead to clearance in this model. In general, the consideration of the limitations of the proposed therapeutic approach, as uncovered by the data, is not present. A more nuanced consideration of the data and its interpretations, including both strengths and limitations, would greatly help to frame the study.

Reviewer #2 (Public Review):

Summary:

The authors conducted research on the role of SerpinE2 and HtrA1 in neural crest migration using Xenopus embryos. The data presented in this study was of high quality and supported the authors' conclusions. The discovery of the potential molecular connection between SerpinE2 and HtrA1 in neural crest cell migration in vivo is significant, as understanding this pathway could potentially lead to treatments for aggressive cancers and pregnancy-related disorders.

Strengths:

Previous research has shown that SerpinE2 and HtrA1 can have both positive and negative effects on cell migration, but their molecular interplay and role in neural crest migration are not well-established. This study is the first to reveal a potential connection between these two proteins in neural crest cell migration in vivo. The authors found that SerpinE2 promotes neural crest migration by inhibiting HtrA1. Additionally, overexpression of Sdc4 partly alleviates neural crest migration issues caused by SerpinE2 knockdown or HtrA1 overexpression. These findings suggest that the SeprinE2-HtrA1-Sdc4 pathway is crucial for neural crest migration.

Weaknesses:

To further increase the study's credibility, the authors could use techniques like western blotting, qRT-PCR, or in situ hybridization to verify the efficiency of SerpinE2 and HtrA1 knockdown and/or overexpression. Furthermore, determining whether the observed craniofacial phenotypes in SerpinE2 and/or HtrA1 mutants were solely due to modified cranial neural crest migration or affected by other factors such as cell proliferation, cell survival, and chondrogenic differentiation could provide more clarity. Lastly, it is unclear whether the SeprinE2-HtrA1-Sdc4 pathway is constant in both cranial and trunk neural crest migration.

Reviewer #2 (Public Review):

Summary:<br /> The study provides valuable and compelling evidence that while activation of the mTOR cascade confers some similarities in alterations in cell size, mTOR pathway activation, cortical lamination, baseline firing properties, and synaptic activity, there are distinctions that could account for clinical differences in seizure and epilepsy phenotypes in patients harboring these mutations. These findings could have important implications going forward as we design clinical therapeutic strategies to modulate mTOR activity in these individuals to treat seizures.

This study presents a valuable finding on the role that distinct mTOR pathway genes play in altered cell shape, cortical laminar migration, and cellular excitability in the mouse medial prefrontal cortex (mPFC). The evidence supporting the claims of the authors is solid, although analysis of the role of the mTORC2 pathway and consideration of distinct metabolic states i.e., amino acid levels would have strengthened the study. The work will be of interest to neuroscientists working on human epilepsy. These genes have each been assayed in previous independent studies and thus the direct comparison is what provides the innovation and interest.

The manuscript by Nguyen and colleagues attempts to define both the common and differential roles of mTOR pathway genes, both by gene knockout (KO) and activation, on cortical neuronal size, cortical lamination, and excitability. They focused on 5 genes that have been linked to human malformations of cortical development (MCD) and epilepsy: RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO. The RhebY35L, mTORS2215Y are known and pathogenic human gain-of-function variants. Each of these genes is known to modulate the activity of mTORC1 and either KO or activation will lead to abnormal and persistent hyperactivation of mTOR activity. Using in utero electroporation they transfected plasmids containing these gene constructs into fetal mouse brains at E15.5 and then assessed neuronal shape and size, laminar positioning, spontaneous activity, synaptic activity, and expression of a novel voltage-gated potassium channel (HCN4) at varying time postnatally e.g., P7-9 (neonates) and P28-43 (young adults).

The study clearly achieves its stated aims i.e., that disruption of each of five distinct mTOR pathway genes, Rheb, mTOR, Depdc5, Pten, and Tsc1, individually impacts pyramidal neuron development and electrophysiological function in the mouse mPFC. The data from each of the 5 genes provides strong support to the notion that mTOR pathway gene mutations yield the unifying clinical parcellation of mTORopathies, likely as a consequence of mTOR pathway activation. The data also provide interesting evidence that subtle or even overt differences in clinical phenotypes between RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO in humans could be due to effects of these genes either on mTOR or on yet to be defined alternative pathways. Assuredly follow-up studies to examine how Rheb, mTOR, Dedpdc5, Pten, and Tsc1 engage with other protein binding partners or other pathways will be warranted in future studies.

Strengths:<br /> The investigators demonstrate that gene KO or activation leads to common changes in cell size (enlargement) though with different effects across each gene subtype suggesting distinct genetic effects despite a common effect on mTOR signaling. The major effect was seen in forebrain neurons expressing mTORS2215Y. They also report gene-specific effects of each mTOR pathway gene on cortical lamination. For example, while RhebY35L, mTORS2215Y, Dedpdc5KO, and Tsc1KO significantly disrupted laminar positioning of neurons in layer 2/3, PtenKO had minimal effects on laminar positioning. This finding is intriguing since it means that simply activating mTOR during fetal brain development will not necessarily alter cortical lamination and that an increase in cell size by itself doesn't disrupt laminar fidelity. To verify that the expression of plasmids led to mTORC1 hyperactivation, phosphorylated levels of S6 (i.e., p-S6), a downstream substrate of mTORC1, were assayed by immunohistochemistry in P28-43 mice. Expression of the RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO plasmids all led to significantly increased p-S6 staining intensity, supporting that the expression of each of these plasmids leads to increased mTORC1 signaling.

Whole-cell current- and voltage-clamp recordings were performed in P25-P51 mice in acute brain slice preparations. Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to decreased depolarization-induced excitability, but only RhebY35L, mTORS2215Y, and Tsc1KO expression led to depolarized resting membrane potentials. Interestingly, expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to the abnormal presence of HCN4 channels with variations in functional expression suggesting a common cellular mechanism that could confer excitability. Treatment with rapamycin, an mTOR inhibitor, reversed the expression changes in HCN4. Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to different impacts on sEPSC properties. Effects of treatment with the selective HCN channel blocker zatebradine on hyperpolarization-induced inward currents in mTORS2215Y neurons confirmed the identity of ΔI as Ih.

Overall the data presented provides a convincing and compelling direct comparison of the roles that select mTOR pathway genes play on brain development and network excitability. It is critical to directly compare these gene effects in mouse models because although these genes are part of the mTOR pathway and clearly cause augmentation of mTOR activation, there are mechanistic differences in how these gees modify mTOR and how they interact with other proteins and phenotypic differences in humans harboring mutations in these same genes.

Weaknesses:<br /> There are a few limitations to an otherwise solid study. First, the authors postulate that all the findings are dependent on mTORC1-related effects but don't assess whether some of the differences could be due to effects on mTORC2 signaling. mTORC2 is an important and poorly understood alternative isoform of mTOR (due to rictor binding) that has effects on distinct cell signaling pathways and in particular actin polymerization. This doesn't diminish the effects of the current analysis of mTORC1 but could explain genotypic differences in each variable. A few prior studies have assessed the role of mTORC2 in epileptogenesis and Cortical malformations (Chen et al., 2019)

Second, the slice recordings were performed in the usual recording aCSF buffer conditions but there is no assessment of the role of amino acids or nutrients in the bath. While it is clear that valuable and viable acute slice recordings can be made in aCSF, the role of the mTOR pathway is to modulate cell growth in response to nutrient conditions. Thus, one variable that could be manipulated and assessed currently in this study is the levels of amino acids i.e., leucine and arginine added to the bath since DEPDC5 and TSC1 are responsive to ambient amino acid levels.

Third, the analysis concedes that the role of somatic mutations in cortical malformations may depend not only on genotypic effects but also on allelic load and cellular subtype affected by the mutation. Thus, it would interesting to see if electroporation either at E14 or E16, thereby affecting a distinct pool of progenitors, would mitigate or accentuate differences between mTOR pathway genes.

Treatment with rapamycin and zatebradine in each condition would have added to the strength of the findings to determine the mTOR-dependence and reversibility of HCN4 effects.

Reviewer #2 (Public Review):

Summary:

The authors present here a mathematical and computational study of the topological/graph theory requirements to obtain sustained oscillations in neural network models. A first approach mathematically demonstrates that, a given network of interconnected neural populations (understood in the sense of dynamical systems) requires an odd number of inhibitory populations to sustain oscillations. The authors extend this result via numerical simulations of (i) a simplified set of Wilson-Cowan networks, (ii) a simplified circuit of the cortico-basal ganglia network, and (iii) a more complex, spike-based neural network of basal ganglia network, which provides insight on experimental findings regarding abnormal synchrony levels in Parkinson's Disease (PD).

Strengths:

The work elegantly and effectively combines solid mathematical proof with careful numerical simulations at different levels of description, which is uncommon and provides additional layers of confidence to the study. Furthermore, the authors included detailed sections to provide intuition about the mathematical proof, which will be helpful for readers less inclined to the perusal of mathematical derivations. Its insightful and well-informed connection with a practical neuroscience problem, the presence of strong beta rhythms in PD, elevates the potential influence of the study and provides testable predictions.

Weaknesses:

In its current form, the study lacks a more careful consideration of the role of delays in the emergence of oscillations. Although they are addressed at certain points during the second part of the study, there are sections in which this could have been done more carefully, perhaps with additional simulations to solidify the authors' claims. Furthermore, there are several results reported in the main figures which are not explained in the main text. From what I can infer, these are interesting and relevant results and should be covered. Finally, the text would significantly benefit from a revision of the grammar, to improve the general readability at certain sections. I consider that all these issues are solvable and this would make the study more complete.

Reviewer #2 (Public Review):

This is a very interesting paper about the coupling of Slack and Nav1.6 and the insight this brings to the effects of quinidine to treat some epilepsy syndromes.

Slack is a sodium-activated potassium channel that is important to hyperpolarization of neurons after an action potential. Slack is encoded by KNCT1 which has mutations in some epilepsy syndromes. These types of epilepsy are treated with quinidine but this is an atypical antiseizure drug, not used for other types of epilepsy. For sufficient sodium to activate Slack, Slack needs to be close to a channel that allows robust sodium entry, like Nav channels or AMPA receptors. but more mechanistic information is not available. Of particular interest to the authors is what allows quinidine to be effective in reducing Slack.

In the manuscript, the authors show that Nav, not AMPA receptors, are responsible for Slack's sensitization to quinidine blockade, at least in cultured neurons (HeK293, primary cortical neurons). Most of the paper focuses on the evidence that Nav1.6 promotes Slack sensitivity to quinidine.

Reviewer #2 (Public Review):

Summary:

This work by Kadeřábková et al. demonstrates the importance of a specific protein folding system to effectively folding β-lactamase proteins, which are responsible for resistance to β-lactam antibiotics, and shows that inhibition of this system sensitize multidrug-resistant pathogens to β-lactam treatment. In addition, the authors extend these observations to a two-species co-culture model where β-lactamases provided by one pathogen can protect another, sensitive pathogen from β-lactam treatment. In this model, disrupting the protein folding system also disrupted protection of the sensitive pathogen from antibiotic killing. Overall, the data presented provide a solid foundation for subsequent investigations and development of inhibitors for β-lactamases and other resistance determinants. This and similar strategies may have particular application to polymicrobial contexts, but the present state of knowledge regarding the existence and clinical effects of microbial interactions in disease, both specifically regarding S. maltophilia and P. aeruginosa as well as generally, is significantly overstated.

Strengths:

The authors use clear and reliable molecular biology strategies to show that β-lactamase proteins from P. aeruginosa and Burkholderia species, expressed in E. coli in the absence of the dsbA protein folding system, are variably less capable of resisting the effects of different β-lactam antibiotics compared to the dsbA-competent parent strain (Figure 1). The appropriate control is included in the supplemental materials to demonstrate that this effect is specifically dependent on dsbA, since complementing the mutant with an intact dsbA gene restores antibiotic resistance (Figure S1). The authors subsequently show that this lack of activity can be explained by significantly reduced protein levels and loss-of-function protein misfolding in the dsbA mutant background (Figure 2). These data support the importance of this protein folding mechanism in the activity of multiple clinically relevant β-lactamases.

Native bacterial species are used for subsequent experiments, and the authors provide important context for their antibiotic choices and concentrations by referencing the breakpoints that guide clinical practice. In Figure 4, the authors show that loss of the DsbA system in P. aeruginosa significantly sensitizes clinical isolates expressing different classes of β-lactamases to clinically relevant antibiotics. The appropriate control showing that the dsbA1 mutation does not result in sensitivity to a non-β-lactam antibiotic is included in Figure S2. The authors further show, using an in vivo model for antibiotic treatment, that treatment of a dsbA1 mutant results in moderate and near-complete survival of the infected organisms. The importance of this system in S. maltophilia is then investigated similarly (Figure 5), showing that a dsbA dsbL mutant is also sensitive to β-lactams and colistin, another antibiotic whose resistance mechanism is dependent on the DsbA protein folding system. Importantly, the authors show that a small-molecule inhibitor that disrupts the DsbA system, rather than genetic mutations, is also capable of sensitizing S. maltophilia to these antibiotics. It should be noted that while the sensitization is less pronounced, this molecule has not been optimized for S. maltophilia and would be expected to increase in efficacy once this is done. Together, the data support that interference with the DsbA system in native hosts can sensitize otherwise resistant pathogens to clinically relevant antibiotic therapy.

Finally, the authors investigate the effects of co-culturing S. maltophilia and P. aeruginosa (Figure 5E). These assays are performed in synthetic cystic fibrosis sputum medium (SCFM), which provides a nutritional context similar to that in CF but without the presence of more complex components such as mucin. The authors show that while P. aeruginosa alone is sensitive to the antibiotic, it can survive moderate concentrations in the presence of S. maltophilia and even grow in higher concentrations where S. maltophilia appears to overproduce its β-lactamases. However, this protection is lost in S. maltophilia without the DsbA protein folding system, showing that the protective effect depends on functional production of β-lactamase. The data support a protective role for DsbA-dependent β-lactamase under these co-culture conditions.

Weaknesses:

While Figure 5E demonstrates a protective effect of DsbA-dependent β-lactamase, the omission of CFU data for S. maltophilia makes it difficult to assess the applicability of the polymicrobial strategy. Since S. maltophilia is pre-cultured prior to the addition of P. aeruginosa and antibiotics, it is unclear whether the protective effect is dependent on high S. maltophilia CFU. It is also unclear what the fate of the S. maltophilia dsbA dsbL mutant is under these conditions. If DsbA-deficient S. maltophilia CFU is not impacted, then this treatment will result in the eradication of only one of the pathogens of interest. If the mutant is lost during treatment, then it is not clear whether the loss of protection is due specifically to the production of non-functional β-lactamase or simply the absence of S. maltophilia.

The alleged clinical relevance and immediate, theoretical application of this approach should be properly contextualized. At multiple junctures, the authors state or suggest that interactions between S. maltophilia and P. aeruginosa are known to occur in disease or have known clinical relevance related to treatment failure and disease states. For instance, the citations provided for S. maltophilia protection of P. aeruginosa in the CF lung environment both describe simplified laboratory experiments rather than clinical or in vivo observations. Similarly, the citations provided for both the role of S. maltophilia in treatment failure and CF disease severity do not support either claim. The role of S. maltophilia in CF is currently unsettled, with more recent work reporting conflicting results that support S. maltophilia as a marker, rather than cause, of severe disease. These citations also do not support the suggestion that S. maltophilia specifically contributes to treatment failure. While it is reasonable to pursue these ideas as a hypothesis or potential concern, there is no evidence provided that these specific interactions occur in vivo or that they have clinical relevance.

Reviewer #2 (Public Review):

In this study, Dietmar Funck and colleagues have made a significant breakthrough by identifying three isoforms of plant 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) as homo/arginine-6-hydroxylases, catalyzing the degradation of 6-hydroxyhomoarginine into 2-aminoadipate-6-semialdehyde (AASA) and guanidine. This discovery marks the very first confirmation of plant or eukaryotic enzymes capable of guanidine production.

The authors selected three plant 2-ODD-C23 enzymes with the highest sequence similarity to bacterial guanidine-producing (EFE) enzymes. They proceeded to clone and express the recombinant enzymes in E coli, demonstrating capacity of all three Arabidopsis isoforms to produce guanidine. Additionally, by precise biochemical experiments, the authors established these three 2-ODD-C23 enzymes as homoarginine-6-hydroxylases (and arginine-hydroxylase for one of them). Furthermore, the authors utilized transgenic plants expressing GFP fusion proteins to show the cytoplasmic localization of all three 2-ODD-C23 enzymes. Most notably, using T-DNA mutant lines and CRISPR/Cas9-generated lines, along with combinations of them, they demonstrate the guanidine-producing capacity of each enzyme isoform in planta. These results provide robust evidence that these three 2-ODD-C23 Arabidopsis isoforms are indeed homoarginine-6-hydroxylases responsible for guanidine generation.<br /> The findings presented in this manuscript are a significant contribution for our understanding of plant biology, particularly given that this work is the first demonstration of enzymatic guanidine production in eukaryotic cells. However, there are a couple of concerns and potential ways for further investigation that the authors should (consider) incorporate.

Firstly, the observation of cytoplasmic and nuclear GFP signals in the transgenic plants may also indicate cleaved GFP from the fusion proteins. Thus, the authors should perform Western blot analysis to confirm the correct size of the 2-ODD-C23 fusion proteins in the transgenic protoplasts.

Secondly, it may be worth measuring pipecolate (and proline?) levels under biotic stress conditions (particularly those that induce transcript changes of these enzymes, Fig S8). Given the results suggesting a potential regulation of the pathway by biotic stress conditions (eg. meJA), these experiments could provide valuable insights into the physiological role of guanidine-producing enzymes in plants. This additional analysis may give a significance of these enzymes in plant defense mechanisms.