Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Brotherton et al. describes a structural study of connexin-26 (Cx26) gap junction channel mutant K125E, which is designed to mimic the CO2-inhibited form of the channel. In the wild-type Cx26, exposure to CO2 is presumed to close the channel through carbamylation of the residue K125. The authors mutated K125 to a negatively charged residue to mimic this effect, and they observed by cryo-EM analysis of the mutated channel that the pore of the channel is constricted. The authors were able to observe conformations of the channel with resolved density for the cytoplasmic loop (in which K125 is located). Based on the observed conformations and on the position of the N-terminal helix, which is involved in channel gating and in controlling the size of the pore, the authors propose the mechanisms of Cx26 regulation.

Strengths:<br /> This is a very interesting and timely study, and the observations provide a lot of new information on connexin channel regulation. The authors use the state of the art cryo-EM analysis and 3D classification approaches to tease out the conformations of the channel that can be interpreted as "inhibited", with important implications for our understanding of how the conformations of the connexin channels controlled.

Weaknesses:<br /> My fundamental question to the premise of this study is: to what extent can K125 carbamylation by recapitulated by a simple K125E mutation? Lysine has a large side chain, and its carbamylation would make it even slightly larger. While the authors make a compelling case for E125-induced conformational changes focusing primarily on the negative charge, I wonder whether they considered the extent to which their observation with this mutant may translate to the carbamoylated lysine in the wild-type Cx26, considering not only the charge but also the size of the modified side-chain.

Reviewer #2 (Public Review):

In this manuscript, the authors set out to identify transporters that must exist in Stramenophiles due to the fact that the second half of glycolysis appears to be conducted in the mitochondria. They hypothesize that a Stramenophile-specific clade of transporters related to the dicarboxylate carriers is likely the relevant family and then go on to test two proteins from Blastocystis due to the infectious disease relevance of this organism. They show rather convincingly that these two proteins are expressed and are localized to the mitochondria in the native organism. The purified proteins bind to glycolytic intermediates and one of them, GIC-2, transports several glycolytic intermediates in vitro. This is a very solid and well-executed study that clearly demonstrates that bCIC-2 can transport glycolytic intermediates.

1. The major weakness is that the authors aren't able to show that this protein actually has this function in the native organism. This could be impossible due to the lack of genetic tools in Blastocystis, but it leaves us without absolute confidence that bGIC-2 is the important glycolytic intermediate mitochondrial transporter (or even that it has this function in vivo).

2. It's atypical that the figures and figure panels don't really follow the order of their citation in the text. It's not a big deal, but mildly annoying to have to skip around in the figures (e.g. Figure 3D-E are described in the same paragraph as Figure 5). In addition, to facilitate the flow and a proper understanding I would encourage a reordering between figures 5D and 6 since Figure 6 is needed to understand the results shown in panel 5D, which may lead to confusion.

3. My impression is that the authors under-emphasize the fact that the hDIC also binds (and is stabilized by) glycolytic intermediates (G3P and 3PG). In the opinion of this reviewer, this might change the interpretation about the uniqueness of the bGIC proteins. They act on additional glycolytic intermediates, but it's not unique.

Reviewer #2 (Public Review):

This manuscript reports the discovery and analysis of a large protein complex that controls mating type and sexual reproduction of the model ciliate Tetrahymena thermophila. In contrast to many organisms that have two mating types or two sexes, Tetrahymena is multi-sexual with 7 distinct mating types. Previous studies identified the mating type locus, which encodes two transmembrane proteins called MTA and MTB that determine the specificity of mating type interactions. In this study, mutants are generated in the MTA and MTB genes and mutant isolates are studied for mating properties. Cells missing either MTA or MTB failed to co-stimulate wild-type cells of different mating types. Moreover, a mixture of mutants lacking MTA or MTB also failed to stimulate. These observations support the conclusion that MTA and MTB may form a complex that directs mating-type identity. To address this, the proteins were epitope-tagged and subjected to IP-MS analysis. This revealed that MTA and MTB are in a physical complex, and also revealed a series of 6 other proteins (MRC1-6) that together with MTA/B form the mating type recognition complex (MTRC). All 8 proteins feature predicted transmembrane domains, three feature GFR domains, and two are predicted to function as calcium transporters. The authors went on to demonstrate that components of the MTRC are localized on the cell surface but not in the cilia. They also presented findings that support the conclusion that the mating type-specific region of the MTA and MTB genes can influence both self- and non-self-recognition in mating.

Taken together, the findings presented are interesting and extend our understanding of how organisms with more than two mating types/sexes may be specified. The identification of the six-protein MRC complex is quite intriguing. It would seem important that the function of at least one of these subunits be analyzed by gene deletion and phenotyping, similar to the findings presented here for the MTA and MTB mutants. A straightforward prediction might be that a deletion of any subunit of the MRC complex would result in a sterile phenotype. The manuscript was very well written and a pleasure to read.

Reviewer #2 (Public Review):

The current manuscript undoubtedly demonstrates that JAG1 can induce osteogenesis via non-canonical signaling. Using the mouse-calvarial critical defect model, the authors have clearly shown the anabolic regenerative effect of JAG1 via non-canonical pathways. Exploring the molecular mechanisms, the authors have shown that non-canonically JAG1 regulates multiple pathways including STAT5, AKT, P38, JNK, NF-ĸB, and p70 S6K, which together possibly culminate in the activation of p70 S6K. More analysis is required to strongly conclude the role of the JAG1-p70 S6K pathway in the process. In summary, these findings have significant implications for designing new approaches for bone regenerative research.

Reviewer #2 (Public Review):

This study elucidates the toxic effects of the lipid aldehyde trans-2-hexadecenal (t-2-hex). The authors show convincingly that t-2-hex induces a strong transcriptional response, leads to proteotoxic stress, and causes the accumulation of mitochondrial precursor proteins in the cytosol.<br /> The data shown are of high quality and well controlled. The genetic screen for mutants that are hyper-and hypo-sensitive to t-2-hex is elegant and interesting, even if the mechanistic insights from the screen are rather limited. The last part of the study is less convincing. The authors show evidence that t-2-hex affects subunits of the TOM complex. However, they do not formally demonstrate that the lipidation of a TOM subunit is responsible for the toxic effect of t-2-hex. A t-2-hex-resistant TOM mutant was not identified. Moreover, it is not clear whether the concentrations of t-2-hex in this study are physiological. This is, however, a critical aspect. The literature is full of studies claiming the toxic effects of compounds such as H2O2; even if such studies are technically sound, they are misleading if non-physiological concentrations of such compounds were used.<br /> Nevertheless, this is an interesting study of high quality. A few specific aspects should be addressed.

Reviewer #2 (Public Review):

In the manuscript by Weber and colleagues, the authors investigated the role of a DEAD-box helicase DDX6 in regulating mRNA stability upon ribosome slowdown in human cells. The authors knocked out DDX6 KO in HEK293T cells and showed that the half-life of a reporter containing a rare codon repeat is elongated in the absence of DDX6. By analogy to the proposed function of fission yeast Dhh1p (DDX6 homolog) as a sensor for slow ribosomes, the authors demonstrated that recombinant DDX6 interacted with human ribosomes. The interaction with the ribosome was mediated by the FDF motif of DDX6 located in its RecA2 domain, and rescue experiments showed that DDX6 requires the FDF motif as well as its interaction with the CCR4-NOT deadenylase complex and ATPase activity for degrading a reporter mRNA with rare codons. To identify endogenous mRNAs regulated by DDX6, they performed RNA-Seq and ribosome footprint profiling. The authors focused on mRNAs whose stability is increased in DDX6 KO cells with high local ribosome density and validated that such mRNA sequences induced mRNA degradation in a DDX6-dependent manner.

The experiments were well-performed, and the results clearly demonstrated the requirement of DDX6 in mRNA degradation induced by slowed ribosomes. However, in some cases, the authors interpreted their data in a biased way, possibly influenced by the yeast study, and drew too strong conclusions. In addition, the authors should have cited important studies about codon optimality in mammalian cells. This lack of information hinders placing their important discoveries in a correct context.

1) Although the authors concluded that DDX6 acts as a sensor of the slowed ribosome, it is not clear if DDX6 indeed senses the ribosome speed. What the authors showed is a requirement of DDX6 for mRNA decay induced by rare codons, and DDX6 binds to the ribosome to exert this role. For example, DDX6 may bridge the sensor and decay machinery on the ribosome. Without structural or biochemical data on the recognition of the slowed ribosome by DDX6, the role of DDX6 as a sensor remains one of the possible models. It should be described in the discussion section.

2) It is not clear if DDX6 directly binds the ribosome. The authors used ribosomes purified by sucrose cushion, but ribosome-associating and FDF motif-interacting factors might remain on ribosomes, even after RNaseI treatment. Without structural or biochemical data of the direct interaction between the ribosome and DDX6, the authors should avoid description as if DDX6 directly binds to the ribosome.

3) Although the authors performed rigorous reporter assays recapitulating the effect of ribosome-retardation sequences on mRNA stability, this is not the first report showing that codon optimality determines mRNA stability in human cells. The authors did not cite important previous studies, such as Wu et al., 2019 (PMID: 31012849), Hia et al., 2019 (PMID: 31482640), Narula et al., 2019 (PMID: 31527111), and Forrest et al., 2020 (PMID: 32053646). These milestone papers should be cited in the Introduction, Results, and Discussion.

4) While both DDX6 and deadenylation by the CCR4-NOT were required for mRNA decay by the slowed ribosome, whether DDX6 is required for deadenylation was not investigated. Given that the CCR4-NOT deadenylate complex directly interacts with the empty ribosome E-site in yeast and humans (Buschauer et al., 2020 PMID: 32299921 and Absmeier et al., 2023 PMID: 37653243), whether the loss of DDX6 also affected the action of the CCR4-NOT complex is an important point to investigate, or at least should be discussed in this paper.

Reviewer #3 (Public Review):

Goetz, Akl and Dixit investigated the heterogeneity in the fidelity of sensing the environment by individual cells in a population using computational modeling and analysis of experimental data for two important and well-studied mammalian signaling pathways: (insulin-like growth factor) IGF/FoxO and (epidermal growth factor) EFG/EFGR mammalian pathways. They quantified this heterogeneity using the conditional mutual information between the input (eg. level of IGF) and output (eg. level of FoxO in the nucleus), conditioned on the "state" variables which characterize the signaling pathway (such as abundances of key proteins, reaction rates, etc.) First, using a toy stochastic model of a receptor-ligand system - which constitutes the first step of both signaling pathways - they constructed the population average of the mutual information conditioned on the number of receptors and maximized over the input distribution and showed that it is always greater than or equal to the usual or "cell state agnostic" channel capacity. They constructed the probability distribution of cell state dependent mutual information for the two pathways, demonstrating agreement with experimental data in the case of the IGF/FoxO pathway using previously published data. Finally, for the IGF/FoxO pathway, they found the joint distribution of the cell state dependent mutual information and two experimentally accessible state variables: the response range of FoxO and total nuclear FoxO level prior to IGF stimulation. In both cases, the data approximately follow the contour lines of the joint distribution. Interestingly, high nuclear FoxO levels, and therefore lower associated noise in the number of output readout molecules, is not correlated with higher cell state dependent mutual information, as one might expect. This paper contributes to the vibrant body of work on information theoretic characterization of biochemical signaling pathways, using the distribution of cell state dependent mutual information as a metric to highlight the importance of heterogeneity in cell populations. The authors suggest that this metric can be used to infer "bottlenecks" in information transfer in signaling networks, where certain cell state variables have a lower joint distribution with the cell state dependent mutual information.

The utility of a metric based on the conditional mutual information to quantify fidelity of sensing and its heterogeneity (distribution) in a cell population is supported in the comparison with data. Some aspects of the analysis and claims in the main body of the paper and SI need to be clarified and extended.

Remaining Comments:

- I think Review Figure 2 which is currently in the SI would improve the main body of the paper if moved there. In that case, the discussion of this figure in the main text would have to address more than it currently does, namely "the same cell's FoxO responses to the same input were found to have significantly less variation compared to the variation within the population".

Reviewer #2 (Public Review):

Summary: In this study, the authors performed a screening for PDXP inhibitors to identify compounds that could increase levels of pyridoxal 5'- phosphate (PLP), the co-enzymatically active form of vitamin B6. For the screening of inhibitors, they first evaluated a library of about 42,000 compounds for activators and inhibitors of PDXP and secondly, they validated the inhibitor compounds with a counter-screening against PGP, a close PDXP relative. The final narrowing down to 7,8-DHF was done using PLP as a substrate and confirmed the efficacy of this flavonoid as an inhibitor of PDXP function. Physiologically, the authors show that, by acutely treating isolated wild-type hippocampal neurons with 7,8-DHF they could detect an increase in the ratio of PLP/PL compared to control cultures. This effect was not seen in PDXP KO neurons.

Strengths: The screening and validation of the PDXP inhibitors have been done very well because the authors have performed crystallographic analysis, a counter screening, and mutation analysis. This is very important because such rigor has not been applied to the original report of 7,8 DHF as an agonist for TrkB. Which is why there is so much controversy on this finding.

Weaknesses: As mentioned in the summary report the study may benefit from some in vivo analysis of PLP levels following 7,8-DHF treatment, although I acknowledge that it may be challenging because of the working out of the dosage and timing of the procedure.

Reviewer #2 (Public Review):

Summary:

In this study, Kashio et al examined the role of TRPV4 in regulating perspiration in mice. They find coexpression of TRPV4 with the chloride channel ANO1 and aquaporin 5, which implies possible coupling of heat sensing through TRPV4 to ion and water excretion through the latter channels. Calcium imaging of eccrine gland cells revealed that the TRPV4 agonist GSK101 activates these cells in WT mice, but not in TRPV4 KO. This effect is reduced with cold-stimulating menthol treatment. Temperature-dependent perspiration in mouse skin, either with passive heating or with ACh stimulation, was reduced in TRPV4 KO mice. Functional studies in mice - correlating the ability to climb a slippery slope to properly regulate skin moisture levels - reveal potential dysregulation of foot pad perspiration in TRPV4 KO mice, which had fewer successful climbing attempts. Lastly, a correlation of TRPV4 to hypohydrosis in humans was shown, as anhidrotic skin showed reduced levels of TRPV4 expression compared to normohidrotic or control skin.

Strengths:

The functional studies of mice climbing slippery slopes is a novel method to determine mechanisms of functional perspiration in mice. Since mice do not perspire for thermoregulation, other functional readouts are needed to study perspiration in mice.

Weaknesses:

1. The coexpression data needs additional controls. In the TRPV4 KO mice, there appears to be staining with the TRPV4 Ab in TRPV4 KO mice below the epidermis. This pattern appears similar to that of the location of the secretory coils of the sweat glands (Fig 1A). Is the co-staining the authors note later in Figure 1 also seen in TRPV4 KOs? This control should be shown, since the KO staining is not convincing that the Ab doesn't have off-target binding.

2. Are there any other markers besides CGRP for dark cells in mice to support the conclusion that mouse secretory cells have clear cell and dark cell properties?

3. The authors utilize menthol (as a cooling stimulus) in several experiments. In the discussion, they interpret the effect of menthol as potentially disrupting TRPV4-ANO1 interactions independent of TRPM8. Yet, the role of TRPM8, such as in TRPM8 KO mice, is not evaluated in this study.

4. Along those lines, the authors suggest that menthol inhibits eccrine function, which might lead to a cooling sensation. But isn't the cooling sensation of sweating from evaporative cooling? In which case, inhibiting eccrine function may actually impair cooling sensations.

5. The climbing assay is interesting and compelling. The authors note performing this under certain temperature and humidity conditions. Presumably, there is an optimal level of skin moisture, where skin that is too dry has less traction, but skin that is too wet may also have less traction. It would bolster this section of the study to perform this assay under hot conditions (perhaps TRPV4 KO mice, with impaired perspiration, would outperform WT mice with too much sweating?), or with pharmacologic intervention using TRPV4 agonists or antagonists to more rigorously evaluate whether this model correlates to TRPV4 function in the setting of different levels of perspiration.

6. There are other studies (PMID 33085914, PMID 31216445) that have examined the role of TRPV4 in regulating perspiration. The presence of TRPV4 in eccrine glands is not a novel finding. Moreover, these studies noted that TRPV4 was not critical in regulating sweating in human subjects. These prior studies are in contradiction to the mouse data and the correlation to human anhidrotic skin in the present study. Neither of these studies is cited or discussed by the authors, but they should be.

Reviewer #2 (Public Review):

Summary:

In the paper, the authors use a cellular Potts model to investigate muscle regeneration. The model is an attempt to combine many contributors to muscle regeneration into one coherent framework. I believe the resulting model has the potential to be very useful in investigating the complex interplay of multiple actors contributing to muscle regeneration.

Strengths:

The manuscript identified relevant model parameters from a long list of biological studies. This collation of a large amount of literature into one framework has the potential to be very useful to other authors. The mathematical methods used for parameterization and validation are transparent.

Weaknesses:

I have a few concerns which I believe need to be addressed fully.

My main concerns are the following:

1) The model is compared to experimental data in multiple results figures. However, the actual experiments used in these figures are not described. To me as a reviewer, that makes it impossible to judge whether appropriate data was chosen, or whether the model is a suitable descriptor of the chosen experiments. Enough detail needs to be provided so that these judgements can be made.

2) Do I understand it correctly that all simulations are done using the same initial simulation geometry? Would it be possible to test the sensitivity of the paper results to this geometry? Perhaps another histological image could be chosen as the initial condition, or alternative initial conditions could be generated in silico? If changing initial conditions is an unreasonably large request, could the authors discuss this issue in the manuscript?

3) Cytokine knockdowns are simulated by 'adjusting the diffusion and decay parameters' (line 372). Is that the correct simulation of a knockdown? How are these knockdowns achieved experimentally? Wouldn't the correct implementation of a knockdown be that the production or secretion of the cytokine is reduced? I am not sure whether it's possible to design an experimental perturbation which affects both parameters.

4) The premise of the model is to identify optimal treatment strategies for muscle injury (as per the first sentence of the abstract). I am a bit surprised that the implemented experimental perturbations don't seem to address this aim. In Figure 7 of the manuscript, cytokine alterations are explored which affect muscle recovery after injury. This is great, but I don't believe the chosen alterations can be done in experimental or clinical settings. Are there drugs that affect cytokine diffusion? If not, wouldn't it be better to select perturbations that are clinically or experimentally feasible for this analysis? A strength of the model is its versatility, so it seems counterintuitive to me to not use that versatility in a way that has practical relevance. - I may well misunderstand this though, maybe the investigated parameters are indeed possible drug targets.

5) A similar comment applies to Figure 5 and 6: Should I think of these results as experimentally testable predictions? Are any of the results surprising or new, for example in the sense that one would not have expected other cytokines to be affected as described in Figure 6?

6) In figure 4, there were differences between the experiments and the model in two of the rows. Are these differences discussed anywhere in the manuscript?

7) The variation between experimental results is much higher than the variation of results in the model. For example, in Figure 3 the error bars around experimental results are an order of magnitude larger than the simulated confidence interval. Do the authors have any insights into why the model is less variable than the experimental data? Does this have to do with the chosen initial condition, i.e. do you think that the experimental variability is due to variation in the geometries of the measured samples?

8) Is figure 2B described anywhere in the text? I could not find its description.

Reviewer #2 (Public Review):

Summary:

Fertilization is a crucial event in sexual reproduction, but the molecular mechanisms underlying egg-sperm fusion remain elusive. Elofsson et al. used AlphaFold to explore possible synapse-like assemblies between sperm and egg membrane proteins during fertilization. Using a systematic search of protein-protein interactions, the authors proposed a pentameric complex of three sperm (IZUMO1, SPACA6, and TMEM81) and two egg (JUNO and CD9) proteins, providing a new structural model to be used in future structure-function studies.

Strengths:

1. The study uses the AlphaFold algorithm to predict higher-order assemblies. This approach could offer insights into a highly transient protein complex, which is challenging to detect experimentally.<br /> 2. The article predicts a pentameric complex between proteins involved in fertilization, shedding light on the architectural aspects of the egg-sperm fusion synapse.

Weaknesses:

1. The procedures and discriminator scores used to evaluate specific from non-specific complexes were developed previously by the same authors. Therefore, in this manuscript, they are not contributing a new method.<br /> 2. The proposed model, which is a prediction from a modeling algorithm, lacks experimental validation of the identity of the components and the predicted contacts.

It is noteworthy that in an independent study, Deneke et al. provide experimental evidence of the interaction between IZUMO1/SPACA6/TMEM81 in zebrafish. This is an important element that supports the findings presented in this manuscript.

Reviewer #2 (Public Review):

The setting of planar cell polarity in epithelial tissues involves a complex interplay of chemical interactions. While local interactions can spontaneously give rise to cell polarity, planar cell polarity also involves tissue scale gradients whose effects are not clear. To understand their role, the authors built a minimal mechanistic model in considering two atypical cadherins, Fat (Ft) and Dachsous (Ds) which can associate at cell-cell interfaces to form hetero-dimers in which monomers belong to adjacent cells. This association can be seen as a local interaction between cells and is also sensitive to overall concentration gradients. From their model which appears to capture diverse experimental observations, the authors conclude that tissue-scale gradients provide to planar cell polarity a directional cue and some robustness to cellular stochasticity. While this model comes after similar works reaching similar predictions, the quality of this model is in its simplicity, its convenience for experimental testing, and the diversity of experimental observations it recapitulates.

A strength of this work is to recapitulate many experimental observations made on planar cell polarity. It, for example, seems to capture the response of tissues to perturbations such as local downregulation of some important proteins, and the polarity patterns observed in the presence of noise in synthesis or cell-to-cell heterogeneity. It also gives a mechanistic description of planar cell polarity, making its experimental interpretation simple. Finally, the simplicity of the model facilitates its exploration and makes it easily testable because of the reduced amount of free model parameters.

A weakness of this work is that it comes after several models with similar hypotheses and similar predictions. Another weakness is that some conclusions of this work rely on visual appreciation rather than quantification. This is particularly true for what concerns 2D patterns. An argument of the authors is for example that their model reproduces a variety of known spatial patterns, but the comparison with experiments is only visual and would be more convincing in being more quantitative.

Reviewer #2 (Public Review):

In this study, Leiba et al. aim at establishing the developing zebrafish embryo as a suitable infection model to study Salmonella persistence in vivo. Under environmental stress (ex: macrophage phagosomes) a proportion of bacteria switch to a slow/arrested growth state confering increased resistance to antibiotic treatments. Persisters are getting increasingly linked to infection relapses. Understanding how persistent infections emerge and bacteria survive in an organism for long time without replicating before switching back to a replicative state is essential. Zebrafish represents an alternative model to mice offering the possibility to image the whole organism and capture persistency with an amazing spatio-temporal resolution.

In this paper, the authors demonstrate that persistent infections of Salmonella can be reproduced in the developing zebrafish. The kinetics of infection have been well characterized and shows a very nice heterogeneity between animals demonstrating the complex host-pathogen interactions (Fig 1). From the perspective of persistence, the presence of Salmonella survivors to host clearing is reported until 14dpi demonstrating the possibility to induce persistent infection in this model. Through the manuscript, the authors have used a variety of state-of-the-art technics illustrating the flexibility of this model including microscopy and imaging of specific immune populations, various transgenic animals and selective depletion of macrophages or neutrophils to assess their relative contributions. Overall, the conclusions of the authors are well supported by the presented data. This said, the authors should strengthen the conclusions of the paper by providing a better characterization of the infection.

Major comments:<br /> 1- Figure 1: What is the general life-spam of the fish?

2- Figure 2: It would be nice to clearly state what infection scenario we are looking at. Have the authors studied "high proliferation", "infected" or "cleared" zebrafish?

3- Figure 3 and 4: It would be very informative if the authors can tell us what proportion of Salmonella is associated with macrophages and neutrophils. From panel C and D (Figure 3) and Figure 4 C and D and Suppl Fig 1, it seems that a lot of bacteria are extracellular. Maybe an EM image of the tissue would help to understand if the bacteria is "all" intracellular or intracellular.

4- Figure 3 and 4: It would be very useful if the authors can tell us if the intracellular bacteria are mainly found individually (like in Figure 3C) or does host cells harbor many intracellular bacteria. Looking at figure 4G: it is not clear to me how many intracellular bacteria can be counted on this image.

5- Figure 3 and 4: The authors should also perform an experiment with a Salmonella strain harboring a growth reporter to quantify the amount of replicating and non-replicating bacteria. This experiment is not absolutely necessary for the story, but if possible, it would provide a very nice add-up to the story and impact to the paper.

6- Figure 6: The authors should provide in suppl. the flow cytometry scatter plots used to delineate the different subpopulations.

7- Figure 6: A specific characterization of macrophages harboring Salmonella persisters at 4dpi is missing. As shown by the authors in Figure 6, the tnfa- populations of macrophages at 4dpi are very similar for both infected and non-infected larvae. Persisters should indeed reside within tnfa- macrophages but they should also induce a specific signature through the actions of Salmonella effectors. Measuring this signature will allow a direct comparison with published data in mice and assess how accurately the zebrafish model recapitulates the manipulation of macrophages by Salmonella

Reviewer #2 (Public Review):

This paper uses two-photon imaging of mouse ganglion cells responding to chromatic natural scenes along with convolutional neural network (CNN) models fit to the responses of a large set of ganglion cells. The authors analyze CNN models to find the most effective input (MEI) for each ganglion cell as a novel approach to identifying ethological function. From these MEIs they identify chromatic opponent ganglion cells, and then further perform experiments with natural stimuli to interpret the ethological function of those cells. They conclude that a type of chromatic opponent ganglion cell is useful for the detection of the transition from the ground to the sky across the horizon. The experimental techniques, data, and fitting of CNN models are all high quality. However, there are conceptual difficulties with both the use of MEIs to draw conclusions about neural function and the ethological interpretations of experiments and data analyses, as well as a lack of comparison with standard approaches. These bear directly both on the primary conclusions of the paper and on the utility of the new approaches.

1. Claim of feature detection. The color opponent cells are cast as a "feature detector" and the term 'detector' is in the title. However insufficient evidence is given for this, and it seems likely a mischaracterization. An example of a ganglion cell that might qualify as a feature detector is the W3 ganglion cell (Zhang et al., 2012). These cells are mostly silent and only fire if there is differential motion on a mostly featureless background. Although this previous work does not conduct a ROC analysis, the combination of strong nonlinearity and strong selectivity are important here, giving good qualitative support for these cells as participating in the function of detecting differential motion against the sky. In the present case, the color opponent cells respond to many stimuli, not just transitions across the horizon. In addition, for the receiver operator characteristic (ROC) analysis as to whether these cells can discriminate transitions across the horizon, the area under the curve (AUC) is on average 0.68. Although there is not a particular AUC threshold for a detector or diagnostic test to have good discrimination, a value of 0.5 is chance, and values between 0.5 and 0.7 are considered poor discrimination, 'not much better than a coin toss' (Applied Logistic Regression, Hosmer et al., 2013, p. 177). The data in Fig. 6F is also more consistent with a general chromatic opponent cell that is not highly selective. These cells may contribute information to the problem of discriminating sky from ground, but also to many other ethologically relevant visual determinations. Characterizing them as feature detectors seems inappropriate and may distract from other functional roles, although they may participate in feature detection performed at a higher level in the brain.

2. Appropriateness of MEI analysis for interpretations of the neural code. There is a fundamental incompatibility between the need to characterize a system with a complex nonlinear CNN and then characterizing cells with a single MEI. MEIs represent the peak in a complex landscape of a nonlinear function, and that peak may or may not occur under natural conditions. For example, MEIs do not account for On-Off cells, On-Off direction selectivity, nonlinear subunits, object motion sensitivity, and many other nonlinear cell properties where multiple visual features are combined. MEIs may be a useful tool for clustering and distinguishing cells, but there is not a compelling reason to think that they are representative of cell function. This is an open question, and thus it should not be assumed as a foundation for the study. This paper potentially speaks to this issue, but there is more work to support the usefulness of the approach. Neural networks enable a large set of analyses to understand complex nonlinear effects in a neural code, and it is well understood that the single-feature approach is inadequate for a full understanding of sensory coding. A great concern is that the message that the MEI is the most important representative statistic directs the field away from the primary promise of the analysis of neural networks and takes us back to the days when only a single sensory feature is appreciated, now the MEI instead of the linear receptive field. It is appropriate to use MEI analyses to create hypotheses for further experimental testing, and the paper does this (and states as much) but it further takes the point of view that the MEI is generally informative as the single best summary of the neural code. The representation similarity analysis (Fig. 5) acts on the unfounded assumption that MEIs are generally representative and conveys this point of view, but it is not clear whether anything useful can be drawn from this analysis, and therefore this analysis does not support the conclusions about changes in the representational space. Overall this figure detracts from the paper and can safely be removed. In addition, in going from MEI analysis to testing ethological function, it should be made much more clear that MEIs may not generally be representative of the neural code, especially when nonlinearities are present that require the use of more complex models such as CNNs, and thus testing with other stimuli are required.

3. Usefulness of MEI approach over alternatives. It is claimed that analyzing the MEI is a useful approach to discovering novel neural coding properties, but to show the usefulness of a new tool, it is important to compare results to the traditional technique. The more standard approach would be to analyze the linear receptive field, which would usually come from the STA of white noise measurement, but here this could come from the linear (or linear-nonlinear) model fit to the natural scene response, or by computing an average linear filter from the natural scene model. It is important to assess whether the same conclusion about color opponency can come from this standard approach using the linear feature (average effective input), and whether the MEIs are qualitatively different from the linear feature. The linear feature should thus be compared to MEIs for Fig. 3 and 4, and the linear feature should be compared with the effects of natural stimuli in terms of chromatic contrast (Fig. 6b). With respect to the representation analysis (Fig. 5), although I don't believe this is meaningful for MEIs, if this analysis remains it should also be compared to a representation analysis using the linear feature. In fact, a representation analysis would be more meaningful when performed using the average linear feature as it summarizes a wider range of stimuli, although the most meaningful analysis would be directly on a broader range of responses, which is what is usually done.

4. Definition of ethological problem. The ethological problem posed here is the detection of the horizon. The stimuli used do not appear to relate to this problem as they do not include the horizon and only include transitions across the horizon. It is not clear whether these stimuli would ever occur with reasonable frequency, as they would only occur with large vertical saccades, which are less common in mice. More common would be smooth transitions across the horizon, or smaller movements with the horizon present in the image. In this case, cells which have a spatial chromatic opponency (which the authors claim are distinct from the ones studied here) would likely be more important for use in chromatic edge detection or discrimination. Therefore the ethological relevance of any of these analyses remains in question.

It is further not clear if detection is even the correct problem to consider. The horizon is always present, but the problem is to determine its location, a conclusion that will likely come from a population of cells. This is a distinct problem from detecting a small object, such as a small object against the background of the sky, which may be a more relevant problem to consider.

5. Difference in cell type from those previously described. It is claimed that the chromatic opponent cells are different from those previously described based on the MEI analysis, but we cannot conclude this because previous work did not perform an MEI analysis. An analysis should be used that is comparable to previous work, the linear spatiotemporal receptive field should be sufficient. However, there is a concern that because linear features can change with stimulus statistics (Hosoya et al., 2005), a linear feature fit to natural scenes may be different than those from previous studies even for the same cell type. The best approach would likely be presenting a white noise stimulus to the natural scenes model to compute a linear feature, which still carries the assumption that this linear feature from the model fit to a natural stimulus would be comparable to previous studies. If the previous cells have spatial chromatic opponency and the current cells only have chromatic opponency in the center, there should be both types of cells in the current data set. One technical aspect relating to this is that MEIs were space-time separable. Because the center and surround have a different time course, enforcing this separability may suppress sensitivity in the surround. Therefore, it would likely be better if this separability were not enforced in determining whether the current cells are different than previously described cells. As to whether these cells are actually different than those previously described, the authors should consider the following uncited work; (Ekesten Gouras, 2005), which identified chromatic opponent cells in mice in approximate numbers to those here (~ 2%). In addition, (Yin et al., 2009) in guinea pigs and (Michael, 1968) in ground squirrels found color-opponent ganglion cells without effects of a spatial surround as described in the current study.

Reviewer #3 (Public Review):

This study succeeds to highlight and address important gaps in our understanding of plant-insect interactions mediating pest control in a widely known agro-ecological system for sustainable intensification, push-pull agriculture. In particular, the authors present a large amount of data on plant volatile emission, thought to be critical for the functioning of these systems, in reasonable and relevant contexts, as well as on other traits of the plants in the system relevant for pest control. These data come from plants grown both in controlled and field environments, which is unusual. The arguments on mechanism are further supported by insect behavioral assays, which seem to be thoughtfully designed, but also use some non-standard approaches that could be better explained. While most or all of the authors' results pre-date some relevant recent publications in this field, they do incorporate comparisons to current literature in order to better place their findings in the current state of the art.

Reviewer #2 (Public Review):

This study by Du et. al addressed the role and regulation of proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) in Aristolochic acid Nephropthathy (AAN) and immune defense. PSTPIP2 expression is downregulated in AAN. Conditional knock-in of PSTPIP2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Reducing the expression of PSTPIP2 to normal levels in knock-in mouse using shRNA promoted kidney injury. Using in vivo model, the role of PSTPIP2 in AAN injury and renal function, apoptosis, neutrophil infiltration and NET formation is established. Using in vitro models, a PSTPIP2/NFkB-mediated NET formation via IL-19-IL20-beta Receptor pathway is shown to induce inflammation and apoptosis in AAN. The studies are well presented.

Reviewer #2 (Public Review):

For about four decades it has been known that RNA molecules can increase the rate of chemical reactions, just like the much more prevalent protein enzymes. Some have suggested that RNA enzymes, also called "ribozymes" were very important at the beginning of life, but that the importance was mostly erased when ribosomal protein synthesis emerged through evolution. The ribosome and spliceosome are two important examples of modern biological functions known to be catalyzed by RNA. In addition to these large RNA machines, the genomes of humans, and all domains of life, also contain a class of small ribozymes that catalyze self-cleavage of the RNA backbone. However, unlike RNA cleaving proteins that are well studied, there exists little evidence that the self-cleaving of RNA by ribozymes has important downstream consequences. This new paper provides evidence that a ribozyme found in all mammals has an important role in memory formation. The authors found a way to block the ribozyme activity and then observe the effect on memory formation in mice, and in the expression of genes in neurons that are known to underly this memory formation process. The authors found that blocking the ribozyme activity in mouse brains actually improved their performance in a memory task. In addition, they found that blocking the ribozyme changed the expression of the gene in which the ribozyme is found (a gene called CPEB3), suggesting that the way the ribozyme effects memory is through controlling the expression of the gene where it is found. The paper confirms the biological importance of this ribozyme, and encourages further investigation into self-cleaving ribozymes in general. Interestingly, the ribozyme found in humans is in fact slower cleaving than most mammals, similar to the blocked ribozyme in these experiments, which brings up the intriguing possibility that the CPEB3 ribozyme is a part of what makes us human!

Reviewer #2 (Public Review):

The authors examine the use of metformin in the treatment of hepatic ischemia/reperfusion injury (HIRI) and suggest the mechanism of action is mediated in part by the gut microbiota and changes in hepatic ferroptosis. While the concept is intriguing, the experimental approaches are inadequate to support these conclusions.

The histological and imaging studies were considered a strength and reveal a significant impact of metformin post-HIRI.

Weaknesses largely stem from the experimental design. The impact of metformin on the microbiota is profound resulting in changes in bile acid, lipid, and glucose homeostasis. Throughout the manuscript no comparisons are made with metformin alone which would better capture the metformin-specific effects. With the pathology and metabolic disturbances resulting from HIRI, it is important to understand if metformin is providing beneficial effects from reported mechanisms such as changes in bile acid, glucose, and/or lipid metabolism, or are these changes the result of a new unappreciated mechanism. A comparison of the reported and the new pathways is not included.

Overall, while the concept is interesting and has potential to better understand the pleiotropic functions of metformin, the limitations with the experimental design and lack of key controls make it challenging to support the conclusions.

Reviewer #2 (Public Review):

Summary:

This study uses transcriptome sequence from a dioecious plant to compare evolutionary rates between genes with male- and female-biased expression and distinguish between relaxed selection and positive selection as causes for more rapid evolution. These questions have been explored in animals and algae, but few studies have investigated this in dioecious angiosperms, and none have so far identified faster rates of evolution in male-biased genes (though see Hough et al. 2014 https://doi.org/10.1073/pnas.1319227111).

Strengths:

The methods are appropriate to the questions asked. Both the sample size and the depth of sequencing are sufficient, and the methods used to estimate evolutionary rates and the strength of selection are appropriate. The data presented are consistent with faster evolution of genes with male-biased expression, due to both positive and relaxed selection.

This is a useful contribution to understanding the effect of sex-biased expression in genetic evolution in plants. It demonstrates the range of variation in evolutionary rates and selective mechanisms, and provides further context to connect these patterns to potential explanatory factors in plant diversity such as the age of sex chromosomes and the developmental trajectories of male and female flowers.

Weaknesses:

The presence of sex chromosomes is a potential confounding factor, since there are different evolutionary expectations for X-linked, Y-linked, and autosomal genes. Attempting to distinguish transcripts on the sex chromosomes from autosomal transcripts could provide additional insight into the relative contributions of positive and relaxed selection.

Reviewer #2 (Public Review):

In this study, Kajtor et al investigated the use of a single-animal trial-based behavioral assay for the assessment of subtle changes in the locomotor behavior of different genetic models of Parkinson's disease of Drosophila. Different genotypes used in this study were Ddc-GAL4>UAS-Parkin-275W and UAS- α-Syn-A53T. The authors measured Drosophila's response to predator-mimicking passing shadow as a threatening stimulus. Along with these, various dopamine (DA) receptor mutants, Dop1R1, Dop1R2 and DopEcR were also tested.<br /> The behavior was measured in a custom-designed apparatus that allows simultaneous testing of 13 individual flies in a plexiglass arena. The inter-trial intervals were randomized for 40 trials within 40 minutes duration and fly responses were defined into freezing, slowing down, and running by hierarchical clustering. Most of the mutant flies showed decreased reactivity to threatening stimuli, but the speed-response behavior was genotype invariant.<br /> These data nicely show that measuring responses to the predator-mimicking passing shadows could be used to assess the subtle differences in the locomotion parameters in various genetic models of Drosophila.

The understanding of the manifestation of various neuronal disorders is a topic of active research. Many of the neuronal disorders start by presenting subtle changes in neuronal circuits and quantification and measurement of these subtle behavior responses could help one delineate the mechanisms involved. The data from the present study nicely uses the behavioral response to predator-mimicking passing shadows to measure subtle changes in behavior. However, there are a few important points that would help establish the robustness of this study.<br /> 1) The visual threat stimulus for measuring response behavior in Drosophila is previously established for both single and multiple flies in an arena. A comparative analysis of data and the pros and cons of the previously established techniques (for example, Gibson et al., 2015) with the technique presented in this study would be important to establish the current assay as an important advancement.<br /> 2) Parkinson's disease mutants should be validated with other GAL-4 drivers along with Ddc-GAL4, such as NP6510-Gal4 (Riemensperger et al., 2013). This would be important to delineate the behavioral differences due to dopaminergic neurons and serotonergic neurons and establish the Parkinson's disease phenotype robustly.<br /> 3) The DopEcR mutant genotype used for behavior analysis is w1118; PBac{PB}DopEcRc02142/TM6B, Tb1. Balancer chromosomes, such as TM6B,Tb can have undesirable and uncharacterised behavioral effects. This could be addressed by removing the balancer and testing the DopEcR mutant in homozygous (if viable) or heterozygous conditions.<br /> 4) The height of the arena is restricted to 1mm. However, for the wild-type flies (Canton-S) and many other mutants, the height is usually more than 1mm. Also, a 1 mm height could restrict the fly movement. For example, it might not allow the flies to flip upside down in the arena easily. This could introduce some unwanted behavioral changes. A simple experiment with an arena of height at least 2.5mm could be used to verify the effect of 1mm height.<br /> 5) The detailed model for Monte Carlo simulation for speed-response simulation is not described. The simulation model and its hyperparameters need to be described in more depth and with proper justification.<br /> 6) The statistical analysis in different experiments needs revisiting. It wasn't clear to me if the authors checked if the data is normally distributed. A simple remedy to this would be to check the normality of data using the Shapiro-Wilk test or Kolmogorov-Smirnov test. Based on the normality check, data should be further analyzed using either parametric or non-parametric statistical tests. Further, the statistical test for the age-dependent behavior response needs revisiting as well. Using two-way ANOVA is not justified given the complexity of the experimental design. Again, after checking for the normality of data, a more rigorous statistical test, such as split-plot ANOVA or a generalized linear model could be used.<br /> 7) The dopamine receptor mutants used in this study are well characterized for learning and memory deficits. In the Parkinson's disease model of Drosophila, there is a loss of DA neurons in specific pockets in the central brain. Hence, it would be apt to use whole animal DA receptor mutants as general DA mutants rather than the Parkinson's disease model. The authors may want to rework the title to reflect the same.

Reviewer #2 (Public Review):

Summary:

The generation of functional extranumerary inner hair cells (IHCs) in postnatal mice, particularly with virus-mediated knockdown of Cldn9 mRNA expression in the neonatal cochlear duct, is an important observation. It is significant because not many studies exist that report molecular manipulations of the neonatal organ of Corti that result in the generation of new hair cells that remain functional and appear to be intact for an extended time, here more than one year. Overall, this is a carefully conducted study; the observations are clear, and the methods are solid. Two independent methods for reducing the expression of Cldn9 mRNA were used: a conditional transgenic model and AAV-mediated knockdown with shRNA. The lack of a functional explanation of how the reduced expression of Cldn9 specifically leads to the formation of extranumerary IHCs leaves open questions. For example, it is not clear whether there is indeed a fate change happening and whether Cldn9 reduction affects developmental processes. The discussion of how Cldn9 reduction potentially affects Notch signaling, without hard evidence, is handwaving.

Strengths:

It is a very interesting observation and somewhat unexpected in its specificity for inner hair cells. Using two different approaches to manipulate Cldn9 expression provides a strong experimental foundation. The study is conducted quantitatively and with care.

Weaknesses:

The lack of mechanistic insight results in an open-ended story where at least the potential interaction of Cldn9 reduction with known and well-characterized signaling pathway components should have been investigated. This missed opportunity limits the scope of the study and should be addressed: How does Cldn9 downregulation affect the expression levels of other known genes linked to hair cell production and cell fate decisions? Quantitative RT-PCR is working well for the authors, and a comparison of the expression of Notch or other known pathway components could provide mechanistic insight.

It is unclear how P21 inner hair cells were identified for the patch clamp experiments shown in Fig 4E-H. This is a challenging endeavor without the possibility of using specific markers.

Please also address the numerous minor points outlined below; it will improve the paper's readability.

Please include page numbers and line numbers in a revised manuscript.

Reviewer #2 (Public Review):

Summary,<br /> The paper aimed to examine the effect of co-ablating Substance P and CGRPα peptides on pain using Tac1 and Calca double knockout (DKO) mice. The authors observed no significant changes in acute, inflammatory, and neuropathic pain. These results suggest that Substance P and CGRPα peptides do not play a major role in mediating pain in mice. Moreover, they reveal that the lack of behavioral phenotype cannot be explained by the redundancy between the two peptides, which are often co-expressed in the same neuron

Strengths,<br /> The paper uses a straightforward approach to address a significant question in the field. The authors confirm the absence of Substance P and CGRPα peptides at the levels of DRG, spinal cord, and midbrain. Subsequently, they employ a comprehensive battery of behavioral tests to examine pain phenotypes, including acute, inflammatory, and neuropathic pain. Additionally, they evaluate neurogenic inflammation by measuring edema and extravasation, revealing no changes in DKO mice. The data are compelling, and the study's conclusions are well-supported by the results. The manuscript is succinct and well-presented.

Reviewer #2 (Public Review):

Summary<br /> This study examines the construct of "cognitive spaces" as they relate to neural coding schemes present in response conflict tasks. The authors use a novel experimental design in which different types of response conflict (spatial Stroop, Simon) are parametrically manipulated. These conflict types are hypothesized to be encoded jointly, within an abstract "cognitive space", in which distances between task conditions depend only on the similarity of conflict types (i.e., where conditions with similar relative proportions of spatial-Stroop versus Simon conflicts are represented with similar activity patterns). Authors contrast such a representational scheme for conflict with several other conceptually distinct schemes, including a domain-general, domain-specific, and two task-specific schemes. The authors conduct a behavioral and fMRI study to test whether prefrontal cortex activity is correlated to one of these coding schemes. Replicating the authors' prior work, this study demonstrates that sequential behavioral adjustments (the congruency sequence effect) are modulated as a function of the similarity between conflict types. In fMRI data, univariate analyses identified activation in left prefrontal and dorsomedial frontal cortex that was modulated by the amount of Stroop or Simon conflict present, and representational similarity analyses that identified coding of conflict similarity, as predicted under the cognitive space model, in right lateral prefrontal cortex.

Strengths

This study addresses an important question regarding how conflict or difficulty might be encoded in the brain within a computationally efficient representational format. Relative to the other models reported in the paper, the evidence in support of the cognitive space model is solid. The ideas postulated by the authors are interesting and valuable ones, worthy of follow-up work that provides additional necessary scrutiny of the cognitive-space account.

Weaknesses

Future, within-subject experiments will be necessary to disentangle the cognitive space model from confounded task variables. A between-subjects manipulation of stimulus orientation/location renders the results difficult to interpret, as the source and spatial scale of the conflict encoding on cortex may differ from more rigorous (and more typical) within-subject manipulations.

Results are also difficult to interpret because Stroop and Simon conflict are confounded with each other. For interpretability, these two sources of conflict need to be manipulated orthogonally, so that each source of conflict (as well as their interaction) could be separately estimated and compared in terms of neural encoding. For example, it is therefore not clear whether the RSA results are due to encoding of only one type of conflict (Stroop or Simon), to a combination of both, and/or to interactive effects.

Finally, the motivation for the use of the term "cognitive space" to describe results is unclear. Evidence for the mere presence of a graded/parametric neural encoding (i.e., the reported conflict RSA effects) would not seem to be sufficient. Indeed, it is discussed in the manuscript that cognitive spaces/maps allow for flexibility through inference and generalization. Future work should therefore focus on linking neural conflict encoding to inference and generalization more directly.

Reviewer #2 (Public Review):

Summary:<br /> Sullivan and Bashaw delve into the mechanisms that drive neural circuit assembly, and specifically, into the regulation of cell surface proteins that mediate axon pathfinding. During nervous system development, axons must traverse a molecularly and physically complex extracellular milieu to reach their synaptic targets. A fundamental, conserved repulsive signaling pathway is initiated by the Slit-Robo ligand-receptor pair. Robo, expressed on axon growth cones, binds Slit, secreted by midline cells, to prevent "pre-crossing" and "re-crossing" of axons at the midline. To control this repulsion, Robo surface levels are tightly regulated. In Drosophila, Commissureless (Comm) downregulates Robo surface levels and is required for axon crossing at the midline. Several studies suggest that PY motifs in Comm are required to localize Robo to endosomes. PY motifs have been shown to bind WW-domain containing proteins including the ubiquitin ligase Nedd4 family, so the authors propose that Comm may regulate Robo through Nedd4 interactions. Previous studies have hinted at a role for Nedd4-mediated ubiquitination of Comm in the regulation of Robo localization, but there have also been conflicting data. For example, Comm mutants that are unable to be ubiquitinated mimic wild-type Comm, suggesting that ubiquitination of Comm is not required for regulation of Robo. The current study utilizes a suite of genetic analyses in Drosophila to resolve discrepancies pertaining to the mode of Comm-dependent regulation of Robo1 and proposes that Comm acts as an adapter for the Nedd4 ubiquitin ligase to recognize Robo1 as a substrate. The authors also demonstrate that Nedd4 is indeed required for midline crossing.

Strengths:<br /> While this work is more incremental rather than field-shifting, it is nonetheless an excellent example of a rigorous, thorough analysis that culminates in enriching our mechanistic understanding of how neurons regulate cell-surface receptors in a spatiotemporal manner to control fundamental steps of circuit wiring. The experimental approach is thorough, and the manuscript is extremely well-written.

Weaknesses:<br /> Some key experiments (eg. complex formation) were performed in cell culture in an overexpression background. Also, there was a missed opportunity to bolster the model proposed by using Comm PY mutants in several experiments. Finally, Comm PY domains are required for proper Comm localization in neurons, but corresponding Robo localization was not analyzed.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, L&S investigates the important general question of how humans achieve invariant behavior over stimuli belonging to one category given the widely varying input representation of those stimuli and more specifically, how they do that in arbitrary abstract domains. The authors start with the hypothesis that this is achieved by invariance transformations that observers use for interpreting different entries and furthermore, that these transformations in an arbitrary domain emerge with the help of the transformations (e.g. translation, rotation) within the spatial domain by using those as "scaffolding" during transformation learning. To provide the missing evidence for this hypothesis, L&S used behavioral category learning studies within and across the spatial, auditory, and visual domains, where rotated and translated 4-element token sequences had to be learned to categorize and then the learned transformation had to be applied in new feature dimensions within the given domain. Through single- and multiple-day supervised training and unsupervised tests, L&S demonstrated by standard computational analyses that in such setups, space and spatial transformations can, indeed, help with developing and using appropriate rotational mapping whereas the visual domain cannot fulfill such a scaffolding role.

Strengths:<br /> The overall problem definition and the context of spatial mapping-driven solution to the problem is timely. The general design of testing the scaffolding effect across different domains is more advanced than any previous attempts clarifying the relevance of spatial coding to any other type of representational codes. Once the formulation of the general problem in a specific scientific framework is done, the following steps are clearly and logically defined and executed. The obtained results are well interpretable, and they could serve as a good stepping stone for deeper investigations. The analytical tools used for the interpretations are adequate. The paper is relatively clearly written.

Weaknesses:<br /> Some additional effort to clarify the exact contribution of the paper, the link between analyses and the claims of the paper, and its link to previous proposals would be necessary to better assess the significance of the results and the true nature of the proposed mechanism of abstract generalization.

1) Insufficient conceptual setup: The original theoretical proposal (the Tolman-Eichenbaum-Machine, Whittington et al., Cell 2020) that L&S relate their work to proposes that just as in the case of memory for spatial navigation, humans and animals create their flexible relational memory system of any abstract representation by a conjunction code that combines on the one hand, sensory representation and on the other hand, a general structural representation or relational transformation. The TEM also suggests that the structural representation could contain any graph-interpretable spatial relations, albeit in their demonstration 2D neighbor relations were used. The goal of L&S's paper is to provide behavioral evidence for this suggestion by showing that humans use representational codes that are invariant to relational transformations of non-spatial abstract stimuli and moreover, that humans obtain these invariances by developing invariance transformers with the help of available spatial transformers. To obtain such evidence, L&S use the rotational transformation. However, the actual procedure they use actually solved an alternative task: instead of interrogating how humans develop generalizations in abstract spaces, they demonstrated that if one defines rotation in an abstract feature space embedded in a visual or auditory modality that is similar to the 2D space (i.e. has two independent dimensions that are clearly segregable and continuous), humans cannot learn to apply rotation of 4-piece temporal sequences in those spaces while they can do it in 2D space, and with co-associating a one-to-one mapping between locations in those feature spaces with locations in the 2D space an appropriate shaping mapping training will lead to the successful application of rotation in the given task (and in some other feature spaces in the given domain). While this is an interesting and challenging demonstration, it does not shed light on how humans learn and generalize, only that humans CAN do learning and generalization in this, highly constrained scenario. This result is a demonstration of how a stepwise learning regiment can make use of one structure for mapping a complex input into a desired output. The results neither clarify how generalizations would develop in abstract spaces nor the question of whether this generalization uses transformations developed in the abstract space. The specific training procedure ensures success in the presented experiments but the availability and feasibility of an equivalent procedure in a natural setting is a crucial part of validating the original claim and that has not been done in the paper.

2) Missing controls: The asymptotic performance in experiment 1 after training in the three tasks was quite different in the three tasks (intercepts 2.9, 1.9, 1.6 for spatial, visual, and auditory, respectively; p. 5. para. 1, Fig 2BFJ). It seems that the statement "However, our main question was how participants would generalise learning to novel, rotated exemplars of the same concept." assumes that learning and generalization are independent. Wouldn't it be possible, though, that the level of generalization depends on the level of acquiring a good representation of the "concept" and after obtaining an adequate level of this knowledge, generalization would kick in without scaffolding? If so, a missing control is to equate the levels of asymptotic learning and see whether there is a significant difference in generalization. A related issue is that we have no information on what kind of learning in the three different domains was performed, albeit we probably suspect that in space the 2D representation was dominant while in the auditory and visual domains not so much. Thus, a second missing piece of evidence is the model-fitting results of the ⦰ condition that would show which way the original sequences were encoded (similar to Fig 2 CGK and DHL). If the reason for lower performance is not individual stimulus difficulty but the natural tendency to encode the given stimulus type by a combo of random + 1D strategy that would clarify that the result of the cross-training is, indeed, transferring the 2D-mapping strategy.

Reviewer #2 (Public Review):

Summary:<br /> This is an unusual, but interesting approach to link the "taste" of plants and plant extracts to their therapeutic use in ancient Graeco-Roman culture. The authors used a panel of 11 trained tasters to test ~700 different medicinal plants and describe them in terms of 22 "taste" descriptors. They correlated these descriptors with the plant's medical use as reported in the De Materia Medica (DMM 1st Century, CE). Correcting for some of the plants' evolutionary phylogenetic relationships, the authors found that taste descriptors along with intensity measures were correlated with the "versatility" and/or a specific therapeutic use of the medicine. For example, simple but intense tastes were correlated with versatility of a medicine. Specific intense tastes were linked to versatility while others were not; intense bitter, starchy, musky, sweet, cooling and soapy were associated with versatility, but sour and woody were negatively associated. Also some specific tastes could be associated with specific uses - both positive and negative associations. Some of these findings make sense immediately, but others are somewhat surprising, and the authors propose some links between taste and medicinal use (both historical and modern use) in the discussion. The authors state that this study allows for a re-evaluation of pre-scientific knowledge, pointing toward a central role for taste in medicine.

Strengths:<br /> The real strength of this study is the novelty of this approach - using modern day tasters to evaluate ancient medicinal plants to understand the potential relationships between taste and therapeutic use, lending some support to the idea that the "taste" of a medicine is linked to its effectiveness as a treatment.

Weaknesses:<br /> Because of the limitations of time and the type of botanicals being tested, there is an inherent difficulty in assessing taste intensity. However, because these botanicals are tested by multiple panelists and sometimes tested repeatedly by individual panelists, this helps support the author's analyses.

Reviewer #2 (Public Review):

Erk2 is an essential element of the MAP kinase signaling cascade and directly controls cell proliferation, migration, and survival. Therefore, it is one of the most important drug targets for cancer therapy. The catalytic subunit of Erk2 has a bilobal architecture, with the small lobe harboring the nucleotide-binding pocket and the large lobe harboring the substrate-binding cleft. Several studies by the Ahn group revealed that the catalytic domain hops between (at least) two conformational states: active (R) and inactive (L), which exchange in the millisecond time scale based on the chemical shift mapping. The R state is a signature of the double phosphorylated Erk2 (2P-Erk2), while the L state has been associated with the unphosphorylated kinase (0P-Erk2). Interestingly, the X-ray structures reveal only minimal differences between these two states, a feature that led to the conclusion that active and inactive states are structurally similar but dynamically very different. The Ahn group also found that ATP-competitive inhibitors can steer the populations of Erk2 either toward the R or the L state, depending on their chemical nature. The latter opens up the possibility of modulating the activity of this kinase by changing the chemistry of the ATP-competitive inhibitor. To prove this point, the authors present a set of nineteen compounds with diverse chemical substituents. From their combined NMR and HDX-Mass Spec analyses, fourteen inhibitors drive the kinase toward the R state, while four compounds keep the kinase hopping between the R and L states. Based on these data, the authors rationalize the effects of these inhibitors and the importance of the nature of the substituents on the central scaffold to steer the kinase activity. While all these inhibitors target the ATP binding pocket, they display diverse structural and dynamic effects on the kinase, selecting a specific structural state. Although the inhibited kinase is no longer able to phosphorylate substrates, it can initiate signaling events functioning as scaffolds for other proteins. Therefore, by changing the chemistry of the inhibitors it may be possible to affect the MAP cascade in a predictable manner. This concept, recently introduced as proof of principle, finds here its significance and practical implications. The design of the next-generation inhibitors must be taken into account for these design principles.<br /> The research is well executed, and the data support the author's conclusions.

Reviewer #2 (Public Review):

In this manuscript the authors present and characterize LOVdeg, a modified version of the blue-light sensitive AsLOV2 protein, which functions as a light-inducible degron in Escherichia coli. Light has been shown to be a powerful inducer in biological systems as it is often orthogonal and can be controlled in both space and time. Many optogenetic systems target regulation of transcription, however in this manuscript the authors target protein degradation to control protein levels in bacteria. This is an important advance in bacteria, as inducible protein degradation systems in bacteria have lagged behind eukaryotic systems due to protein targeting in bacteria being primarily dependent on primary amino acid sequence and thus more difficult to engineer. In this manuscript, the authors exploit the fact that the J-alpha helix of AsLOV2, which unwinds into a disordered domain in response to blue light, contains an E-A-A amino acid sequence which is very similar to the C-terminal L-A-A sequence in the SsrA tag which is targeted by the unfoldases ClpA and ClpX. They truncate AsLOV2 to create AsLOV2(543) and combine this truncation with a mutation that stabilizes the dark state to generate AsLOV2*(543) which, when fused to the C-terminus of mCherry, confers light-induced degradation. The authors do not verify the mechanism of degradation due to LOVdeg, but evidence from deletion mutants contained in the supplemental material hints that there is a ClpA dominated mechanism. The LOVdeg is able to target mCherry for protein degradation across different phases of bacterial growth, which is important for regulating processes at stationary phase and a potential additional advantage over transcriptional repression systems. They demonstrate modularity of this LOVdeg by using it to degrade the LacI repressor, CRISPRa activation through degradation of MCP-SoxS, and the AcrB protein which is part of the AcrAB-TolC multidrug efflux pump. In all cases, measurement of the effect of the LOVdeg is indirect as the authors measure reduction in LacI repression, reduction in CRISPRa activation, and drug resistance rather than directly measuring protein levels. Nevertheless the evidence is convincing, although seemingly less effective than in the case of mCherry degradation, although it is hard to compare due to the different endpoints being measured. The authors further modify LOVdeg to contain a known photocycle mutation that slows its reversion time in the dark, so that LOVdeg is more sensitive to short pulses of light which could be useful in low light conditions or for very light sensitive organisms. They also demonstrate that combining LOVdeg with a blue-light transcriptional repression system (EL222) can decrease protein levels an additional 23-fold (relative to 7-fold with LOVdeg alone). Finally, the authors apply LOVdeg to a metabolic engineering task, namely reducing expression of octanoic acid by regulating the enzyme CpFatB1, an acyl-ACP thioesterase. The authors show that tagging CpFatB1 with LOVdeg allows light induced reduction in octanoic acid titer over a 24 hour fermentation. In particular, by comparing control of CpFatB1 with EL222 transcriptional repression alone, LOVdeg, or both the authors show that light-induced protein degradation is more effective than light-induced transcriptional repression. The authors suggest that this is because transcriptional repression is not effective when cells are at stationary phase (and thus there is no protein dilution due to cell division). Overall, the authors have generated a modular, light-activated degron tag for use in Escherichia coli that is likely to be a useful tool in the synthetic biology and metabolic engineering toolkit.

Reviewer #2 (Public Review):

In this study, the authors hypothesized that the balance of mTOR complexes in Sertoli cells may also play a significant role in age-dependent changes in the sperm epigenome. To test this hypothesis, the authors use transgenic mice with manipulated activity of mTOR complexes in Sertoli cells. These results suggest that the mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.

The authors attempt to demonstrate that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. The authors have effectively met their research objectives, and their conclusions are supported by the data presented.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors work to extend their previous observation that galectin-9 interacts with arabinogalactans of Mtb in their EMBO reports 2021 manuscript. Here they provide evidence that the CARD2 domain of galectin-9 can inhibit the growth of Mtb in culture. In addition, antibodies that also bind to AG appear to inhibit Mtb growth in culture. These data indicate that independent of the common cell-associated responses to galectin-9 and antibodies, the interaction of these proteins with AG of mycobacteria may have consequences for bacterial growth.

Strengths:

The authors provided several lines of evidence in culture media that the introduction of galectin-9 proteins and antibodies inhibits the growth rate of Mtb.

Weaknesses:

In light of other observations that cleaved galectin-9 levels in the plasma is a biomarker for severe infection (Padilla A et al Biomolecules 2021 and Iwasaki-Hozumi H et al. Biomoleucles 2021) it is difficult to reconcile the author's interpretation that the elevated gal-9 in Active TB patients (Figure 1E) contributes to the maintenance of latent infection in humans. The authors should consider incorporating these observations in the interpretation of their own results.

The anti-AG titers were measured only in individuals with active TB (Figure 3C), generally thought to be a less protective immunological state. The speculation that individuals with anti-AG titers have some protection is not founded. Further only 2 mAbs were tested to demonstrate restriction of Mtb in culture. It is possible that clones of different affinities for AG present within a patient's polyclonal AG-antibody responses may or may not display a direct growth restriction pressure on Mtb in culture. The authors should soften the claims about the presence of AG-titers in TB patients being indicative of protection.

Reviewer #2 (Public Review):

Summary:

The authors generated a DNA methylation score in cord blood for detecting exposure to cigarette smoke during pregnancy. They then asked if it could be used to predict height, weight, BMI, adiposity, and WHR throughout early childhood.

Strengths:

The study included two cohorts of European ancestry and one of South Asian ancestry.

Weaknesses:

1. The number of mothers who self-reported any smoking was very low, much lower than in the general population and practically non-existent in the South Asian population. As a result, all analyses appeared to have been underpowered. It is possibly for this reason that the authors chose to generate their DNA methylation model using previously published summary statistics. The resulting score is not of great value in itself due to the low-powered dataset used to estimate covariance between CpG sites. In fact, a score was generated for a much larger, better-powered dataset several years ago (Reese, EHP, 2017, PMID 27323799).

2. The conclusion that "even minimal smoking exposure in South Asian mothers who were not active smokers showed a DNAm signature of small body size and low birthweight in newborns" is not warranted because no analyses were performed to show that the association between DNA methylation and birth size/weight was driven by maternal smoking.

3. Although it was likely that some mothers were exposed to second-hand smoke and/or pollution, data on this was either non-existent or not included in this study. Including this would have allowed a more novel investigation of the effects of smoke exposure on the pregnancies of non-smoking mothers.

4. One of the European cohorts and half of the South Asian cohort had DNA methylation measured on only 2500 CpG sites. This set of sites included only 125 sites previously linked to prenatal smoking. The resulting model of prenatal smoking was small (only 11 CpG sites). It is possible that a large model may have been more powerful.

5. The health outcomes investigated are potentially interesting but there are other possibly more important outcomes of interest such as birth complications, asthma, and intellectual impairment which are known to be associated with prenatal smoking.

Reviewer #2 (Public Review):

Summary:<br /> Peled et al identified HER2 mutations in connection with resistance to the anti-HER2 antibody Pertuzumab-mediated therapy. After constructing a yeast display library of Pertuzumab variants with 3.86×1011 sequences for targeted screening of variant combinations in chosen 6 out of 14 residues, the authors performed experimental screening to obtain the clones that bind to HER2 WT and/or mutants (S310Y and S310F), and then combined new variations to obtain antibodies with a broad spectrum binding to both WT and two HER2 mutants. These are interesting studies of clinical impact and translational potential.

Strengths:<br /> 1. Deep computational analyses of large datasets of clinical data provide useful information about HER2 mutations and their potential relevance to antibody therapy resistance.

2. There is valuable information analyzing the residues within or near the interface between the antigen HER2 and the Pertuzumab antibody (heavy chain). The experimental antibody library screening obtained 90+ clones from 3.86×1011 sequences for further functional validation.

Weaknesses:<br /> 1. There is a lack of assessment for antibody variant functions in cancer cell phenotypes in vitro (proliferation, cell death, motility) or in vivo (tumor growth and animal survival). The only assay was the western blotting of phosphopho-HER3 in Figure 4. However, HER2 levels and phosphor-HER2 were not analyzed.

2. There is a misleading impression from the title of computational engineering of a therapeutic antibody and the statement in the abstract "we designed a multi-specific version of Pertuzumab that retains original function while also bindings these HER2 variants" for a few reasons:<br /> a. The primary method used for variant antibody identification for HER2 mutant binding is rather traditional experimental screening based on yeast display instead of the computational design of a multi-specific version of Pertuzumab.<br /> b. There is insufficient or lack of computational power in the antibody design or prioritization in choosing variant residues for the library construction of 3.86×1011 sequences. It seems random combinations from 6 residues out of 4 groups with 20 amino acid options.<br /> c. The final version of the tri-binding variant is a combination of screened antibody clones instead of computation design from scratch.<br /> d. There is incomplete experimental evidence about the therapeutic values of newly obtained antibody clones.

3. Figures can be improved with better labeling and organization. Some essential pieces of data such as Supplementary Figure 1B on HER2 mutations in S310 that abrogated its binding to Pertuzumab should be placed in the main figures.

4. It is recommended to provide a clear rationale or flowchart overview into the main Figure 1. Figure 2A can be combined with Figure 1 to the list of targeted residues.

5. The quality of Figures such as Figure 2B-C flow data needs to be improved.

Reviewer #2 (Public Review):

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

Major comments:

- The similar protective effect of FMT with fecal samples, A. muciniphila or B. fragilis is perhaps not that surprising, considering that FMT likely restores microbiota-mediated colonization resistance against ETEC infection. While FMT with fecal samples increases SCFAs, it is unclear whether/how FMT with A. muciniphila or B. fragilis alter the microbiota composition/abundance as well as metabolites in the current models in a way that offers protection.

- Does ETEC infection in piglets/mice cause histological damage in the intestines? These data should be shown.

- Line 447, "ETEC adheres to intestinal epithelial cells". However, there is no data showing the adherence (or invasion) of ETEC to intestinal epithelial cells, irrespective of piglets/mouse/organoids.

- In both basal-out and apical-out intestinal organoid models, A. muciniphila protects organoids against ETEC infection. Did ETEC enter into intestinal epithelial cells at all after only one hour of infection? Is the protection through certain A. muciniphila metabolites?

Reviewer #2 (Public Review):

In this manuscript, Tiedje and colleagues longitudinally track changes in parasite numbers across four time points as a way of assessing the effect of malaria control interventions in Ghana. Some of the study results have been reported previously, and in this publication, the authors focus on age-stratification of the results. Malaria prevalence was lower in all age groups after IRS. Follow-up with SMC, however, maintained lower parasite prevalence in the targeted age group but not the population as a whole. Additionally, they observe that diversity measures rebounds more slowly than prevalence measures. Overall, I found these results clear, convincing, and well-presented. They add to a growing literature that demonstrates the relevance of asymptomatic reservoirs.

There is growing interest in developing an expanded toolkit for genomic epidemiology in malaria, and detecting changes in transmission intensity is one major application. As the authors summarize, there is no one-size-fits-all approach, and the Bayesian MOIvar estimate developed here has the potential to complement currently used methods. I find its extension to a calculation of absolute parasite numbers appealing as this could serve as both a conceptually straightforward and biologically meaningful metric. However, I am not fully convinced the current implementation will be applied meaningfully across additional studies.

1. I find the term "census population size" problematic as the groups being analyzed (hosts grouped by age at a single time point) do not delineate distinct parasite populations. Separate parasite lineages are not moving through time within these host bins. Rather, there is a single parasite population that is stochastically divided across hosts at each time point. I find this distinction important for interpreting the results and remaining mindful that the 2,000 samples at each time point comprise a subsample of the true population. Instead of "census population size", I suggest simplifying it to "census count" or "parasite lineage count".

It would be fascinating to use the obtained results to model absolute parasite numbers at the whole population level (taking into account, for instance, the age structure of the population), and I do hope this group takes that on at some point even if it remains outside the scope of this paper. Such work could enable calculations of absolute---rather than relative---fitness and help us further understand parasite distributions across hosts.

2. I'm uncertain how to contextualize the diversity results without taking into account the total number of samples analyzed in each group. Because of this, I would like a further explanation as to why the authors consider absolute parasite count more relevant than the combined MOI distribution itself (which would have sample count as a denominator). It seems to me that the "per host" component is needed to compare across age groups and time points---let alone different studies.

3. Thinking about the applicability of this approach to other studies, I would be interested in a larger treatment of how overlapping DBLa repertoires would impact MOIvar estimates. Is there a definable upper bound above which the method is unreliable? Alternatively, can repertoire overlap be incorporated into the MOI estimator?

Smaller comments:<br /> - Figure 1 provides confidence intervals for the prevalence estimates, but these aren't carried through on the other plots (and Figure 5 has lost CIs for both metrics). The relationship between prevalence and diversity is one of the interesting points in this paper, and it would be helpful to have CIs for both metrics when they are directly compared.

Reviewer #2 (Public Review):

Summary:<br /> In the manuscript by Luo et al, the authors investigated the nature and function of TRAIL-HS binding for the regulation of TRAIL-mediated apoptosis in cancer cells. The authors discovered that TRAIL binds to 12mer HS and identified the amino acid residues critical for the binding. The authors further nicely showed that 12mer HS binds to TRAIL homotrimer and larger HS can further promote the formation of larger TRAIL oligomers. Structural analyses were conducted to characterize the binding of TRAIL/HS complexes. At functional level, the authors demonstrated that HS promotes the cell surface binding of TRAIL to enhance TRAIL-mediated apoptosis in a variety of cancer cells. Moreover, the ability of TRAIL to induce apoptosis is correlated with cell surface HS level. Lastly, the authors showed that HS forms complex with TRAIL and its receptor DR5 and promotes DR5 internalization.

Strengths:<br /> Overall, this is a nicely executed study providing both mechanistic and functional insight for TRAIL-mediated apoptosis. It conducted detailed characterization on the direct binding between HS and TRAIL and provided solid evidence supporting the role of such interaction for the regulation of TRAIL-induced apoptosis. The experiments were well-designed with proper controls included. The data interpretation is accurate. The manuscript was clearly written and easy to follow by general readers.

Weaknesses:<br /> There is no major weakness identified from this study. As the authors pointed out, the current relationship between cell surface HS level and sensitivity to TRAIL-mediated apoptosis is still correlative and will need further investigation in the future.

Reviewer #2 (Public Review):

In this study, the authors seek to answer two main questions: 1) Whether interfering with lactate availability in hepatocytes through depletion of hepatocyte specific MCT-1 depletion would reduce steatosis, and 2) Whether MCT-1 in stellate cells promote fibrogenesis. While the first question is based on the observation that haploinsufficiency of MCT-1 makes mice resistant to steatosis, the rationale behind how MCT-1 could impact fibrogenesis in stellate cells is not clear. A more detailed discussion regarding how lactate availability would regulate two different processes in two different cell types would be helpful. The authors employ several mouse models and in vitro systems to show that MCT1 inhibition in hepatic stellate cells reduces the expression of COL-1.

The authors have sufficiently addressed prior comments and added new experiments to provide details on possible mechanisms.

Reviewer #2 (Public Review):

Summary:

In their manuscript, the authors address the question of whether the inversion polymorphism in D. melanogaster can be explained by sexually antagonistic selection. They designed a new simulation tool to perform computer simulations, which confirmed their hypothesis. They also show a tradeoff between male reproduction and survival. Furthermore, some inversions display sex-specific survival.

Strengths:<br /> It is an interesting idea on how chromosomal inversions may be maintained

Weaknesses:<br /> General points:<br /> The manuscript lacks clarity of writing. It is impossible to fully grasp what the authors did in this study and how they reached their conclusions. Therefore, I will highlight some cases that I found problematic.<br /> Although this is an interesting idea, it clearly cannot explain the apparent influence of seasonal and clinal variation on inversion frequencies.

Specific points:<br /> The simulations are highly specific and make very strong assumptions, which are not well-justified.

Reviewer #2 (Public Review):

Ehring et al. analyze contributions of Dispatched, Scube2, serum lipoproteins and Sonic Hedgehog lipid modifications to the generation of different Shh release forms. Hedgehog proteins are anchored in cellular membranes by N-terminal palmitate and C-terminal cholesterol modifications, yet spread through tissues and are released into the circulation. How Hedgehog proteins can be released, and in which form, remains controversial. The authors systematically dissect contributions of several previously identified factors, and present evidence that Disp, Scube2 and lipoproteins concertedly act to release a novel Shh variant that is cholesterol-modified but not palmitoylated. The results provide new insights into the function of Disp and Scube2 in Hedgehog release. The findings concerning the function of lipoproteins and cholesterol in Hedgehog release are largely confirmatory (PMID 23554573, 20685986). However, in light of the multitude of competing models for Hedgehog release, the present study is a valuable contribution that provides further insights into the relevance of lipoproteins in this process.

A novel and surprising finding of the present study is the differential removal of Shh N- or C-terminal lipid anchors depending on the presence of HDL and/or Disp. In particular, the identification of a non-palmitoylated but cholesterol-modified Shh variant that associates with lipoproteins is potentially important. The authors use RP-HPLC and defined controls to assess the properties of processed Shh forms, but their precise molecular identify remains to be defined. A caveat is the strong reliance on over-expression of Shh in a single cell line. The authors detect Shh variants that are released independently of Disp and Scube2 in secretion assays, which however are excluded from interpretation as experimental artifacts. Thus, it would be important to demonstrate key findings in cells that secrete Shh endogenously.

Reviewer #2 (Public Review):

By mapping the sites of the Mcm2-7 replicative helicase loading across the budding yeast genome using high-resolution chromatin endogenous cleavage or ChEC, Bedalov and colleagues find that these markers for origins of DNA replication are much more broadly distributed than previously appreciated. Interestingly, this is consistent with early reconstituted biochemical studies that showed that the ACS was not essential for helicase loading in vitro (e.g. Remus et al., 2009, PMID: 19896182). To accomplish this, they combined the results of 12 independent assays to gain exceptionally deep coverage of Mcm2-7 binding sites. By comparing these sites to previous studies mapping ssDNA generated during replication initiation, they provide evidence that at least a fraction of the 1600 most robustly Mcm2-7-bound sequences act as origins. A weakness of the paper is that the group-based (as opposed to analyzing individual Mcm2-7 binding sites) nature of the analysis prevents the authors from concluding that all of the 1,600 sites mentioned in the title act as origins. The authors also show that the location of Mcm2-7 location after loading are highly similar in the top 500 binding sites, although the mobile nature of loaded Mcm2-7 double hexamers prevents any conclusions about the location of initial loading. Interestingly, by comparing subsets of the Mcm2-7 binding sites, they find that there is a propensity of at least a subset of these sites to be nucleosome depleted, to overlap with at least a partial match to the ACS sequence (found at all of the most well-characterized budding yeast origins), and a GC-skew centered around the site of Mcm loading. Each of these characteristics is related to previously characterized S. cerevisiae origins of replication.

Overall, this manuscript greatly broadens the number of sites that are capable of loading Mcm2-7 in budding yeast cells and shows that a subset of these additional sites act as replication origins. Although these studies show that the sequence specificity of S. cerevisiae replication origins still sets it apart from metazoan origins, the ability to license and initiate replication from sites with increasing sequence divergence suggests a previously unappreciated versatility.

Reviewer #2 (Public Review):

This is a thorough and convincing body of work that represents an incremental but significant improvement on iterations of this method of CRISPR-based Sterile Insect Technique ('pgSIT'). In this version, compared to previous, the authors target more genes than previously, in order to induce both female inviability (targeting the genes intersex and doublesex, compared to fem-myo previously) and male sterility (targeting a beta-tubulin, as previously in the release generation.

The characterization of the lines is extensive and this data will be useful to the field. However, what is lacking is some context as to how this formulation compares to the previous iteration. Mention is made of the possible advantage of removing most females, compared to just making them flightless (as previously) but there is no direct comparison, either experimental, or theoretical i.e. imputing the life history traits into a model. For me this is a weakness, yet easily addressed. In a similar vein, much is made in alluding to the 'safety concerns of gene drive' and how this is a more palatable half-way house, just because it has CRISPR component within it; it is not. It would be much more sensible, and more informative, to compare this pgSIT technology to RIDL (release of insects carrying a dominant lethal), which is essentially a transgene-based version of the Sterile Insect Technique, as is the work presented here.

The authors achieve impressive results and show that these strains, under a scenario of high levels of release ratios compared to WT, could achieve significant local suppression of mosquito populations. The sensitivity analysis that examines the effect of changing different biological or release parameters is well performed and very informative.

The authors are honest in acknowledging that there are still challenges in bringing this to field release, namely in developing sexing strains and optimizing release strategies.

Reviewer #2 (Public Review):

In this work, Vollenweider et al. examine the effectiveness of using natural products, specifically molecules that chelate iron, to treat infectious agents. Through the purification of 320 environmental isolates, 25 potential candidates were identified from natural products based on inhibition assays and were further screened. The structural information and chemical composition were determined.

The paper is well-structured and thorough; targeting virulence factors in this manner is a great idea. My enthusiasm is dampened by the mediocre effects of the compounds. The lack of a dose-response curve in the survivability assays suggests a limited scope for these molecules. While it is encouraging that the best survivability occurred at the lowest toxicity level, it opens questions as to how effective such molecules can be. Either the reduction in mortality was offset by using higher concentrations, which was not observed in the compound-alone test, or there is no dose-response curve. The latter would suggest to me that the variation in survivability is not due to the addition of siderophores.

I would also like to see how these molecules compare to other iron-chelating molecules. Desferoxamine is a bacteria-derived siderophore that is FDA-approved. However, it is not used to treat infections. Would the author consider comparing their candidate molecules to well-studied molecules? This also raises questions about the novelty of this work; I think the authors could rephrase the discussion to better reflect that bioprospecting for iron-chelating molecules has previously occurred and been successful.

Finally, I am concerned about the few mutations reported in the resistance study. Looking at the SI, it appears that very few mutations were seen. It is unclear what filtering the authors used to arrive at such a low number of mutations. Even filtering against mutations that were selected by adaptation to the media, it seems low that only a handful of clones had distinct mutations.

This paper has a lot of strengths. The workflow is logical and well-executed; the only significant weakness is the effect of the molecules and the lack of an explanation for a dose-response curve in the survivability assay, especially when compared to the data reported in Figure 3. As the authors describe in lines 214-217.

Reviewer #2 (Public Review):

Summary:<br /> Dr. Khulman and colleagues present a very interesting experimental and mathematical modeling work on IS608 transposition. The system has a number of unique advantages that create experimental possibilities utilized here to investigate transposition dynamics. This kind of approach is badly missing from the field and I believe the experiments and modeling work shown here and the type of results that can be derived are groundbreaking and certainly deserve high visibility.

Strengths:<br /> The attempt to measure and model transposition dynamics in cells.

Weaknesses:<br /> - Lack of controls using an active site mutant of TnpB.<br /> - Lack of control in RecA- cells as transposon restoration is proposed to be dependent on homologous recombination.<br /> - Lack of consideration of the levels of ωRNA present.

Reviewer #2 (Public Review):

Hagihara et al. conducted a study investigating the correlation between decreased brain pH, increased brain lactate, and poor working memory. They found altered brain pH and lactate levels in animal models of neuropsychiatric and neurodegenerative disorders. Their study suggests that poor working memory performance may predict higher brain lactate levels.

However, the study has some significant limitations. One major concern is that the authors examined whole-brain pH and lactate levels, which might not fully represent the complexity of disease states. Different brain regions and cell types may have distinct protein and metabolite profiles, leading to diverse disease outcomes. For instance, certain brain regions like the hippocampus and nucleus accumbens exhibit opposite protein/signaling pathways in neuropsychiatric disease models.

Moreover, the memory tests used in the study are specific to certain brain regions, but the authors did not measure lactate levels in those regions. Without making lactate measurements in brain-regions and cell types involved in these diseases, any conclusions regarding the role of lactate in CNS diseases is premature.

Additionally, evidence suggests that exogenous treatment with lactate has positive effects, such as antidepressant effects in multiple disease models (Carrard et al., 2018, Carrard et al., 2021, Karnib et al., 2019, Shaif et al., 2018). It also promotes learning, memory formation, neurogenesis, and synaptic plasticity (Suzuki et al., 2011, Yang et al., 2014, Weitian et al., 2015, Dong et al., 2017, El Hayek et al. 2019, Wang et al., 2019, Lu et al., 2019, Lev-Vachnish et a.l, 2019, Descalzi G et al., 2019, Herrera-López et al., 2020, Ikeda et al., 2021, Zhou et al., 2021,Roumes et al., 2021, Frame et al., 2023, Akter et al., 2023).

In conclusion, the relevance of total brain pH and lactate levels as indicators of the observed correlations is controversial, and evidence points towards lactate having more positive rather than negative effects. It is important that the authors perform studies looking at brain-region-specific concentrations of lactate and that they modulate lactate levels (decrease) in animal models of disease to validate their conclusions. It is also important to consider the above-mentioned studies before concluding that "altered brain pH and lactate levels are rather involved in the underlying pathophysiology of some patients with neuropsychiatric disorders" and that "lactate can serve as a potential therapeutic target for neuropsychiatric disorders".

Reviewer #2 (Public Review):

The study is novel and valuable to the field and provides new and important insights into the role of lipid mediators in VML injuries. By expanding our understanding of the mechanisms that regulate muscle regeneration following VML injuries, the study has the potential to guide the development of novel therapeutic interventions that promote tissue repair and recovery. The data presented in the manuscript is of good quality. The findings and conclusions are supported by a variety of different analyses (e.g., gene expression, histology, flow cytometry).

Despite the strengths of the study, some limitations are identified. Specifically, the impact of maresin 1 on macrophage phenotypes (M1/M2) could have been explored in more detail using histological or protein expression analysis. Moreover, additional data are needed to substantiate the claims about increased muscle regeneration. Lastly, the study does not address myofiber innervation, myofiber-type transitions, or motor unit remodeling.

Reviewer #2 (Public Review):

In this study, the goal is to leverage the power of Bayesian inference to estimate online the probability that any given arbitrarily chosen strategy is being used by the decision-maker. By computing the trial-by-trial MAP and variance of the posterior distribution for each candidate strategy, the authors can not only see which strategy is primarily being used at every given time during the task and when strategy changes occur but also detect when the target rule of a learning task becomes the front-running strategy, i.e., when successful learning occurs.

Strengths:<br /> 1. The proposed approach adds to recent methods for capturing the dynamics of decision-making at finer temporal resolution (trials) (Roy et al., 2021; Ashwood et al., 2022) but it is novel and differs from these in that it is suited especially well for analyzing when learning occurs, or when a rule switches and learning must recommence, and it does not necessitate large numbers of trials.

2. The manuscript starts with a validation of the approach using synthetic data and then is applied to datasets of trial-based two-alternative forced choice tasks ranging from rodent to non-human primate to human, providing solid evidence of its utility.

3. Compared to classic procedures for identifying when an animal has learned a contingency which typically needs to be conservative in favor of better accuracy, this method retrieves signs of learning happening earlier (~30 trials earlier on average). This is achieved by identifying the moment (trial) when the posterior probability of the correct "target" rule surpasses the probability of all other strategies. Having greater temporal precision in detecting when learning happens may have a very significant impact on studies of the neural mechanisms of learning.

4. This approach seems amenable to testing many different strategies depending on the purpose of the analysis. In the manuscript, the authors test target versus non-target strategies (correct versus incorrect) and also in another version of the analysis, they test what they call "exploratory" strategies.

5. One of the main appeals of this method is its apparent computational simplicity. It necessitates only updating on every trial the parameters of a beta distribution (prior distribution for a given strategy) with the evidence that the behavior on trial was either consistent or inconsistent with the strategy. Two scalars, the mode of the posterior (MAP) and the inverse of the variance, are all that are required for identifying the decision criterion (highest MAP and if tied lowest variance) and the learning criterion (first trial where MAP for target strategy is higher than chance).

Weaknesses:<br /> 1. It seems like a limitation of this approach is that the candidate strategies to arbitrate between must be known ex-ante. It is not clear how this approach could be applied to uncover latent strategies that are not mixtures of the strategies selected.

2. Different strategies may be indistinguishable from each other and thus it may not be possible to distinguish between them. Similarly, the fact that two strategies seem to be competing for the highest MAP doesn't necessarily mean that those are correct strategies and perhaps interchangeable as the manuscript seems to suggest.

3. The decay parameter is a necessary component to make the strategy selection non-stationary and accommodate data sets where the rules are changing throughout the task. However, the choice of the decay parameter value bounds does not seem very principled. Having this parameter as a free-parameter adds a flexibility that seems to have significant effects on when the strategy switch is detected and how stable the detected switch is.

4. This method is a useful approach for arbitrating between strategies and describing the behavior with a temporal precision that may prove important for studies attempting to tie these precise events to changes in neural activity. However, it seems limited in its explanatory power. In its current form, this method does not provide a prediction of the probability to transition from one strategy to another. And, because the MAP of different strategies may be close at any given moment, it is hard to imagine using this approach to tease out the different "mental states" that represent each strategy being at play.

Reviewer #2 (Public Review):

Working memory is not error free. Behavioral reports of items held in working memory display several types of bias, including contraction bias and serial dependence. Recent work from Akrami and colleagues demonstrates that inactivating rodent PPC reduces both forms of bias, raising the possibility of a common cause.

In the present study, Boboeva, Pezotta, Clopath, and Akrami introduce circuit and descriptive variants of a model in which the contents of working memory can be replaced samples from recent sensory history. This volatility manifests as contraction bias and serial dependence in simulated behavior, parsimoniously explaining both sources of bias. The authors validate their model by showing that it can recapitulate previously published and novel behavioral results in rodents and neurotypical and atypical humans.

Both the modeling and the experimental work is rigorous, providing convincing evidence that a model of working memory in which reports sometimes sample past experience can produce both contraction bias and serial dependence, and that this model is consistent with behavioral observations across rodents and humans in the parametric working memory (PWM) task.

These efforts constitute an admirable initial validation of the proposed model, and the authors present several novel predictions that will allow for further tests in future experiments. First, the authors note that their circuit model predicts a bimodal error distribution in delayed estimation paradigms (due to noisy sampling of sensory history on a subset of trials) that merges into a unimodal distribution when recent sensory history and the current to-be-reported stimulus have very similar values (Fig. 5c). Analysis of extent delayed estimation datasets (e.g., https://osf.io/jmkc9/) or new experiments will provide the opportunity for a straightforward test of this hypothesis.

Second, the bulk of the modeling efforts presented here are devoted to a circuit-level description of how putative posterior parietal cortex (PPC) and working-memory (WM) related networks may interact to produce such volatility and biases in memory. This effort is extremely useful because it allows the model to be constrained by neural observations and manipulations in addition to behavior, and the authors begin this line of inquiry here (by showing that the circuit model can account for effects of optogenetic inactivation of rodent PPC). As the authors note, population electrophysiology in PPC and WM-related areas and single-trial analyses will play an important role in the ultimate validation of this model.

Finally, it is noteworthy that, in the spirit of moving away from an overreliance on p-values (e.g., Amrhein et al., PeerJ 2017), the authors eschew conventional hypothesis testing when reporting their experimental results. The p-values aren't missed, in large part thanks to excellent visualizations and apparently large effect sizes. It's unclear how well this approach would generalize to other questions and datasets; nevertheless, this study provides an interesting data point in the ongoing conversation around reproducibility and rigor.

Reviewer #2 (Public Review):

Schmid et al present a lovely study looking at the effect of passive auditory exposure on learning a categorization task.<br /> The authors utilize a two-alternative choice task where mice have to discriminate between upward and downward moving frequency sweeps. Once mice learn to discriminate easy stimuli, the task is made psychometric and additional intermediate stimuli are introduced (as is standard in the literature). The authors introduce an additional two groups of animals, one that was passively exposed to the task stimuli before any behavioral shaping, and one that had passive exposure interleaved with learning. The major behavioral finding is that passive exposure to sounds improves learning speed. The authors show this in a number of ways through linear fits to the learning curves. Additionally, by breaking down performance based on the "extreme" vs "psychometric" stimuli, the authors show that passive exposure can influence responses to sounds that were not present during the initial training period. One limitation here is that the presented analysis is somewhat simplistic, does not include any detailed psychometric analysis (bias, lapse rates etc), and primarily focuses on learning speed. Ultimately though, the behavioral results are interesting and seem supported by the data.

To investigate the neural mechanisms that may underlie their behavioral findings, the authors turn to a family of artificial neural network models and evaluate the consequences of different learning algorithms and schedules, network architectures, and stimulus distributions, on the learning outcomes. The authors work through five different architectures that fail to recapitulate the primary behavior findings before settling on a final model, utilizing a combination of supervised and unsupervised learning, that was capable of reproducing the key aspects of the experiments. Ultimately, the behavioral results presented are consistent with network models that build latent representations of task-relevant features that are determined by statistical properties of the input distribution.

Reviewer #2 (Public Review):

This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however the biological insight gained is rather limited.

Reviewer #2 (Public Review):

The authors attempt to address a long-standing controversy in the study of the neural correlates of visual awareness, namely whether neurons in prefrontal cortex are necessarily involved in conscious perception. Several leading theories of consciousness propose a necessary role for (at least some sub-regions of) PFC in basic perceptual awareness (e.g., global neuronal workspace theory, higher order theories), while several other leading theories posit that much of the previously reported PFC contributions to perceptual awareness may have been confounded by task-based cognition that co-varied between the aware and unaware reports (e.g., recurrent processing theory, integrated information theory). By employing intracranial EEG in human patients and a threshold detection task on low-contrast visual stimuli, the authors assessed the timing and location of neural populations in PFC that are differentially activated by stimuli that are consciously perceived vs. not perceived. Overall, the reported results support the view that certain regions of PFC do contribute to visual awareness, but at time-points earlier than traditionally predicted by GNWT and HOTs.

Major strengths of this paper include the straightforward visual threshold detection task including the careful calibration of the stimuli and the separate set of healthy control subjects used for validation of the behavioral and eye tracking results, the high quality of the neural data in six epilepsy patients, the clear patterns of differential high gamma activity and temporal generalization of decoding for seen versus unseen stimuli, and the authors' interpretation of these results within the larger research literature on this topic. This study appears to have been carefully conducted, the data were analyzed appropriately, and the overall conclusions seem warranted given the main patterns of results.

Weaknesses include the saccadic reaction time results and the potential flaws in the design of the reporting task. As the authors acknowledge, this is not a "no report" paradigm, rather, it's a paradigm aimed at balancing the post-perceptual cognitive and motor requirements between the seen and unseen trials. On each trial, subjects/patients either perceived the stimulus or not, and had to briefly maintain this "yes/no" judgment until a fixation cross changed color, and the color change indicated how to respond (saccade to the left or right). Differences in saccadic RTs (measured from the time of the fixation color change to moving the eyes to the left or right response square) were evident between the seen and unseen trials (faster for seen). In the discussion, the authors summarize several alternative explanations of the saccade results and limitations of their report paradigm that will help guide future research.

The current results help advance our understanding of the contribution of PFC to visual awareness. These results, when situated within the larger context of the rapidly developing literature on this topic provide converging evidence that some sub-regions of PFC contribute to visual awareness, but at latencies earlier than originally predicted by proponents of, especially, global neuronal workspace theory. Three recent studies that used "no report paradigms", but with clearly visible stimuli, reported very similar results in PFC (Vishne et al., 2023; Broday-Dvir et al., 2023; Cogitate et al., 2023). The current study uses a report paradigm, but with consciously seen vs. unseen conditions, to fill the gap left by these previous studies, i.e., it remained unclear whether the PFC results from the previous studies were related to conscious or unconscious processing. Taken as a whole, evidence appears to be converging for a limited and early-in-time (200-300ms) contribution of PFC to visual awareness, after task and motor confounds are minimized.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediators of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from post-mortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the pre-symptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied with the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function. This is an expected result, given the fairly focal approach of ephrinB2 knockdown in C3-C5 spinal segments.

The major limitation of the study is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many other that comprise the ventral gray matter, have not been investigated in this study. Nonetheless, there is convincing evidence to suggest astrocytes play a significant role, as compared to oligodendrocytes in promoting ALS pathogenesis.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript by Martin-Flores et al. examines the role of DKK3 in Alzheimer's disease, focusing on the regulation of synaptic number. Using human AD brain databases and tissue samples, the authors demonstrate increased levels of DKK3 protein and mRNA in the brains of AD patients. DKK3 is expressed in excitatory neurons in WT mouse brains and accumulates at atrophic neurites around amyloid plaques in AD mouse brains. Interestingly, the secretion of DKK3 appears to be regulated by NMDAR antagonists, as well as chemical LTD. Through gain and loss of function studies, the authors reveal that DKK3 regulates the number of both excitatory and inhibitory synapses with distinct downstream pathways. Finally, the authors investigate the contribution of DKK3 to synaptic changes in AD and find that DKK3 loss of function rescues both excitatory and inhibitory synaptic defects, resulting in improved memory function in J20 mice.

Strengths:<br /> Overall, the data is clearly presented and deals with the novel roles of DKK3 in controlling excitatory and inhibitory synapses. The finding that shRNA expression of DKK3 in AD model mice rescues synaptic phenotypes and memory impairment is potentially interesting and may provide a new strategy for AD treatment.

Weaknesses:<br /> There are no major weaknesses.

Reviewer #3 (Public Review):

Summary:<br /> Onck and co-workers present in this work the identification of binding partners and sites of polyPR on various nuclear transport components and elucidate how polyPR might potentially influence the transport process. It's interesting to note that some interaction sites on transport components also serve as their inherent/functional binding sites (Figure 3). The difference in the effects between short polyPR (PR7) and long polyPR (PR50) is also evident, although the authors might need to clarify the mechanisms better. Overall, I find this manuscript well organized and concisely written, and it would greatly enhance our understanding of the toxicity induced by polyPR.

Strengths:<br /> The 1-bead per atom force field model used in the study is well-tuned for studying the interactions between polyPR and proteins, as the essential cation-pi interactions (between Arg and Phe/Tyr/Trp) was included using a 8-6 LJ model.

Weaknesses:<br /> To cite the author's response: "At the moment, accurately capturing the binding of NCT components to their native binding targets and the competition with polyPR are best resolved by all-atom molecular dynamics simulations, which come with significant computational demands. This level of detail and computation-intensive analyses is beyond the scope of the current study."

Reviewer #2 (Public Review):

Summary:<br /> Chang et al. investigate neuronal activity firing patterns across various cortical regions in an interesting context-dependent tactile vs visual detection task, developed previously by the authors (Chevee et al., 2021; doi: 10.1016/j.neuron.2021.11.013). The authors report the important involvement of a medial frontal cortical region (MM, probably a similar location to wM2 as described in Esmaeili et al., 2021 & 2022; doi: 10.1016/j.neuron.2021.05.005; doi: 10.1371/journal.pbio.3001667) in mice for determining task rules.

Strengths:<br /> The experiments appear to have been well carried out and the data well analysed. The manuscript clearly describes the motivation for the analyses and reaches clear and well-justified conclusions. I find the manuscript interesting and exciting!

Weaknesses:<br /> I did not find any major weaknesses.

Reviewer #2 (Public Review):

Summary:<br /> The authors' long-term goals are to understand the utility of precisely phased cortex stimulation regimes on recovery of function after spinal cord injury (SCI). In prior work, the authors explored the effects of contralesion cortex stimulation. Here, they explore ipsilesion cortex stimulation in which the corticospinal fibers that cross at the pyramidal decussation are spared. The authors explore the effects of such stimulation in intact rats and rats with a hemisection lesion at the thoracic level ipsilateral to the stimulated cortex. The appropriately phased microstimulation enhances contralateral flexion and ipsilateral extension, presumably through lumbar spinal cord crossed-extension interneuron systems. This microstimulation improves weight bearing in the ipsilesion hindlimb soon after injury, before any normal recovery of function would be seen. The contralateral homologous cortex can be lesioned in intact rats without impacting the microstimulation effect on flexion and extension during gait. In two rats ipsilateral flexion responses are noted, but these are not clearly demonstrated to be independent of the contralateral homologous cortex remaining intact.

Strengths:<br /> This paper adds to prior data on cortical microstimulation by the laboratory in interesting ways. First, the strong effects of the spared crossed fibers from the ipsi-lesional cortex in parts of the ipsi-lesion leg's step cycle and weight support function are solidly demonstrated. This raises the interesting possibility that stimulating the contra-lesion cortex as reported previously may execute some of its effects through callosal coordination with the ipsi-lesion cortex tested here. This is not fully discussed by the authors but may represent a significant aspect of these data. The authors demonstrate solidly that ablation of the contra-lesional cortex does not impede the effects reported here. I believe this has not been shown for the contra-lesional cortex microstimulation effects reported earlier, but I may be wrong.

Effects and neuroprosthetic control of these effects are explored well in the ipsi-lesion cortex tests here.

Weaknesses:<br /> Some data is based on very few rats. For example (N=2) for ipsilateral flexion effects of microstimulation. N=3 for homologous cortex ablation, and only ipsi extension is tested it seems. There is no explicit demonstration that the ipsilateral flexion effects in only 2 rats reported can survive the contra-lateral cortex ablation.

Some improvements in clarity and precision of descriptions are needed, as well as fuller definitions of terms and algorithms.

Likely Impacts:<br /> This data adds in significant ways to prior work by the authors, and an understanding of how phased stimulation in cortical neuroprosthetics may aid in recovery of function after SCI, especially if a few ambiguities in writing and interpretation are fully resolved.

Reviewer #2 (Public Review):

Summary:<br /> This is a fundamental and elegant study showing the role of BMP signaling in cerebellar development. This is an important question because there are multiple diseases, including aggressive childhood cancers, which involve granule cell precursors. Thus understanding of the factors that govern the formation of the granule cell layer is important both from a basic science and a disease perspective.

Overall, the manuscript is clear and well-written. The figures are extremely clear, wonderfully informative, and overall quite beautiful.

Figures 1-3 show the experimental design and report how BMP activity is altered over development in both the chick and the human developing cerebellum. Both data are very impressive and convincing.

They then go on to modulate BMP activity in the developing chick, using a complex electroporation paradigm that allows them to label cells with GFP as well as with cell-specific reporters of BMP activity levels. They bidirectionally modulate BMP levels and then can look at both cell-specific and non-specific alterations in the formation of the external and internal granule cell layer, across different developmental timepoints. These are really elegant and rigorous experiments, as they look at both sagittal and transverse sections to collect this data. This makes the data extremely compelling. With these rigorous techniques, they show that BMP signaling serves more than one function across development: it is involved in the initial tangential migration from the rhombic lip, but at a later time, both up- and down-regulation of BMP activity reduces the density of amplifying cells in the external granule cell layer.

Strengths:<br /> Overall, I think the paper is interesting and important and the data is strong. The use of both chick and human tissue strengthens the findings. They are extremely rigorous, analyzing data from multiple planes at multiple ages, which also really strengthens their findings. The dual electroporation approach is extremely elegant, providing beautiful visual representations of their findings.

Weaknesses:<br /> I find no significant weaknesses.

Reviewer #2 (Public Review):

Summary:

H2S is a gas that is toxic to many animals and causes avoidance in animals such as C. elegans. The authors show that H2S increases the frequency of turning and the speed of locomotion. The response was shown to be modulated by a number of neurons and signaling pathways as well as by ambient oxygen concentrations. The long-term adaptation involved gene expression changes that may be related to iron homeostasis as well as the homeostasis of mitochondria.

Strengths:

Overall, the authors provide many pieces that will be important for solving how H2S signals through neuronal circuits to change gene expression and physiological programs. The experiments rely mostly on a behavioral assay that measures the increase of locomotion speed upon exposure to H2S. This assay is then combined with manipulations of environmental factors, different wild-type strains, and mutants. The mutants analyzed were obtained as candidates from the literature and from transcriptional profiling that the authors carried out in worms that were exposed to H2S. These studies imply several genetic signaling pathways, some neurons, and metabolism-related factors in the response to H2S. Hence the data provided should be useful for the field.

Weaknesses:

On the other hand, many important aspects of the underlying mechanisms remain unsolved and the reader is left with many loose ends. For example, it is not clear how H2S is actually sensed, how sensory neurons are activated and signal to downstream circuits, and what the role of ciliated and RMG neurons is in this circuit. It remains unclear how signals lead to gene expression and physiological changes such as metabolic rewiring. Solving all this would clearly be beyond the scope of a single manuscript. Yet, the manuscript also does not focus on understanding one of these central aspects and rather is all over the place, which makes it harder to understand for readouts that are not in this core field. Multiple additional methods and approaches exist to dig deeper into these mechanisms in the future, such as neuronal calcium imaging, optogenetics, and metabolic analysis. To generate a story that will be interesting to a broad readership substantial additional experimentation would be required. Further, in the current manuscript, it is often difficult to understand the rationales of the experiments, why they were carried out, and how to place them into a context. This could be improved in terms of documentation, narration/explanation, and visualization.

Reviewer #2 (Public Review):

Summary:<br /> Authors hypothesized that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. They test this hypothesis in a mouse model of TBI induced by controlled cortical impact in wild-type and kcnt1 knock-out (KO) mice. The authors performed a series of experiments including behavioral assessment, electrographic recordings in vivo and in vitro, western blotting, and immunofluorescence imaging with the goal of investigating the contributory role of KNa1.1 channel to post-traumatic epileptogenesis.

Strengths:<br /> The hypothesis is innovative, focusing on the specific role of the KNa1.1 channel in the development of PTE post-TBI. The use of a comprehensive set of techniques, including EEG, whole cell patch clamp, western blot, immunofluorescence imaging, and behavioral assessments, provides a diverse data set. The study makes initial steps in correlating specific molecular changes with functional outcomes in TBI models, offering potential pathways for therapeutic intervention.

Weaknesses:<br /> 1) The study presents interesting findings on early changes in protein expression and electrophysiological properties following TBI. However, I would like to draw attention to the timeline of EEG and cellular assessments that require further clarification or consideration. The patch clamp recordings and other assays were conducted within 14 days post-TBI, while EEG recordings with or without PTZ testing were performed at 3 months post-injury. This temporal gap leaves a period where changes in electrophysiological properties and PTE status are not accounted for. Since epileptogenesis post-TBI involves a dynamic process spanning from hyperacute and acute phases to chronic development, capturing these changes continuously or at least at more frequent intervals (on and off bi-weekly) could provide a more comprehensive understanding of this progression. In the current study design, the one-week duration of EEG recordings at the 3-month timepoint raises the possibility that some seizures might have occurred undetected between the early post-injury phase and the EEG recording period. This gap could potentially affect the interpretation of results, especially when correlating early post-injury cellular changes with later seizure activity and thresholds and hence is a significant limitation to data interpretation. Experiments using western blots, immunofluorescence, and patch clamp were done at an early timepoint hence the relevance of these datasets to PTE status outcome is not established.

2) While referencing Nichols et al., 2015, to justify the 14-day timeline for characterizing seizures is understandable, it is important to consider differences in animal models (juvenile rats in Nichols vs. adult mice in the present study) which might influence the generalizability of the findings.

3) Behavior: Authors performed behavioral assays using the rotarod technique evaluating the hanging time of mice with different severity of TBI (mild, moderate, severe). The purpose of the testing is explained as 'to sort out an appropriate TBI model'. The authors also measured mortality rates 'to attain a stable model'. It is not clear what is assumed by the terms 'appropriate' or 'stable' model. Furthermore, the relevance of this to post-traumatic epileptogenesis is unclear. Additionally, the mortality in the Sham group within 2 weeks of craniectomy is not explained.

4) Seizure assessment: authors report seizure severity in PTZ-induced seizures but no mention about the severity of spontaneous seizures between different TBI severity modalities. When characterizing the PTZ-induced seizures, the mild TBI group does not have generalized seizures. Does this mean that al all 6 tested animals in the mild TBI group had exclusively focal seizures? What about the spontaneous seizure occurrence: were all seizures generalized or were any focal too? Did that differ between mild, moderate, and severe TBI?

5) Experiments with KCNT1 KO mice: in all experiments with a mutant mouse line, authors only used them in TBI group. Without the Sham group, it is difficult to discern whether any observed changes in seizure susceptibility in the KCNT1 KO TBI group are due to gene deletion, the TBI, or a combination of both. This group would provide a crucial comparison point to isolate the effects of the KCNT1 knockout from those of TBI. This limits the ability to make comprehensive conclusions about the role of the KCNT1 gene in seizure susceptibility following TBI.

6) While the current study showed interesting data about the KNa1.1 changes early after TBI, the study design and disconnect between early and late electrophysiology experiments timeline, does not establish a correlative or causative link between KNa1.1 and post-traumatic epileptogenesis since it remained unresolved whether KCNT1 KO mice developed no PTE (or less severe/ less frequent seizures at 3 months) compared with WT mice and what are the seizure properties of KCNT1 KO Sham mice compared to WT TBI and Sham groups. The hypothesis was that modulation of KNa1.1 channel specifically in inhibitory interneurons contributes to the hyperactivity of neurons in the peripheral cortex at the lesion site, enhances seizure susceptibility to PTZ-induced seizures, and promotes the occurrence of PTE. The part about 'promotes the occurrence of PTE' was not established.

7) NeuN is not the best marker of neurons in the context of TBI since TBI affects its expression patterns which will influence the interpretation of co-localization results. Unlike NeuN, Nissl staining is less likely to be affected by factors that alter protein expression, such as TBI. Therefore, it can be a more stable marker for identifying neurons in injured brain tissue.

8) Statistics: Authors report only SEM, which shows the precision of the mean and it will decrease as the sample size increases and does not reflect the data variability.

Reviewer #2 (Public Review):

In this study, Leighton et al performed remarkable experiments by combining in-vivo patch-clamp recording with two-photon dendritic Ca2+ imaging. The voltage-clamp mode is a major improvement over the pioneer versions of this combinatorial experiment that has led to major breakthroughs in the neuroscience field for visualizing and understanding synaptic input activities in single cells in-vivo (sharp electrodes: Svoboda et al, Nature 1997, Helmchen et al, Nature Neurosci 1999; whole-cell current-clamp: Jia et al, Nature 2010, Chen et al, Nature 2011. I suggest that these papers would be cited). This is because in voltage-clamp mode, despite the full control of membrane voltage in-vivo not being realistic, is nevertheless most effective in preventing back-propagation action potentials, which would severely confound the measurement of individual synaptically-induced Ca2+ influx events. Furthermore, clamping the cell body at a strongly depolarized potential (here the authors did -30mV) also facilitates the detection of synaptically-induced Ca2+ influx. As a result, the authors successfully recorded high-quality Ca2+ imaging data that can be used for precise analysis. To date, even in view of the rapid progress of voltage-sensitive indicators and relevant imaging technologies in recent years, this very old 'art' of combining single-cell electrophysiology and two-photon imaging (ordinary, raster-scanned, video-rate imaging) of Ca2+ signals still enables measurements of the best-level precision.

On the other hand, the interpretation of data in this study is a bit narrow-minded and lacks a comprehensive picture. Some suggestions to improve the manuscript are as follows:

1. The authors made a segregation of 'spine synapse' and 'shaft synapse' based solely on the two-photon images in-vivo. However, caution shall be taken here, because the optical resolution under in-vivo imaging conditions like this cannot reliably tell apart whether a bright spot within or partially overlapping a segment of the dendrite is a spine on top of (or below) it. Therefore, what the authors consider as a 'shaft synapse' (by detecting Ca2+ hotspots) has an unknown probability of being just a spine on top or below the dendrite. If there is other imaging data of higher axial resolution to validate or calibrate, the authors shall take some further considerations or analysis to check the consistency of their data, as the authors do need such a segregation between spine and shaft synapses to show how they evolve over the brain development stages.

2. The use of terminology 'bursts of spontaneous inputs' for describing voltage-clamp data seems improper. Conventionally, 'burst' refers to suprathreshold spike firing events, but here, the authors use 'burst' to refer to inward synaptic currents collected at the cell body. Not every excitatory synaptic input (or ensemble of inputs) activation will lead to spike firing under naturalistic conditions, therefore, these two concepts are not equivalent. It is recommended to use 'barrage of inputs' instead of 'burst of inputs'. Imagine a full picture of the entire dendritic tree, the fact that the authors could always capture spontaneous Ca2+ events here and there within a few pieces of dendrites within an arbitrary field-of-view suggests that, the whole dendritic tree must have many more such events going on as a barrage while the author's patch electrode picks up the summed current flow from the whole dendritic tree.

3. Following the above issue, an analysis of the temporal correlation between synaptic (not segregating 'spine' or 'shaft') Ca2+ events and EPSCs is absent. Again, the authors drew arbitrary time windows to clump the events for statistical analysis. However, the demonstrated example data already shows that the onset times of individual synaptic Ca2+ events do not necessarily align with the beginning of a 'barrage' inward current event.

4. The authors claim that "these observations indicate that the activity patterns investigated here are not or only slightly affected by low-level anesthesia". It would be nice to show some of the recordings in this work without any anesthesia to support this claim.

Reviewer #2 (Public Review):

Summary: The study by Jaime-Tobon & Moser is a truly major effort to bridge the gap between classical observations on how auditory neurons respond to sounds and the synaptic basis of these phenomena. The so-called spiral ganglion neurons (SGNs) are the primary auditory neurons connecting the brain with hair cells in the cochlea. They all respond to sounds increasing their firing rates, but also present multiple heterogeneities. For instance, some present a low threshold to sound intensity, whereas others have high threshold. This property inversely correlates with the spontaneous rate, i.e., the rate at which each neuron fires in the absence of any acoustic input. These characteristics, along with others, have been studied by many reports over the years. However, the mechanisms that allow the hair cells-SGN synapses to drive these behaviors are not fully understood.

Strengths:<br /> The level of experimental complexity described in this manuscript is unparalleled, producing data that is hardly found elsewhere. The authors provide strong proof for heterogeneity in transmitter release thresholds at individual synapses and they do so in extremely complex experimental settings. In addition, the authors found other specific differences such as in synaptic latency and max EPSCs. A reasonable effort is put into bridging these observations with those extensively reported in in vivo SGNs recordings. Similarities are many and differences are not particularly worrying as experimental conditions cannot be perfectly matched, despite the authors' efforts in minimizing them.

Weaknesses:<br /> Some concern surges in relation to mismatches with previous reports of IHC-SGN synapses function. EPSCs at these synapses present a peculiar distribution of amplitudes, shapes, and rates. These characteristics are well-established and some do not seem to be paralleled in this study. Here, amplitude distributions are drastically shifted to smaller values, and rates of events are very low, all compared with previous evidence. The reasons for these discrepancies are unclear. The rate at which spontaneous EPSCs appear is an especially sensitive matter. A great part of the conclusions relies on the definition of which of the SGNs (or should say synapses) belong to the low end and which to the high end in the spectrum of spontaneous rates. The data presented by the authors seem a bit off and the criteria used to classify recordings are not well justified. The authors should clarify the origin of these differences since they do not seem to come from obvious reasons such as animal ages, recording techniques, mouse strain, or even species.

Reviewer #2 (Public Review):

Summary:

In this paper, Sang et al. set out to identify gustatory receptors involved in salt taste sensation in Drosophila melanogaster. In a two-choice assay screen of 30 Ir mutants, they identified that Ir60b is required for avoidance of high salt. In addition, they demonstrate that activation of Ir60b neurons is sufficient for gustatory avoidance using either optogenetics or TRPV1 to specifically activate Ir60b neurons. Then, using tip recordings of labellar gustatory sensory neurons and proboscis extension response behavioral assays in Ir60b mutants, the authors demonstrate that Ir60b is dispensable for labellar taste neuron responses to high salt and the suppression of proboscis extension by high salt. Since external gustatory receptor neurons (GRNs) are not implicated, they look at Poxn mutants, which lack external chemosensory sensilla but have intact pharyngeal GRNs. High salt avoidance was reduced in Poxn mutants but was still greater than Ir60b mutants, suggesting that pharyngeal gustatory sensory neurons alone are sufficient for high salt avoidance. The authors use a new behavioral assay to demonstrate that Ir60b mutants ingest a higher volume of sucrose mixed with high salt than control flies do, suggesting that the action of Ir60b is to limit high salt ingestion. Finally, they identify that Ir60b functions within a single pair of gustatory sensory neurons in the pharynx, and that these neurons respond to high salt but not bitter tastants.

Strengths:

A great strength of this paper is that it rigorously corroborates previously published studies that have implicated specific Irs in salt taste sensation. It further introduces a new role for Ir60b in limiting high salt ingestion, demonstrating that Ir60b is necessary and sufficient for high salt avoidance and convincingly tracing the action of Ir60b to a particular subset of gustatory receptor neurons. Overall the authors have achieved their aim by identifying a new gustatory receptor involved in limiting high salt ingestion. They use rigorous genetic, imaging, and behavioral studies to achieve this aim, often confirming a given conclusion with multiple experimental approaches. They have further done a great service to the field by replicating published studies and corroborating the roles of a number of other Irs in salt taste sensation. An aspect of this study that merits further investigation is how the same gustatory receptor neurons and Ir in the pharynx can be responsible for regulating the ingestion of both appetitive (sugar) and aversive tastants (high salt).

Weaknesses:

There are several weaknesses that, if addressed, could greatly improve this work.<br /> 1) The authors combine the results and discussion but provide a very limited interpretation of their results. More discussion of the results would help to highlight what this paper contributes, how the authors interpret their results, and areas for future study.<br /> 2) The authors rename previously studied populations of labellar GRNs to arbitrary letters, which makes it difficult to understand the experiments and results in some places. These GRN populations would be better referred to according to the gustatory receptors they are known to express.<br /> 3) The conclusion that GRNs responsible for high salt aversion may be inhibited by those that function in low salt attraction is not well substantiated. This conclusion seems to come from the fact that overexpression of Ir60b in salt attraction and salt aversion sensory neurons still leads to salt aversion, but there need not be any interaction between these two types of sensory neurons if they act oppositely on downstream circuits.<br /> 4) The authors rely heavily on a new Droso-X behavioral apparatus that is not sufficiently described here or in the previous paper the authors cite. This greatly limits the reader's ability to interpret the results.

Reviewer #2 (Public Review):

Summary:<br /> McSwiggen et al present a high throughput platform for SPT that allows them to identify pharmaceutics interactions with the diffusional behavior of receptors and in turn to identify potent new ligands and cellular mechanisms. The manuscript is well written, it provides a solid new mentor and a proper experimental foundation

Strengths:<br /> The method capitalizes and extends to existing high throughput toolboxes and is directly applied to multiple receptors and ligands. The outcomes are important and relevant for society. 10^6 cells and >400 ligands per is a significant achievement.

The method can detect functionally relevant changes in transcription factor dynamics and accurately differentiate the ligand/target specificity directly within the cellular environment. This will be instrumental in screening libraries of compounds to identify starting points for the development of new therapeutics. Identifying hitherto unknown networks of biochemical signaling pathways will propel the field of single-particle live cell and quantitative microscopy in the area of diagnostics. The manuscript is well-written and clearly conveys its message.

Weaknesses:<br /> There are a few elements, that if rectified would improve the claims of the manuscript.

The authors claim that they measure receptor dynamics. In essence, their readout is a variation in diffusional behavior that correlates to ligand binding. While ligand binding can result in altered dynamics or /and shift in conformational equilibrium, SPT is not recording directly protein structural dynamics, but their effect on diffusion. They should correct and elaborate on this.

L 148 What do the authors mean 'No correlation between diffusion and monomeric protein size was observed, highlighting the differences between cellular protein dynamics versus purified systems'. This is not justified by data here or literature reference. How do the authors know these are individual molecules? Intensity distributions or single bleaching steps should be presented.

Along the same lines, the data in Figs 2 and 4 show that not only the immobile fraction is increased but also that the diffusion coefficient of the fast-moving (attributed to free) is reduced. The authors mention this and show an extended Fig 5 but do not provide an explanation. How do potential transient ligand binding and the time-dependent heterogeneity in motion (see comment above) contribute to this? Also, in line 216 the authors write "with no evidence" of transient diffusive states. How do they define transient diffusive states? While there are toolboxes to directly extract the existence and abundance of these either by HMM analysis or temporal segmentation, the authors do not discuss or use them.

The authors discuss the methods for extracting kinetic information of ligand binding by diffusion. They should consider the temporal segmentation of heterogenous diffusion. There are numerous methods published in journals or BioRxiv based on analytical or deep learning tools to perform temporal segmentation. This could elevate their analysis of Kon and Koff.

Reviewer #2 (Public Review):

Summary:

In the present manuscript, Zhang et al utilize single-nuclei RNA-Seq to investigate the heterogeneity of perirenal adipose tissue. The perirenal depot is interesting because it contains both brown and white adipocytes, a subset of which undergo functional "whitening" during early development. While adipocyte thermogenic transdifferentiation has been previously reported, there remain many unanswered questions regarding this phenomenon and the mechanisms by which it is regulated.

Strengths:

The combination of UCP1-lineage tracing with the single nuclei analysis allowed the authors to identify four populations of adipocytes with differing thermogenic potential, including a "whitened" adipocyte (mPRAT-ad2) that retains the capacity to rapidly revert to a brown phenotype upon cold exposure. They also identify two populations of white adipocytes that do not undergo browning with acute cold exposure.

Anatomically distinct adipose depots display interesting functional differences, and this work contributes to our understanding of one of the few brown depots present in humans.

Weaknesses:

The most interesting aspect of this work is the identification of a highly plastic mature adipocyte population with the capacity to switch between a white and brown phenotype. The authors attempt to identify the transcriptional signature of this ad2 subpopulation, however, the limited sequencing depth of single nuclei somewhat lessens the impact of these findings. Furthermore, the lack of any form of mechanistic investigation into the regulation of mPRAT whitening limits the utility of this manuscript. However, the combination of well-executed lineage tracing with comprehensive cross-depot single-nuclei presented in this manuscript could still serve as a useful reference for the field.

Reviewer #2 (Public Review):

This study by Matsuo-Takasaki et al. reported the development of a novel suspension culture system for hiPSC maintenance using Wnt/PKC inhibitors. The authors showed elegantly that inhibition of the Wnt and PKC signaling pathways would repress spontaneous differentiation into neuroectoderm and mesendoderm in hiPSCs, thereby maintaining cell pluripotency in suspension culture. This is a solid study with substantial data to demonstrate the quality of the hiPSC maintained in the suspension culture system, including long-term maintenance in >10 passages, robust effect in multiple hiPSC lines, and a panel of conventional hiPSC QC assays. Notably, large-scale expansion of a clinical grade hiPSC using a bioreactor was also demonstrated, which highlighted the translational value of the findings here. In addition, the author demonstrated a wide range of applications for the IWR1+LY suspension culture system, including support for freezing/thawing and PBMC-iPSC generation in suspension culture format. The novel suspension culture system reported here is exciting, with significant implications in simplifying the current culture method of iPSC and upscaling iPSC manufacturing.

Another potential advantage that perhaps wasn't well discussed in the manuscript is the reported suspension culture system does not require additional ECM to provide biophysical support for iPSC, which differentiates from previous studies using hydrogel and this should further simplify the hiPSC culture protocol.

Interestingly, although several hiPSC suspension media are currently available commercially, the content of these suspension media remained proprietary, as such the signaling that represses differentiation/maintains pluripotency in hiPSC suspension culture remained unclear. This study provided clear evidence that inhibition of the Wnt/PKC pathways is critical to repress spontaneous differentiation in hiPSC suspension culture.

I have several concerns that the authors should address, in particular, it is important to benchmark the reported suspension system with the current conventional culture system (eg adherent feeder-free culture), which will be important to evaluate the usefulness of the reported suspension system. Also, the manuscript lacks a clear description of a consistent robust effect in hiPSC maintenance across multiple cell lines. There are also several minor comments that should be addressed to improve readability, including some modifications to the wording to better reflect the results and conclusions.

Reviewer #2 (Public Review):

Summary:

The authors have developed novel Ovalbumin model (OTII) peptide that can be labeled with a site-specific FlAsH dye to track agonist peptides both in vitro and in vivo. The utility of this tool could allow better tracking of activated polyclonal T cells particularly in novel systems. The authors have provided solid evidence that peptides are functional, capable of activating OTII T cells, and these peptides can undergo trogocytosis by cognate T cells only.

Strengths:<br /> -An extensive array of in vitro and in vivo studies are used to assess peptide functionality.<br /> -Nice use of cutting edge intravital imaging,<br /> -internal controls such as multiple non-cogate T cells were used to improve robustness of the results<br /> -One of the strengths is the direct labeling of the peptide, and the potential utility in other systems.

Weaknesses:<br /> -Peptide labeling specificity and efficiency is not clear. High levels of background labeling. While it was sufficient for demonstrating the system works, it may pose problems depending on the peptide sequence, and/or use at lower dose.<br /> -Only one peptide system was tested, namely OVA323-339 region.<br /> -Limited novel biological findings. This study mostly describes a new tool that may have exciting potential.

Reviewer #2 (Public Review):

Summary:

The goal of this study was to examine the role of FNDC5 in the response of the murine skeleton to either lactation or a calcium-deficient diet. The authors find that female FNDC5 KO mice are somewhat protected from bone loss and osteocyte lacunar enlargement caused by either lactation or a calcium-deficient diet. In contrast, male FNDC5 KO mice lose more bone and have a greater enlargement of osteocyte lacunae than their wild-type controls. Based on these results, the authors conclude that in males irisin protects bone from calcium deficiency but that in females it promotes calcium removal from bone for lactation.

While some of the conclusions of this study are supported by the results, it is not clear that the modest effects of FNDC5 deletion have an impact on calcium homeostasis or milk production.

Specific comments:

1. The authors sometimes refer to FNDC5 and other times to irisin when describing causes for a particular outcome. Because irisin was not measured in any of the experiments, the authors should not conclude that lack of irisin is responsible. Along these lines, is there any evidence that either lactation or a calcium-deficient diet increases the production of irisin in mice?

2. The results of the irisin-rescue experiment shown in figure 2G cannot be appropriately interpreted without normal diet controls. In addition, some evidence that the AAV8-irisin virus actually increased irisin levels in the mice would strengthen the conclusion.

3. There is insufficient evidence to support the idea that the effect of FNDC5 on bone resorption and osteocytic osteolysis is important for the transfer of calcium from bone to milk. Previous studies by others have shown that bone resorption is not required to maintain milk or serum calcium when dietary calcium is sufficient but is critical if dietary calcium is low (Endo. 156:2762-73, 2015). To support the conclusions of the current study, it would be necessary to determine whether FNDC5 is required to maintain calcium levels when lactating mice lack sufficient dietary calcium.

4. The amount of cortical bone loss due to lactation is very similar in both WT and FNDC5 KO mice. The results of the statistical analysis of the data presented in figure 1B are surprising given the very similar effect size of lactation. The key result from the 2-way ANOVA is whether there is an effect of genotype on the effect size of lactation (genotype-lactation interaction). The interaction terms were not provided. Similar concerns are noted for the results shown in figure 1G and H.

5. It is not clear what justifies the term 'primed' or 'activated' for resorption. Is there evidence that a certain level of TRAP expression lowers the threshold for osteocytic osteolysis in response to a stimulus?

Review #2 (Public Review)

In the manuscript Watanuki et al. define the metabolic profile of HSCs in stress/proliferative (myelosuppression with 5-FU), and mitochondrial inhibition and homeostatic conditions. Their conclusions are that during proliferation HSCs rely more on glycolysis (as other cell types) while HSCs in homeostatic conditions are mostly dependent on mitochondrial metabolism. Mitochondrial inhibition is used to demonstrate that blocking mitochondrial metabolism results in similar features of proliferative conditions.

The authors used state-of-the-art technologies that allow metabolic readout in a limited number of cells like rare HSCs. These applications could be of help in the field since one of the major issues in studying HSCs metabolism is the limited sensitivity of the "standard" assays, which make them not suitable for HSC studies.

Reviewer #2 (Public Review):

Summary:<br /> The work by Varadharajan et. al. explored a previously known genetic variant and its pathophysiology in the development of alcohol-associated liver injury. It provides a plausible mechanism for how varying levels of MBOAT7 could impact the lipid metabolomics of the cell, leading to a deleterious phenotype in MBOAT7 knockout. The authors further characterized the impact of the lipidomic changes and raised lysosomal biogenesis and autophagic flux as mechanisms of how MBOAT7 deletion causes the progression of ALD.

Strengths:<br /> Connecting the GWAS data on MBOAT7 variants with plausible pathophysiology greatly enhances the translational relevance of these findings. The global lipidomic profiling of ALD mice is also very informative and may lead to other discoveries related to lipid handling pathways.

Weaknesses:<br /> The rationale of why MBOAT7 metabolites are lower in heavy drinkers than in normal individuals is not well explained. MBOAT7 loss of function drives ALD, but unclear if MBOAT7 deletion also drives preference for alcohol or if alcohol inhibits MBOAT7 function. Presuming most individuals studied here were WT and expressed an appropriate level of MBOAT7?<br /> Also, the discussion of mechanisms of MBOAT7-induced dysregulation of lysosomal biogenesis/autophagy, while very interesting, seems incomplete. It is not clear how MBOAT7 an enzyme involved in membrane phospholipid remodeling increases mTOR which leads to decreased TFEB target gene transcription. Furthermore, given the significant disturbances of global lipidomic profiling in MBOAT7 knockout, many pathways are potentially affected by this deletion. Further in vivo modeling that specifically addresses these pathways (TFEB targeting, mTOR inhibitor) would help strengthen the conclusions of this paper.

Reviewer #2 (Public Review):

Summary:

While a significant portion of immunotherapy research has focused on the pivotal role of T cells in tumor immunity, their effectiveness may be limited by the suppressive nature of the tumor environment. On the other hand, myeloid cells are commonly found within tumors and can withstand these adverse conditions. However, these cells often adopt an immunosuppressive phenotype when infiltrating tumors. Therefore, manipulating myeloid cells could potentially enhance the anti-tumor potential of immunotherapy.

In this manuscript, Farhat-Younes and colleagues have demonstrated that activating the IgM receptor signaling in myeloid cells induces an oxygen burst, the secretion of Granzyme B, and the lysis of adjacent tumor cells. Furthermore, they have outlined a strategy to utilize these features to generate CAR macrophages. However, they have identified a limitation: the expression of scFv in myeloid cells induces ER stress and the degradation of misfolded proteins. To address this issue, chimeric receptors were designed based on the high-affinity FcγRI for IgG. When macrophages transfected with these receptors were exposed to tumor-binding IgG, extensive tumor cell killing, and the release of reactive oxygen species and Granzyme B were observed.

Strengths:<br /> In general, I consider this work to be significant, and the results are compelling. It emphasizes the specific considerations and requirements for successful manipulation in myeloid cells, which could further advance the field of cellular engineering for the benefit of immunotherapy

Weaknesses:

Nevertheless, there are several minor issues that should be addressed:

1- TCR fragments are commonly used to induce ER stress in non-immune cells. Therefore, it would be interesting to investigate whether TCR fragments can be expressed in myeloid cells and if they induce ER stress. Addressing this issue would support the notion that these cells lack the ER chaperones required for folding immunoglobulin variable chains.<br /> 2- It would be valuable to determine whether, after the degradation of scFv fragments by myeloid cells, they are presented on MHC-I and MHC-II.<br /> 3- Some methodological details, such as the vaccination protocol and high-resolution microscopy procedures, are missing from the text.

Reviewer #2 (Public Review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to readers.

The data, for the most part, support the conclusions but I do have concerns about some of the data and the interpretations that should be addressed.

Reviewer #2 (Public Review):

Summary:

This combined experimental-theoretical paper introduces a novel two-domain statistical thermodynamic model (primarily Equation 1) to study allostery in generic systems but focusing here on the tetracycline repressor (TetR) family of transcription factors. This model, building on a function-centric approach, accurately captures induction data, maps mutants with precision, and reveals insights into epistasis between mutations.

Strengths:

The study contributes innovative modeling, successful data fitting, and valuable insights into the interconnectivity of allosteric networks, establishing a flexible and detailed framework for investigating TetR allostery. The manuscript is generally well-structured and communicates key findings effectively.

Weaknesses:

The only minor weakness I found was that I still don't have a better sense into (a) intuition and (b) mathematical derivation of Equation 1, which is so central to the work. I would recommend that the authors provide this early on in the main text.

Reviewer #3 (Public Review):

Summary<br /> In this research advance, the authors purport to show that the unified neutral theory of biodiversity (UNTB) is not a suitable null model for exploring the relationship between macroecological quantities, and additionally that the stochastic logistic growth model (SLM) is a viable replacement. They do this by citing other studies where UNTB was unable to capture individual macroecological quantities, and then demonstrating SLM's strength at predicting the same diversity metrics. They extend this analysis to show SLM's modeling capability at multiple scales of coarse graining, in addition to its failures at predicting these metrics' variances. Finally, authors conduct a similar analysis to Madi et al. (2020) by investigating the relationship between diversity measures within a group and across coarse-grained groups (e.g. genera diversity in one family compared to diversity of families). The authors show that choosing SLM as a null model reveals some previously reported relationships to be no longer "novel", in the sense that the patterns can be adequately captured by the null model. Authors also show that relationships not captured by the null model can be recovered by adding correlations, suggesting interactions are the driving force behind them.

Strengths<br /> 1. Authors make a strong argument that UNTB is not a good null model of macroecological observables and especially relationships between them. Authors convincingly argue that a SLM is a better null since the gamma distribution it predicts is a better description of the empirical Abundance Fluctuation Distributions (AFD).<br /> 2. Authors show that the gamma distribution predicted by SLM is a good fit for the AFD's at many different scales of coarse graining, not just the OTU level as was previously demonstrated. Authors show the same distribution predicted the mean diversity and richness at all scales of coarse graining.<br /> 3. Authors convincingly demonstrate how SLM can be used to test the relevance of interactions to macroecological relationships.

Weaknesses<br /> This reviewer's concerns were convincingly addressed by the revisions.

Overall Impact<br /> The authors present a convincing argument for the use of SLM as a better non-interacting null model for macroecological quantities and relationships.

Reviewer #2 (Public Review):

Summary:

In this study, the authors investigate the effect of ACh on neuronal responses in the auditory cortex of anesthetized rats during an auditory oddball task. The paradigm consisted of two pure tones (selected from the frequency responses at each recording site) presented in a pseudo-random sequence. One tone was presented frequently (the "standard" tone) and the other infrequently (the "deviant" tone). The authors found that ACh enhances the detection of unexpected stimuli in the auditory environment by increasing or decreasing the neuronal responses to deviant and standard tones.

Strengths:

The study includes the use of appropriate and validated methodology in line with the current state-of-the-art, rigorous statistical analysis and the demonstration of the effects of acetylcholine on auditory processing.

Weaknesses:

The study was conducted in anesthetized rats, and further research is needed to determine the behavioral relevance of these findings.

Reviewer #2 (Public Review):

Summary:

This interesting study addresses the ability of Ym1 protein crystals to promote pulmonary type 2 inflammation in vivo, in mice.

Strengths:

The data are extremely high quality, clearly presented, significantly extending previous work from this group on the type 2 immunogenicity of protein crystals.

Weaknesses:

There are no major weaknesses in this study. It would be interesting to see if Ym2 crystals behave similarly to Ym1 crystals in vivo. Some additional text in the Introduction and Discussion would enrich those sections.

Reviewer #2 (Public Review):

Summary:<br /> The work fruitfully adds to the tools to study cannabinoid action and pharmacology specifically, but also this method is applicable to other drugs, in particular, if lipophilic in nature.

Strengths:<br /> The addition of chocolate flavor overcomes aversive reactions which are often experienced in pharmacological treatments, leading to possible caveats in the interpretation of the behavioral outcomes.

Weaknesses:<br /> Certainly, more THC mediated behavioral outcomes could have been tested, but the work presents a proof-of-concept study to investigate acute THC treatment.<br /> It would have been interesting if this application form is also possible for chronic treatment regimen.

Reviewer #3 (Public Review):

Summary: This paper presents evidence of recursive self-embedding in the vocalization structure of orangutans, using fine-grained acoustical analysis. It proves the existence of isochrony nested in isochrony in the motifs produced by a nonhuman vocal system.

Strengths: Very clear written, clear analysis, excellent responses to the Reviewers.

Weaknesses: Jargonous language may be reduced. A video showing the sound as it unfolds and the spectrogram (as in Fig 1A) of the long call could be useful to best exemplify the results.

Reviewer #2 (Public Review):

Summary:<br /> Morucci et al. tested the influence of linguistic prosody long-term priors in forming predictions about simple acoustic rhythmic tone sequences composed of alternating tone duration, by violating context-dependent short-term priors formed during sequence listening. Spanish and Basque participants were selected due to the different rhythmic prosody of the two languages (functor-initial vs. Functor final, respectively), despite a common cultural background. The authors found that neuromagnetic responses to casual tone omissions reflected the linguistic prosody pattern of the participant's dominant language: in Spanish speakers, omission responses were larger to short tones, whereas in Basque speakers, omission responses were larger to long tones. Source localization of these responses revealed this interaction pattern in the left auditory cortex, which the authors interpret as reflecting a perceptual bias due to acoustic cues (inherent linguistic rhythms, rather than linguistic content). Importantly, this pattern was not found when the rhythmic sequence entailed pitch, rather than duration, cues. To my knowledge, this is the first study providing neural signatures of a known behavioral effect linking ambiguous rhythmic tone sequence perceptual organization to linguistic experience.

The conclusions of the study are well supported by the data, albeit weakly by the source analysis, but I have the impression that the rationale of the study and the analyses performed may be missing an important aspect of rhythmic sequence perception, namely the involvement of entrained oscillatory activity to the perceived rhythm, particularly phase alignment to pattern onsets. This view would not change the impact of the results but add depth to their interpretation.

Strengths:<br /> 1) The choice of participants. The bilingual population of the Basque country is perfect for performing studies that need to control for cultural and socio-economic background while having profound linguistic differences. In this sense, having dominant Basque speakers as a sample equates that in Molnar et al. (2016), and thus overcomes the lack of direct behavioral evidence for a difference in rhythmic grouping across linguistic groups. Molnar et al. (2016)'s evidence on the behavioral effect is compelling, and the evidence on neural signatures provided by the present study aligns with it.

2) The experimental paradigm. It is a well-designed acoustic sequence, that considers aspects such as gap length insertion, to be able to analyze omission responses free from subsequent stimulus-driven responses, and which includes a control sequence that uses pitch instead of duration as a cue to rhythmic grouping, which provides a stronger case for the differences found between groups to be due to prosodic duration cues.

3) Data analyses. Sound, state-of-the-art methodology in the event-related field analyses at the sensor level.

Weaknesses:<br /> 1) Despite the evidence provided on neural responses, the main conclusion of the study reflects a known behavioral effect on rhythmic sequence perceptual organization driven by linguistic background (Molnar et al. 2016, particularly). Also, the authors themselves provide a good review of the literature that evidences the influence of long-term priors in neural responses related to predictive activity. Thus, in my opinion, the strength of the statements the authors make on the novelty of the findings may be a bit far-fetched in some instances.

2) Albeit the paradigm is well designed, I fail to see the grounding of the hypotheses laid by the authors as framed under the predictive coding perspective. The study assumes that responses to an omission at the beginning of a perceptual rhythmic pattern will be stronger than at the end. I feel this is unjustified. If anything, omission responses should be larger when the gap occurs at the end of the pattern, as that would be where stronger expectations are placed: if in my language a short sound occurs after a long one, and I perceptually group tone sequences of alternating tone duration accordingly, when I hear a short sound I will expect a long one following; but after a long one, I don't necessarily need to expect a short one, as something else might occur.

3) In this regard, it is my opinion that what is reflected in the data may be better accounted for (or at least, additionally) by a different neural response to an omission depending on the phase of an underlying attentional rhythm (in terms of Large and Jones rhythmic attention theory, for instance) and putative underlying entrained oscillatory neural activity (in terms of Lakatos' studies, for instance). Certainly, the fact that the aligned phase may differ depending on linguistic background is very interesting and would reflect the known behavioral effect.

4) Source localization is performed on sensor-level significant data. The lack of source-level statistics weakens the conclusions that can be extracted. Furthermore, only the source reflecting the interaction pattern is taken into account in detail as supporting their hypotheses, overlooking other sources. Also, the right IFG source activity is not depicted, but looking at whole brain maps seems even stronger than the left. To sum up, source localization data, as informative as it could be, does not strongly support the author's claims in its current state.

Reviewer #2 (Public Review):

Summary:<br /> In this study, Hauser et al investigate the role of amphibian (Xenopus laevis) mast cells in cutaneous immune responses to the ecologically important pathogen Batrachochytrium dendrobatidis (Bd) using novel methods of in vitro differentiation of bone marrow-derived mast cells and in vivo expansion of skin mast cell populations. They find that bone marrow-derived myeloid precursors cultured in the presence of recombinant X. laevis Stem Cell Factor (rSCF) differentiate into cells that display hallmark characteristics of mast cells. They inject their novel (r)SCF reagent into the skin of X. laevis and find that this stimulates the expansion of cutaneous mast cell populations in vivo. They then apply this model of cutaneous mast cell expansion in the setting of Bd infection and find that mast cell expansion attenuates the skin burden of Bd zoospores and pathologic features including epithelial thickness and improves protective mucus production and transcriptional markers of barrier function. Utilizing their prior expertise with expanding neutrophil populations in X. laevis, the authors compare mast cell expansion using (r)SCF to neutrophil expansion using recombinant colony-stimulating factor 3 (rCSF3) and find that neutrophil expansion in Bd infection leads to greater burden of zoospores and worse skin pathology.

Strengths:<br /> The authors report a novel method of expanding amphibian mast cells utilizing their custom-made rSCF reagent. They rigorously characterize expanded mast cells in vitro and in vivo using histologic, morphologic, transcriptional, and functional assays. This establishes solid footing with which to then study the role of rSCF-stimulated mast cell expansion in the Bd infection model. This appears to be the first demonstration of the exogenous use of rSCF in amphibians to expand mast cell populations and may set a foundation for future mechanistic studies of mast cells in the X. laevis model organism.

Weaknesses:<br /> The conclusions regarding the role of mast cell expansion in controlling Bd infection would be stronger with a more rigorous evaluation of the model, as there are some key gaps and remaining questions regarding the data. For example:

1. Granulocyte expansion is carefully quantified in the initial time courses of rSCF and rCSF3 injections, but similar quantification is not provided in the disease models (Figures 3E, 4G, 5D-G). A key implication of the opposing effects of mast cell vs neutrophil expansion is that mast cells may suppress neutrophil recruitment or function. Alternatively, mast cells also express notable levels of csfr3 (Figure 2) and previous work from this group (Hauser et al, Facets 2020) showed rG-CSF-stimulated peritoneal granulocytes express mast cell markers including kit and tpsab1, raising the question of what effect rCSF3 might have on mast cell populations in the skin. Considering these points, it would be helpful if both mast cells and neutrophils were quantified histologically (based on Figure 1, they can be readily distinguished by SE or Giemsa stain) in the Bd infection models.

2. Epithelial thickness and inflammation in Bd infection are reported to be reduced by rSCF treatment (Figure 3E, 5A-B) or increased by rCSF3 treatment (Figure 4G) but quantification of these critical readouts is not shown.

3. Critical time points in the Bd model are incompletely characterized. Mast cell expansion decreases zoospore burden at 21 dpi, while there is no difference at 7 dpi (Figure 3E). Conversely, neutrophil expansion increases zoospore burden at 7 dpi, but no corresponding 21 dpi data is shown for comparison (Figure 4G). Microbiota analysis is performed at a third time point,10 dpi (Figure 5D-G), making it difficult to compare with the data from the 7 dpi and 21 dpi time points. Reporting consistent readouts at these three time points is important to draw solid conclusions about the relationship of mast cell expansion to Bd infection and shifts in microbiota.

4. Although the effect of rSCF treatment on Bd zoospores is significant at 21 dpi (Figure 3E), bacterial microbiota changes at 21 dpi are not (Figure S3B-C). This discrepancy, how it relates to the bacterial microbiota changes at 10 dpi, and why 7, 10, and 21 dpi time points were chosen for these different readouts (Figure 5F-G), is not discussed.

5. The time course of rSCF or rCSF3 treatments relative to Bd infection in the experiments is not clear. Were the treatments given 12 hours prior to the final analysis point to maximize the effect? For example, in Figure 3E, were rSCF injections given at 6.5 dpi and 20.5 dpi? Or were treatments administered on day 0 of the infection model? If the latter, how do the authors explain the effects at 7 dpi or 21 dpi given mast cell and neutrophil numbers return to baseline within 24 hours after rSCF or rCSF3 treatment, respectively?

The title of the manuscript may be mildly overstated. Although Bd infection can indeed be deadly, mortality was not a readout in this study, and it is not clear from the data reported that expanding skin mast cells would ultimately prevent progression to death in Bd infections.

Reviewer #2 (Public Review):

Summary:<br /> This study focuses on the 5-HT3 serotonin receptor, a pentameric ligand-gated ion channel important in chemical neurotransmission. There are many cryo-EM structures of this receptor with diverse ligands bound, however assignment of functional states to the structures remains incomplete. The team applies voltage-clamp fluorometry to measure, at once, both changes in ion channel activity, and changes in fluorescence. Four cysteine mutants were selected for fluorophore labeling, two near the neurotransmitter site, one in the ECD vestibule, and one at the ECD-TMD junction. Agonists, partial agonists, and antagonists were all found to yield similar changes in fluorescence, a proxy for conformational change, near the neurotransmitter site. The strength of the agonist correlated to a degree with propagation of this fluorescence change beyond the local site of neurotransmitter binding. Antagonists failed to elicit a change in fluorescence in the vestibular the ECD-TMD junction sites. The VCF results further turned up evidence supporting intermediate (likely pre-active) states.

Strengths:<br /> The experiments appear rigorous, the problem the team tackles is timely and important, the writing and the figures are for the most part very clear. We sorely need approaches orthogonal to structural biology to annotate conformational states and observe conformational transitions in real membranes- this approach, and this study, get right to the heart of what is missing.

Weaknesses:<br /> The weaknesses in the study itself are overall minor, I only suggest improvements geared toward clarity. What we are still missing is application of an approach like this to annotate the conformation of the part of the receptor buried in the membrane; there is important debate about which structure represents which state, and that is not addressed in the current study.

Reviewer #2 (Public Review):

Summary:<br /> The authors have developed a comprehensive set of tools to describe dynamics within a single time-series or across multiple time-series. The motivation is to better understand interacting networks within the human brain. The time-series used here are from direct estimates of the brain's electrical activity; however the tools have been used with other metrics of brain function and would be applicable to many other fields.

Strengths:<br /> The methods described are principled, based on generative probabilistic models.<br /> This makes them compact descriptors of the complex time-frequency data.<br /> Few initial assumptions are necessary in order to reveal this compact description.<br /> The methods are well described and demonstrated within multiple peer reviewed articles.<br /> This toolbox will be a great asset to the brain imaging community.

Weaknesses:<br /> The only question I had (originally) was how to objectively/quantitatively compare different network models. This has now been addressed by the authors in the latest revision.

Reviewer #2 (Public Review):

Summary:<br /> The authors build a gene expression model based on histone post-translational modifications and find that H3K27ac is correlated with gene expression. They proceed to perturb H3K27ac at 8 gene promoters, and measure gene expression changes to test their model.

Strengths:<br /> The combination of multiple methods to model expression, along with utilizing 6 histone datasets in 13 cell types allowed the authors to build a model that correlates between 0.7-0.79 with gene expression. This group also utilized a tool they are experts in, dCas9-p300 fusions to perturb H3K27ac and monitor gene expression to test their model. Ranked correlations showed some support for the predictions after the perturbation of H3K27ac.

Weaknesses:<br /> The perturbation of only 8 genes, and the only readout being qPCR-based gene expression, as opposed to including H3K27ac, weakened their validation of the computational model. Likewise, the use of six genes that were not expressed being most activated by dCas9-p300 might weaken the correlations vs. looking at a broad range of different gene expressions as the original model was trained on.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript characterizes a chemoattractant response to human serum by pathogenic bacteria, focusing on pathogenic strains of Salmonella enterica (Se). The researchers conducted the chemotaxis assays using a micropipette injection method that allows real-time tracking of bacterial population densities. They found that clinical isolates of several Se strains present a chemoattractant response to human serum. The specific chemoattractant within the serum is identified as L-serine, a highly characterized and ubiquitous chemoattractant, that is sensed by the Tsr receptor. They further show that chemoattraction to serum is impaired with a mutant strain devoid of Tsr. X-ray crystallography is then used to determine the structure of L-serine in the Se Tsr ligand binding domain, which differs slightly from a previously determined structure of a homologous domain. They went on to identify other pathogens that have a Tsr domain through a bioinformatics approach and show that these identified species also present a chemoattractant response to serum.

Strengths and Weaknesses:<br /> This study is well executed and the experiments are clearly presented. These novel chemotaxis assays provide advantages in terms of temporal resolution and the ability to detect responses from small concentrations. That said, it is perhaps not surprising these bacteria respond to serum as it is known to contain high levels of known chemoattractants, serine certainly, but also aspartate. In fact, the bacteria are shown to respond to aspartate and the tsr mutant is still chemotactic. The authors do not adequately support their decision to focus exclusively on the Tsr receptor. Tsr is one of the chemoreceptors responsible for observed attraction to serum, but perhaps, not the receptor. Furthermore, the verification of chemotaxis to serum is a useful finding, but the work does not establish the physiological relevance of the behavior or associate it with any type of disease progression. I would expect that a majority of chemotactic bacteria would be attracted to it under some conditions. Hence the impact of this finding on the chemotaxis or medical fields is uncertain.

The authors also state that "Our inability to substantiate a structure-function relationship for NE/DHMA signaling indicates these neurotransmitters are not ligands of Tsr." Both norepinephrine (NE) and DHMA have been shown previously by other groups to be strong chemoattractants for E. coli (Ec), and this behavior was mediated by Tsr (e.g. single residue changes in the Tsr binding pocket block the response). Given the 82% sequence identity between the Se and Ec Tsr, this finding is unexpected (and potentially quite interesting). To validate this contradictory result the authors should test E. coli chemotaxis to DHMA in their assay. It may be possible that Ec responds to NE and DHMA and Se doesn't. However, currently, the data is not strong enough to rule out Tsr as a receptor to these ligands in all cases. At the very least the supporting data for Tsr being a receptor for NE/DHMA needs to be discussed.

The authors also determine a crystal structure of the Se Tsr periplasmic ligand binding domain bound to L-Ser and note that the orientation of the ligand is different than that modeled in a previously determined structure of lower resolution. I agree that the SeTsr ligand binding mode in the new structure is well-defined and unambiguous, but I think it is too strong to imply that the pose of the ligand in the previous structure is wrong. The two conformations are in fact quite similar to one another and the resolution of the older structure, is, in my view, insufficient to distinguish them. It is possible that there are real differences between the two structures. The domains do have different sequences and, moreover, the crystal forms and cryo-cooling conditions are different in each case. It's become increasingly apparent that temperature, as manifested in differential cooling conditions here, can affect ligand binding modes. It's also notable that full-length MCPs show negative cooperativity in binding ligands, which is typically lost in the isolated periplasmic domains. Hence ligand binding is sensitive to the environment of a given domain. In short, the current data is not convincing enough to say that a previous "misconception" is being corrected.

Reviewer #2 (Public Review):

Greve et al. investigated the effects of a disease-associated gamma-actin mutation (E334Q) on actin filament polymerization, association of selected actin-binding proteins, and myosin activity. Recombinant wildtype and mutant proteins expressed in sf9 cells were found to be folded and stable, and the presence of the mutation altered a number of activities. Given the location of the mutation, it is not surprising that there are changes in polymerization and interactions with actin binding proteins. Nevertheless, it is important to quantify the effects of the mutation to better understand disease etiology. Some weaknesses were identified in the paper as discussed below.

Throughout the paper, the authors report average values and the standard-error-of-the-mean (SEM) for groups of three experiments. Reporting the SEM is not appropriate or useful for so few points, as it does not reflect the distribution of the data points. When only three points are available, it would be better to just show the three different points. Otherwise, plot the average and the range of the three points.

The description and characterization of the recombinant actin is incomplete. Please show gels of purified proteins. This is especially important with this preparation since the chymotrypsin step could result in internally cleaved proteins and altered properties, as shown by Ceron et al (2022). The authors should also comment on N-terminal acetylation of actin.

The authors do not use the best technique to assess actin polymerization parameters. Although the TIRF assay is excellent for some measurements, it is not as good as the standard pyrene-actin assays that provide critical concentration, nucleation, and polymerization parameters. The authors use pyrene-actin in other parts of the paper, so it is not clear why they don't do the assays that are the standard in the actin field.

The authors' data suggest that, while the binding of cofilin-1 to both the WT and mutant actins remains similar, the major defect of the E334Q actin is that it is not as readily severed/disassembled by cofilin. What is missing is a direct measurement of the severing rate (number of breaks per second) as measured in TIRF.

Figure 4 shows that the E334Q mutation increases rather than decreases the number of filaments that spontaneously assemble in the TIRF assay, but it is unclear how reduced severing would lead to increased filament numbers, rather, the opposite would be expected. A more straightforward approach would be to perform experiments where severing leads to more nuclei and therefore enhances the net bulk assembly rate.

Figure 5 A: in the pyrene disassembly assay, where actin is diluted below its critical concentration, cofilin enhances the rate of depolymerization by generating more free ends. The E334Q mutation leads to decreased cofilin-induced severing and therefore lower depolymerization. While these data seem convincing, it would be better to present them as an XY plot and fit the data to lines for comparison of the slopes.

Figure 5 B and C: the cosedimentation data do not seem to help elucidate the underlying mechanism. While the authors report statistical significance, differences are small, especially for gel densitometry measurements where the error is high, which suggests that there may be little biological significance. Importantly, example gels from these experiments should be shown, if not the complete set included in the supplement. In B, the higher cofilin concentrations would be expected to stabilize the filaments and thus the curve should be U-shaped.

Figure 5 D: these data show that the binding of cofilin to WT and E334Q actin is approximately the same, with the mutant binding slightly more weakly. It would be clearer if the two plots were normalized to their respective plateaus since the difference in arbitrary units distracts from the conclusion of the figure. If the difference in the plateaus is meaningful, please explain.

Figure 6: It is assumed that the authors are trying to show in this figure that cofilin binds both actins approximately the same but does not sever as readily for E334Q actin. The numerous parameters measured do not directly address what the authors are actually trying to show, which presumably is that the rate of severing is lower for E334Q than WT. It is therefore puzzling why no measurement of severing events per second per micron of actin in TIRF is made, which would give a more precise account of the underlying mechanism.

Actin-activated steady-state ATPase data of the NM2A with mutant and WT actin would have been extremely useful and informative. The authors show the ability to make these types of measurements in the paper (NADH assay), and it is surprising that they are not included for assessing the myosin activity. It may be because of limited actin quantities. If this is the case, it should be indicated.

(line 310) The authors state that they "noticed increased rapid dissociation and association events for E334Q filaments" in the motility assay. This observation motivates the authors to assess actin affinities of NM2A-HMM. Although differences in rigor and AM.ADP affinities are found between mutant and wt actins, the actin attachment lifetimes (many minutes) are unlikely to be related to the rapid association and dissociation event seen in the motility assay. Rather, this jiggling is more likely to be related to a lower duty ratio of the myosins, which appears to be the conclusion reached for the myosin-V data. These points should be clarified in the text.

(line 327) The authors report that the 1/K1 value is unchanged. There are no descriptions of this experiment in the paper. I am assuming the authors measured the ATP-induced dissociation of actomyosin and determined ATP affinity (K1) from this experiment. If this is the case, they should describe the experiment and show the data, provide a second-order rate constate for ATP binding, and report the max rate of dissociation (k2). This is a kinetic experiment done frequently by this group, so the absence of these details is surprising.

Reviewer #2 (Public Review):

Summary:<br /> In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibited form is stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

Strengths:<br /> The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gbeta/gamma subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

Reviewer #2 (Public Review):

Summary:<br /> The study by Yeo and co-authors addresses a long-lasting issue about botulinum neurotoxin (BoNT) intoxication. The current view is that the toxin binds to its receptors at the axon terminus by its HCc domain and is internalized in recycled neuromediator vesicles just after the release of the neuromediators. Then, the HCn domain assists the translocation of the catalytic light chain (LC) of the toxin through the membrane of these endocytic vesicles into the cytosol of the axon terminus. There, the LC cleaves its SNARE substrate and blocks neurosecretion. However, other views involving kinetic aspects of intoxication suggest that the toxin follows the retrograde axonal transport up to the nerve cell body and then back to the nerve terminus before cleaving its substrate.

In the current study, the authors claim that the BoNT/A (isotype A of BoNT) not only progresses to the cell body but once there, follows the retrograde transport trafficking pathway in a retromer-dependent fashion, through the Golgi apparatus, until reaching the endoplasmic reticulum. Next, the LC dissociates from the HC (a process not studied here) and uses the translocon Sec61 machinery to retro-translocate into the cytosol. Only then, does the LC traffic back to the nerve terminus following the anterograde axonal transport. Once there, LC cleaves its SNARE substrate (SNAP25 in the case of BoTN/A) and blocks neurosecretion.

To reach their conclusion, Yeo and co-authors use a combination of engineered tools: a cell line able to differentiate into neurons (ReNcell VN), a reporter dual fluorescent protein derived from SNAP25, the substrate of BoNT/A (called SNAPR), the use of either native BoNT/A or a toxin to which three fragment 11 of the reporter fluorescent protein Neon Green (mNG) are fused to the N-terminus of the LC (BoNT/A-mNG11x3), and finally ReNcell VN transfected with mNG1-10 (a protein consisting of the first 10 beta strands of the mNG).

SNAPR is stably expressed all over in the ReNcell VN. SNAPR is yellow (red and green) when intact and becomes red only when cleaved by BoNT/A LC, the green tip being degraded by the cell. When the LC of BoNT/A-mNG11x3 reaches the cytosol in ReNcell VN transfected by mNG1-10, the complete mNG is reconstituted and emits a green fluorescence.

In the first experiment, the authors show that the catalytic activity of the LC appears first in the cell body of neurons where SNAPR is cleaved first. This phenomenon starts 24 hours after intoxication and progresses along the axon towards the nerve terminus during an additional 24 hours. In a second experiment, the authors intoxicate the ReNcell VN transfected by mNG1-10 using the BoNT/A-mNG11x3. The fluorescence appears also first in the soma of neurons, then diffuses in the neurites in 48 hours. The conclusion of these two experiments is that translocation occurs first in the cell body and that the LC diffuses in the cytosol of the axon in an anterograde fashion.

In the second part of the study, the authors perform a siRNA screen to identify regulators of BoNT/A intoxication. Their aim is to identify genes involved in intracellular trafficking of the toxin and translocation of the LC. Interestingly, they found positive and negative regulators of intoxication. Regulators could be regrouped according to the sequential events of intoxication. Genes affecting binding to the cell-surface receptor (SV2) and internalization. Genes involved in intracellular trafficking. Genes involved in translocation such as reduction of the disulfide bond linking the LC to the HC and refolding in the cytosol. Genes involved in signaling such as tyrosine kinases and phosphatases. All these groups of genes may be consistent with the current view of BoNT intoxication within the nerve terminus. However, two sets of genes were particularly significant to reach the main conclusion of the work and definitely constitute an original finding important to the field. One set of genes consists of those of the retromer, and the other relates to the Sec61 translocon. This should indicate that once endocytosed, the BoNT traffics from the endosomes to the Golgi apparatus, and then to the ER. Ultimately, the LC should translocate from the ER lumen to the cytosol using the Sec61 translocon. The authors further control that the SV2 receptor for the BoNT/A traffics along the axon in a retromer-dependent fashion and that BoNT/A-mNG11x3 traverses the Golgi apparatus by fusing the mNG1-10 to a Golgi resident protein.

Strengths:<br /> The findings in this work are convincing. The experiments are carefully done and are properly controlled. In the first part of the study, both the activity of the LC is monitored together with the physical presence of the toxin. In the second part of the work, the most relevant genes that came out of the siRNA screen are checked individually in the ReNcell VN / BoNT/A reporter system to confirm their role in BoNT/A trafficking and retro-translocation.

These findings are important to the fields of toxinology and medical treatment of neuromuscular diseases by BoNTs. They may explain some aspects of intoxication such as slow symptom onset, aggravation, and appearance of central effects.

Weaknesses:<br /> The findings antagonize the current view of the intoxication pathway that is sustained by a vast amount of observations. The findings are certainly valid, but their generalization as the sole mechanism of BoNT intoxication should be tempered. These observations are restricted to one particular neuronal model and engineered protein tools. Other models such as isolated nerve/muscle preparations display nerve terminus paralysis within minutes rather than days. Also, the tetanus neurotoxin (TeNT), whose mechanism of action involving axonal transport to the posterior ganglia in the spinal cord is well described, takes between 5 and 15 days. It is thus possible that different intoxication mechanisms co-exist for BoNTs or even vary depending on the type of neurons.

Although the siRNA experiments are convincing, it would be nice to reach the same observations with drugs affecting the endocytic to Golgi to ER transport (such as Retro-2, golgicide or brefeldin A) and the Sec61 retrotranslocation (such as mycolactone). Then, it would be nice to check other neuronal systems for the same observations.

Reviewer #2 (Public Review):

Summary:<br /> This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:<br /> The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:<br /> The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

1) At the lower level, we first need to understand what the individual species do and "like" (their environmental niche). That information is not presented, and the methods suggest that the representation of each species in the JSDM is likely to be extremely poor.

2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the model is not set up this way, and the authors thus do a lot of indirect exploration, rather than direct hypothesis testing.

Even when the focus is not the individual species, but rather their association, we need to formulate what the expectation is. The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one can then add the layer of interspecific associations.

The change in activity and space use can be analysed much simpler, by looking at the activity times and spatial distribution directly. It remains unclear what the contribution of the JSDM is, unless it is able to represent this activity and spatial information, and put it in a testable interaction with human disturbance.

The topic is actually rather complicated. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

Then, in the next step, one would have to formulate specific hypotheses about which species are likely to change their associations more, and which less (based e.g. on predator-prey or competitive interactions). The data and analyses presented do not answer any of these issues.

Another more substantial point is that, according to my understanding of the methods, the per-species models are very inappropriate: the predictors are only linear, and there are no statistical interactions (L374). There is no conceivable species in the world whose niche would be described by such an oversimplified model.

We have no idea of even the most basic characteristics of the per-species models: prevalences, coefficient estimates, D2 of the model, and analysis of the temporal and spatial autocorrelation of the residuals, although they form the basis for the association analysis! Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

Also, all correlations among species should be shown for the raw data and for the model residuals: how much does that actually change and can thus be explained by the niche models?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. I failed to see any advance in ecological understanding at the community level.

Reviewer #2 (Public Review):

Allison Coté et al. investigated the ordering and spatial distribution of nascent transcripts in several cells using smFISH, expansion microscopy, and live-cell imaging. They find that pre-mRNA splicing occurs post-transcriptionally at the clouds around the transcription start site, termed the transcription site proximal zone. They show that pre-mRNA may undergo continuous splicing when they pass through the zone after transcription. These data suggest a unifying model for explaining previously reported co-transcriptional splicing events and provide a direction for further study of the nature of the slow-moving zone around the transcription start site.

This paper is well-written. The findings are very important, and the data supports the conclusions well. However, some aspects of the image and description need to be clarified and revised.

1) The sentence "By distinguishing the separate fluorescent signals from probes bound to exons and introns, we could visualize splicing intermediates (represented by colocalized intron and exon spots) relative to the site of transcription (represented by bright colocalized intron and exon spots) and fully spliced products (represented by exon spots alone)." is accidentally repeated twice, one of them should be deleted.<br /> 2) The authors describe Figure 4E and 4F results in the main text as that "we performed RNA FISH simultaneously with immunofluorescence for SC35, a component of speckles, and saw that these compartmentalized pre-mRNA did indeed appear near nuclear speckles both before (Supplementary Figure 6C) and after (Figure 4E) splicing inhibition." However, no SC35 staining is shown in the Figure 4E. A similar situation happened in describing Figure 4F.

Reviewer #2 (Public Review):

Lin et al attempt to examine the role of lncRNAs in human evolution in this manuscript. They apply a suite of population genetics and functional genomics analyses that leverage existing data sets and public tools, some of which were previously built by the authors, who clearly have experience with lncRNA binding prediction. However, I worry that there is a lack of suitable methods and/or relevant controls at many points and that the interpretation is too quick to infer selection. While I don't doubt that lnc RNAs contribute to the evolution of modern humans, and certainly agree that this is a question worth asking, I think this paper would benefit from a more rigorous approach to tackling it.

I thank the authors for their revisions to the manuscript; however, I find that the bulk of my comments have not been addressed to my satisfaction. As such, I am afraid I cannot say much more than what I said last time, emphasising some of my concerns with regards to the robustness of some of the analyses presented. I appreciate the new data generated to address some questions, but think it could be better incorporated into the text - not in the discussion, but in the results.

Reviewer #2 (Public Review):

In this manuscript the authors generate an annotated brain atlas for the prairie vole, which is a widely studied organism. This species has a suite of social behaviors that are difficult or impossible to study in conventional rodents, and has attracted a large community of researchers. The atlas is impressive and will be a fantastic resource. The authors use this atlas to examine brain-wide c-fos expression in prairie voles that were paired with same sex or opposite sex vole across multiple timepoints. In some sense the design resembles PET studies done in primates that take whole brain scans after an important behavioral experience. The authors observed increased c-fos expression across a network of brain regions that largely corresponds with the previous literature. The study design captured several novel observations including that c-fos expression in some regions correlate strongly between males and females during pair bond formation and mating, suggesting synchrony in neural activity. The authors address an important caveat that c-fos provides a snapshot of neural activity and that important populations of neurons could be active and not express c-fos. Thus observed correlations are likely to be robust, but that the absence of differences (in say accumbens) may just reflect the limits of c-fos estimation of neural activity. Similarly, highly coordinated neural activity between males and females might still be driven by different mechanisms if different cell types were activated within a specific region. The creation of this resource and it's use in a well designed study is an important accomplishment.

Reviewer #2 (Public Review):

Summary:<br /> The authors describe a novel ML approach to predict binding between MHC-bound peptides and T-Cell receptors. Such approaches are particularly useful for predicting the binding of peptide sequences with low similarity when compared to existing data sets. The authors focus on improving dataset quality and optimizing model architecture to achieve a pan-specific predictive model in hopes of achieving a high-precision model for novel peptide sequences.

Strengths:<br /> Since assuring the quality of training datasets is the first major step in any ML training project, the extensive human curation, computational analysis, and enhancements made in this manuscript represent a major contribution to the field. Moreover, the systematic approach to testing redundancy reduction and data augmentation is exemplary, and will significantly help future research in the field.

The authors also highlight how their model can identify outliers and how that can be used to improve the model around known sequences, which can help the creation and optimization of future datasets for peptide binding.

The new models presented here are novel and built using paired α/β TCR sequence data to predict peptide-specific TCR binding, and have been extensively and rigorously tested.

Weaknesses:<br /> Achieving an accurate pan-specific model is an ambitious goal, and the authors have significant difficulties when trying to achieve non-random performance for the prediction of TCR binding to novel peptides. This is the most challenging task for this kind of model, but also the most desirable when applying such models to biotechnological and bioengineering projects.

The manuscript is a highly technical and extremely detailed computational work, which can make the achievements and impact of the work hard to parse for application-oriented researchers.<br /> The authors briefly mention real-world use cases for TCR specificity predictions, but do not contextualize the work into possible applications.

Reviewer #2 (Public Review):

Summary:

This paper nicely introduces WormPsyQi, an imaging analysis pipeline that effectively quantifies synaptically localized fluorescent signals in C. elegans through high-throughput automation. This toolkit is particularly valuable for the analysis of densely packed regions in 3D space, such as the nerve ring. The authors applied WormPsyQi to various aspects, including the examination of sexually dimorphic synaptic connectivity, presynaptic markers in eight head neurons, five GRASP reporters, electrical synapses, the enteric nervous system, and developmental synapse comparisons. Furthermore, they validated WormPsyQi's accuracy by comparing its results to manual analysis.

Strengths:

Overall, the experiments are well done, and their toolkit demonstrates significant potential and offers a valuable resource to the C. elegans community. This will expand the range of possibilities for studying synapses in C. elegans.

Reviewer #2 (Public Review):

Summary:<br /> Developing a mechanical model of C. elegans is difficult to do from basic principles because it moves at a low (but not very small) Reynolds number, is itself visco-elastic, and often is measured moving at a solid/liquid interface. The ElegansBot is a good first step at a kinetic model that reproduces a wide range of C. elegans motiliy behavior.

Strengths:<br /> The model is general due to its simplicity and likely useful for various undulatory movements. The model reproduces experimental movement data using realistic physical parameters (e.g. drags, forces, etc). The model is predictive (semi?) as shown in the liquid-to-solid gait transition. The model is straightforward in implementation and so likely is adaptable to modification and addition of control circuits.

Weaknesses:<br /> Since the inputs to the model are the actual shape changes in time, parameterized as angles (or curvature), the ability of the model to reproduce a realistic facsimile of C. elegans motion is not really a huge surprise.

The authors do not include some important physical parameters in the model and should explain in the text these assumptions. 1) The cuticle stiffness is significant and has been measured [1]. 2) The body of C. elegans is under high hydrostatic pressure which adds an additional stiffness [2]. 3) The visco-elasticity of C. elegans body has been measured. [3]

There is only a very brief mention of proprioception. The lack of inclusion of proprioception in the model should be mentioned and referenced in more detail in my opinion.

These are just suggested references. There may be more relevant ones available.

1. Rahimi M, Sohrabi S, Murphy CT. Novel elasticity measurements reveal C. elegans cuticle stiffens with age and in a long-lived mutant. Biophys J. 2022 Feb 15;121(4):515-524. doi: 10.1016/j.bpj.2022.01.013. Epub 2022 Jan 19. PMID: 35065051; PMCID: PMC8874029.

2. Park SJ, Goodman MB, Pruitt BL. Analysis of nematode mechanics by piezoresistive displacement clamp. Proc Natl Acad Sci U S A. 2007 Oct 30;104(44):17376-81. doi: 10.1073/pnas.0702138104. Epub 2007 Oct 25. PMID: 17962419; PMCID: PMC2077264.

3. Backholm M, Ryu WS, Dalnoki-Veress K. Viscoelastic properties of the nematode Caenorhabditis elegans, a self-similar, shear-thinning worm. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):4528-33. doi: 10.1073/pnas.1219965110. Epub 2013 Mar 4. PMID: 23460699; PMCID: PMC3607018.

Reviewer #2 (Public Review):

In this study, Hernandez-Hernandez et al developed a gender-dependent mathematical model of arterial myocytes based on a previous model and new experimental data. The ionic currents of the model and its sex difference were formulated based on patch clamp experimental data, and the model properties were compared with single cell and tissue scale experimental results. This is a study that is of importance for the modeling field as well as for experimental physiology.

Reviewer #2 (Public Review):

Summary:<br /> The authors are studying the behavioral response to pathogen exposure. They and others have previously describe the role that the G-protein coupled receptors in the nervous system plays in detecting pathogens, and initiating behavioral patterns (e.g. avoidance/learned avoidance) that minimize contact. The authors study this problem in C. elegans, which is amenable to genetic and cellular manipulations and allow the authors to define cellular and signaling mechanisms. This paper extends the original idea to now implicate signaling and transcriptional pathways within a particular neuron (ASJ) and the gut in mediating avoidance behaviour.

Strengths:<br /> The work is rigorous and elegant and the data are convincing. The authors make superb use of mutant strains in C. elegans, as well tissue specific gene inactivation and expression and genetic methods of cell ablation. to demonstrate how a gene, NPR15 controls behavioral changes in pathogen infection. The results suggest that ASJ neurons and the gut mediate such effects. I expect the paper will constitute an important contribution to our understanding of how the nervous system coordinates immune and behavioral responses to infection.

Reviewer #2 (Public Review):

This is a nice story about auricular chondrocyte maintenance, its molecular mechanism, and the role of the BMP 1 receptor in microtia disease conditions. A detailed analysis of different parts of the ear, their proliferation, and their differentiation condition with histological and immunofluorescence analysis strengthens the evidence. Further validation with patient sample RNA-Seq also helps the study end with an informative story.

From the public point of view, I want to say that the authors want to explain how auricular chondrocytes differ from growth plates or other chondrocytes. The authors show that Prxx1 is a good marker to differentiate auricular chondrocytes from different types of chondrocytes, which I doubt because other chondrocytes have an expression of Prxx1 at a lower level.

Another thing the authors mention is that microtia conditions develop through reduced size without affecting proliferation and apoptosis. The authors never provide any evidence about how the ablation of Bmpr1a affects the size, protein trafficking, and ECM organization.

Crosstalk between BMP-PKA in auricular chondrocytes and switching the chondrocytes' cell fate in osteoblast cells are not entirely stable by these studies for physiological functions.

Reviewer #3 (Public Review):

Summary:<br /> The authors set out to characterize the anatomical connectivity profile and the functional responses of chandelier cells (ChCs) in the mouse primary visual cortex. Using retrograde rabies tracing, optogenetics, and in vitro electrophysiology, they found that the primary source of input to ChCs are local layer 5 pyramidal cells, as well as long-range thalamic and cortical connections. ChCs provided input to local layer 2/3 pyramidal neurons, but did not receive reciprocal connections.

With two-photon calcium imaging recordings during passive viewing of drifting gratings, the authors showed that ChCs exhibit weakly selective visual responses, high correlations within their own population, and strong responses during periods of arousal (assessed by locomotion and pupil size). These results were replicated and extended in experiments with natural images and prediction of receptive field structure using a convolutional neural network.

Furthermore, the authors employed a learned visuomotor task in a virtual corridor to show that ChCs exhibit strong responses to mismatches between visual flow and locomotion, locomotion-related activation (similar to what was shown above), and visually-evoked suppression. They also showed the existence of two clusters of pyramidal neurons with functionally different responses - a cluster with "classically visual" responses and a cluster with locomotion- and mismatch-driven responses (the latter more correlated with ChCs). Comparing naive and trained mice, the authors found that visual responses of ChCs are suppressed following task learning, accompanied by a shortening of the axon initial segment (AIS) of pyramidal cells and an increase in the proportion of AIS contacted by ChCs. However, additional controls would be required to identify which component(s) of the experimental paradigm led to the functional and anatomical changes observed.

Strengths:<br /> The authors bring a comprehensive, state-of-the-art methodology to bear, including rabies tracing, in vivo two-photon calcium imaging, in vitro electrophysiology, optogenetics and chemogenetics, and deep neural networks. Their analyses and statistical tests are sound and for the most part, support their claims. Their results are in line with previous findings and extend them to the primary visual cortex.

Weaknesses:<br /> - Some of the results (e.g. arousal-related responses) are not entirely surprising given that similar results exist in other cortical areas.

Reviewer #2 (Public Review):

Summary:<br /> Nakai-Futatsugi et al. present a novel method to analyze the correlation between the degree of pigmentation and the gene expression profile of human-induced pluripotent stem cell-derived RPE (iPSC-RPE) cells at the single cell level. This was achieved with the use of ALPS (Automated Live imaging and cell Picking System), an invention developed by the same authors. Briefly, it allows one to choose and photograph a specific cell from a culture dish and proceed to single-cell digital RNA-seq. The authors identify clusters of cells that present differential gene expression, but this shows no association with the degree of pigmentation of the cells. Further data analysis allowed the authors to correlate the degree of pigmentation to some degree with the expression of complement and lysosome-related genes.

Strengths:<br /> An important amount of data related to gene expression and heterogeneity of the iPSC-RPE population has been generated in this work.

Weaknesses:<br /> However, the justification of the analysis, and the physiological relevance of the hypothesis and the findings could be strengthened.

Importantly, I fail to grasp from the introduction what is the previous evidence that leads to the hypothesis. Why would color intensity be related to the quality of cell transplantation? In fact, cell transplantation is not evaluated at all in this work. The authors mention "quality metrics for clinical use", but this concept is not further explained. Neither is the concept of "sufficient degree of pigmentation" explained.

On the other hand, the positive correlation of cluster formation with complement and lysosome-related genes is not discussed.

As a consequence, it is very difficult to evaluate the impact of these findings on the field.

Reviewer #2 (Public Review):

This paper introduces a new model that aims to explain the generators of temporal decoding matrices (TGMs) in terms of underlying signal properties. This is important because TGMs are regularly used to investigate neural mechanisms underlying cognitive processes, but their interpretation in terms of underlying signals often remains unclear. Furthermore, neural signals are often variant over different instances of stimulation despite behaviour being relatively stable. The author aims to tackle these concerns by developing a generative model of electrophysiological data and then showing how different parameterizations can explain different features of TGMs. The developed technique is able to capture empirical observations in terms of fundamental signal properties. Specifically, the model shows that complexity is necessary in terms of spatial configuration, frequencies and latencies to obtain a TGM that is comparable to empirical data.

The major strength of the paper is that the novel technique has the potential to further our understanding of the generators of electrophysiological signals which are an important way to understand brain function. The paper clearly outlines how the method can be used to capture empirical data. Furthermore, the used techniques are state-of-the-art and the developed model is publicly shared in open source code.

On the other hand, there is no unambiguous mapping between neurobiological mechanisms and different signal generators, making it hard to draw firm conclusions about neural underpinnings based on this analysis.

Reviewer #2 (Public Review):

This manuscript by Petersen and colleagues investigates the mechanistic underpinnings of activation of the ion channel TREK-1 by mechanical inputs (fluid shear or membrane stretch) applied to cells. Using a combination of super-resolution microscopy, pair correlation analysis and electrophysiology, the authors convincingly show that the application of shear to a cell can lead to changes in the distribution of TREK-1 and the enzyme PhospholipaseD2 (PLD2), relative to lipid domains defined by either GM1 or PIP2. The activation of TREK-1 by mechanical stimuli was shown to be sensitized by the presence of PLD2, but not a catalytically inactive xPLD2 mutant. In addition, the activity of PLD2 is increased when the molecule is more associated with PIP2, rather than GM1 defined lipid domains. The presented data do not exclude direct mechanical activation of TREK-1, rather suggest a modulation of TREK-1 activity, increasing sensitivity to mechanical inputs, through an inherent mechanosensitivity of PLD2 activity. The authors additionally demonstrate that cellular uptake of cholesterol inhibits TREK-1 activation and, in ex vivo studies, that depletion of cholesterol from astrocytes reduces correlation of TREK-1 and G1 lipids in mouse brain slices. In vivo studies, using Drosophila melanogaster behavioural assays, were used to demonstrate that disrupting PLD2 altered behavioural responses to mechanical and electrical inputs. These data demonstrate that manipulation of PLD2 analogue in the fly can alter sensory transduction, suggesting that PLD functions to regulate sensitivity to mechanical force. However, as the authors note, there is no TREK-1 homologue in this organism: thus the identity of the downstream effectors of PLD in D. melanogaster remain unknown. This work will be of interest to the growing community of scientists investigating the myriad mechanisms that can tune mechanical sensitivity of cells, providing valuable insight into the role of functional PLD2 in sensitizing TREK-1 activation in response to mechanical inputs, in some cellular systems.

The authors convincingly demonstrate that, post application of shear, an alteration in the distribution of TREK-1 and mPLD2 (in HEK293T cells) from being correlated with GM1 defined domains (no shear) to increased correlation with PIP2 defined membrane domains (post shear). The association of TREK-1 with PIP2 required functional mPLD2. These data were generated using super-resolution microscopy to visualise, at sub diffraction resolution, the localisation of labelled protein, compared to labelled lipids. The use of super-resolution imaging enabled the authors to visualise changes in cluster association that would not have been achievable with diffraction limited microscopy.

This work provides further evidence of the astounding flexibility of mechanical sensing in cells. By outlining how mechanical activation of TREK-1 can be sensitised by mechanical regulation of PLD2 activity, the authors highlight a mechanism by which TREK-1 sensitivity could be regulated under distinct physiological conditions.

Reviewer #2 (Public Review):

In their submitted manuscript, Blin et al. describe differences in the olfactory-driven behaviors of river-dwelling surface forms and cave-dwelling blind forms of the Mexican tetra, Astyanax mexicanus. They provide a dataset of unprecedented detail, that compares not only the behaviors of the two morphs but also that of a significant number of F2 hybrids, therefore also demonstrating that many of the differences observed between the two populations have a clear (and probably relatively simple) genetic underpinning.

To complete the monumental task of behaviorally testing 425 six-week-old Astyanax larvae, the authors created a setup that allows for the simultaneous behavioral monitoring of multiple larvae and the infusion of different odorants without introducing physical perturbations into the system, thus biasing the responses of cavefish that are particularly fine-tuned for this sensory modality. During the optimization of their protocol, the authors also found that for cave-dwelling forms one hour of habituation was insufficient and a full 24 hours were necessary to allow them to revert to their natural behavior. It is also noteworthy that this extremely large dataset can help us see that population averages of different morphs can mask quite significant variations in individual behaviors.

Testing with different amino-acids (applied as relevant food-related odorant cues) shows that cavefish are alanine- and histidine-specialists, while surface fish elicit the strongest behavioral responses to cysteine. It is interesting that the two forms also react differently after odor detection: while cave-dwelling fish decrease their locomotory activity, surface fish increase it. These differences are probably related to different foraging strategies used by the two populations, although, as the observations were made in the dark, it would be also interesting to see if surface fish elicit the same changes in light as well.

Further work will be needed to pinpoint the exact nature of the genetic changes that underlie the differences between the two forms. Such experimental work will also reveal how natural selection acted on existing behavioral variations already present in the SF population.

It will be equally interesting, however, to understand what lies behind the large individual variation of behaviors observed both in the case surface and cave populations. Are these differences purely genetic, or perhaps environmental cues also contribute to their development? Does stochasticity provided by the developmental process has also a role in this? Answering these questions will reveal if the evolvability of Astyanax behavior was an important factor in the repeated successful colonization of underground caves.

Reviewer #2 (Public Review):

The mechanisms that mediate female aggression remain poorly understood. Chiu, Schretter, and colleagues, employed circuit dissection techniques to tease apart the specific roles of particular doublesex and fruitless expressing neurons in the fly Drosophila in generating a persistent aggressive state. They find that activating the fruitless positive alPg neurons, generated an aggressive state that persisted for >10min after the stimulation ended. Similarly, activating the doublesex positive pC1de neurons also generated a peristent state. Activating pC1d or pC1e individually did not induce a persistent state. Interestingly, while neural activation of alPGs and pC1d+e neurons induced a persistent behavioural states it did not induce persistent activity in the neurons being activated.

The authors have revised the manuscript in accordance with comments of the reviewers. The conclusions of this paper are by and large well supported by the data. These data will be a useful addition to the literature on the circuit basis of female aggression, and open up intriguing avenues for further studies to explore.

Reviewer #2 (Public Review):

Summary:<br /> In a proof-of-concept study with the aspiration of developing a treatment to delay HD onset, Choi et al. design and test an A>G DNA base editing strategy to exploit the recently established inverse relationship between the number of uninterrupted CAG repeats in polyglutamine repeat expansions and the age-of-onset of Huntington's Disease (HD). Most of the study is devoted to optimizing a base editing strategy typified by BE4max and gRNA2. The base editing is performed in human HEK293 cells engineered with a 51 CAG canonical repeat and in HD knock-in mice harboring 105+ CAG repeats.

Weaknesses:<br /> Genotypic data on DNA editing are not portrayed in a clear manner consistent with the study's goal, namely reducing the number of uninterrupted CAG repeats by a clinically relevant amount according to the authors' least square approximated mean age-at-onset. No phenotypic data are presented to show that editing performed in either model would lead to reduced hallmarks of HD onset.

More evidence is needed to support the central claims and therapeutic potential needs to be more adequate.

Reviewer #2 (Public Review):

In this study, the authors describe a pipeline to sequence expressed var genes from RNA sequencing that improves on a previous one that they had developed. Importantly, they use this approach to determine how var gene expression changes with short-term culture. Their finding of shifts in the expression of particular var genes is compelling and casts some doubt on the comparability of gene expression in short-term culture versus var expression at the time of participant sampling.

Other studies have relied on short-term culture to understand var gene expression in clinical malaria studies. This study indicates the need for caution in over-interpreting findings from these studies.

We appreciate the careful attention of the authors to our comments and the edits that have been made. One additional suggestion that would be helpful to readers is to include in Table S1 the new approach described in the manuscript. This will provide the reader a direct means of comparing what the authors have done to past work.

Reviewer #2 (Public Review):

In this study, a minimalist setup was used to investigate the selectivity of the nuclear pore complex as a function of its diameter. To this end, a series of solid-state pores in a free-standing palladium membrane were designed and attached to a PDMS-based fluid cell that could be mounted on a confocal microscope. In this way, the frequency of translocation events could be measured in an unbiased manner. Furthermore, the pores were designed to exhibit the key properties of the nuclear pore complex: (i) the size of the pore, (ii) disordered FG Nups specifically located in the central channel; (ii) transport receptors that can shuttle through the central channel by binding to the FG-Nups. Additionally, such system offered the advantage of monitoring the translocation of multiple fluorescently labeled molecules (e.g. Kap95 and BSA) simultaneously and under well-controlled conditions.

The authors were able to demonstrate convincingly that the pore selectivity depends on the pore diameter, the FG Nup layer organization within the pore and the transport receptors concentration that can specifically interact with FG Nups. It was shown that the pores coated with FG Nups (e.g. Nsp1 in this case) and smaller than 50-60 nm are highly selective and such selectivity is increasing with the decrease of the pore diameter. Also, it was shown that the pore selectivity moderately enhances at the high Kap95 concentration (1 µM). Importantly, it was also shown that the selectivity is becoming negligible for the pores, which are larger than 60-75 nm.

The experimental data are well supported by coarse-grained modelling of Nsp1-coated pores, and the theoretical prediction correlates qualitatively with the experimentally obtained data.

Reviewer #2 (Public Review):

This study explores the breadth of effects of one important metabolite, azelaic acid, on marine microbes, and reveals in-depth its pathway of uptake and catabolism in one model bacterial strain. This compound is known to be widely produced by phytoplankton and plants, and to have complex effects on associated microbiomes.

This work uses transcriptomics to assay the response of two strains that show contrasting responses to the metabolite: one catabolizes the compound and assimilates the carbon, while the other shows growth inhibition and stress response. A highly induced TRAP transporter, adjacent to a previously identified regulator, is inferred to be the specific uptake system for azelaic acid, though this function was not directly tested via genetic or biochemical methods. Nevertheless, this is a significant finding that will be useful for exploring the distribution of azelaic acid uptake capability across metagenomes and other bacteria.

The authors use pulse-chase style metabolomics experiments to beautifully demonstrate the fate of azelaic acid through catabolic pathways. They also measure an assimilation rate per cell, though it remains unclear how this measured rate relates to natural systems. The metabolomics approach is an elegant way to show carbon flux through cells, and could serve as a model for future studies.

The study seeks to extend the results from two model strains to complex communities, using seawater mesocosm experiments and soil/Arabidopsis experiments. The seawater experiments show a community shift in mesocosms with added azelaic acid. The mechanisms for the shift were not determined in this study; further work is necessary to demonstrate which community members are directly assimilating the compound, benefitting indirectly, or experiencing inhibition. The authors also took the unusual and creative step of performing similar experiments in a soil - Arabidopsis system. I admire the authors' desire to identify unifying themes across ecosystems. The parallels are intriguing, and future experiments could determine the different modes of action in aquatic vs terrestrial microbial communities.

This work is a nice illustration of how we can begin to tease apart the effects of chemical currencies on marine ecosystems. A key strength of this work is the combination of transcriptomics and metabolomics methods, along with assaying the impacts of the metabolite on model strains of bacteria and whole communities. Given the sheer number of compounds that probably play critical roles in community interactions, a key challenge for the field will be navigating the tradeoffs between breadth and depth in future studies of metabolite impacts. This study offers a good compromise and will be a useful model for future studies.

Reviewer #2 (Public Review):

In this study, the authors utilize a compendium of public genomic data to identify transcription factors (TF) that can identify their DNA binding motifs in the presence of nuclosome-wrapped chromatin and convert the chromatin to open chromatin. This class of TFs are termed Pioneer TFs (PTFs). A major strength of the study is the concept, whose premise is that motifs bound by PTFs (assessed by ChIP-seq for the respective TFs) should be present in both "closed" nucleosome wrapped DNA regions (measured by MNase-seq) as well as open regions (measured by DNAseI-seq) because the PTFs are able to open the chromatin. Use of multiple ENCODE cell lines, including the H1 stem cell line, enabled the authors to assess if binding at motifs changes from closed to open. Typical, non-PTF TFs are expected to only bind motifs in open chromatin regions (measured by DNaseI-seq) and not in regions closed in any cell type. This study contributes to the field a validation of PTFs that are already known to have pioneering activity and presents an interesting approach to quantify PTF activity.

For this reviewer, there were a few notable limitations. One was the uncertainty regarding whether expression of the respective TFs across cell types was taken into account. This would help inform if a TF would be able to open chromatin. Another limitation was the cell types used. While understandable that these cell types were used, because of their deep epigenetic phenotyping and public availability, they are mostly transformed and do not bear close similarity to lineages in a healthy organism. Next, the methods used to identify PTFs were not made available in an easy-to-use tool for other researchers who may seek to identify PTFs in their cell type(s) of interest. Lastly, some terms used were not defined explicitly (e.g., meaning of dyads) and the language in the manuscript was often difficult to follow and contained improper English grammar.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Yu and colleagues sought to identify new susceptibility genes for adolescent idiopathic scoliosis (AIS). The significance of this work is high, especially given the still large knowledge gap of the mechanistic underpinnings for AIS. In this multidisciplinary body of work, the authors first performed a genetic association study of AIS case-control cohorts (combined 9,161 cases and 80,731 controls) which leveraged common SNPs in 1027 previously defined matrisome genes. Two nonsynonymous variants were found to be significantly associated with AIS: MMP14 p.Asp273Asn and COL11A1 p.Pro1153Leu, the latter of which had the more robust association and remained significant when females were tested independent of males. Next, the authors followed a series of functional validation experiments to support biological involvement of COL11A1 p.Pro1153Leu in AIS through expression, biochemical, and histological studies in physiologically relevant cell and mouse models. Together, the authors propose a hitherto unreported model for AIS that involves the interplay of the COL11A1 susceptibility locus with estrogen signaling to alter a Pax1-Col11a1-Mmp3 signaling axis at the growth plate.

Strengths:

The manuscript is clearly written and follows a series of logical steps toward connecting multiple matrisome genes and putative AIS effectors in a new framework of pathomechanism. The multidisciplinary nature of the work makes it a strong body of work wherein multiple models offer multiple lines of supportive data. Thus, this manuscript remains an important multidisciplinary study of the genetic and functional basis of adolescent idiopathic scoliosis (AIS). To the benefit of the overall manuscript quality, the reviewers have addressed most concerns to satisfaction. Please include the list of three rare missense variants mentioned in the response to reviewers as a supplementary table. Please also include methods for the SKATO rare variant burden analysis.

Reviewer #2 (Public Review):

Summary:<br /> In Ferrareti et al. they identify adaptively introgressed genes using VolcanoFinder and then identify pathways enriched for adaptively introgressed genes. They also use a signet to identify pathways that are enriched for Denisovan alleles. The authors find that angiogenesis and nitric oxide induction are enriched for archaic introgression.

Strengths:<br /> Most papers that have studied the genetic basis of high altitude (HA) adaptation in Tibet have highly emphasized the role of a few genes (e.g. EPAS1, EGLN1), and in this paper, the authors look for more subtle signals in other genes (e.g EP300, NOS 2) to investigate how archaic introgression may be enriched at the pathway level.

Looking into the biological functions enriched for Denisovan introgression in Tibetans is important for characterizing the impact of Denisovan introgression.

Weaknesses:<br /> The manuscript lacks details or justification about how/why some of the analyses were performed. Below are some examples where the authors could provide additional details.

The authors made specific choices in their window analysis. These choices are not justified or there is no comment as to how results might change if these choices were perturbed. For example, in the methods, the authors write "Then, the genome was divided into 200 kb windows with an overlap of 50 kb and for each of them we calculated the ratio between the number of significant SNVs and the total number of variants."

Additional information is needed for clarity. For example, "we considered only protein-protein interactions showing confidence scores {greater than or equal to} 0.7 and the obtained protein frameworks were integrated using information available in the literature regarding the functional role of the related genes and their possible involvement in high-altitude adaptation." What do the confidence scores mean? Why 0.7?

In the method section (Identifying gene networks enriched for Denisovan-like derived alleles), the authors write "To validate VolcanoFinder results by using an independent approach". Does this mean that for signet the authors do not use the regions identified as adaptively introgressed using volcanofinder? I thought in the original signet paper, the authors used a summary describing the amount of introgression of a given region.

Later, the authors write "To do so, we first compared the Tibetan and Denisovan genomes to assess which SNVs were present in both modern and archaic sequences. These loci were further compared with the ancestral reconstructed reference human genome sequence (1000 Genomes Project Consortium et al., 2015) to discard those presenting an ancestral state (i.e., that we have in common with several primate species)." It is not clear why the authors are citing the 1000 genomes project. Are they comparing with the reference human genome reference or with all populations in the 1000 genomes project? Also, are the authors allowing derived alleles that are shared with Africans? Typically, populations from Africa are used as controls since the Denisovan introgression occurred in Eurasia.

The methods section for Figures 4B, 4C, and 4D is a little hard to understand. What is the x-axis on these plots? Is it the number of pairwise differences to Denisovan? The caption is not clear here. The authors mention that "Conversely, for non-introgressed loci (e.g., EGLN1), we might expect a remarkably different pattern of haplotypes distribution, with almost all haplotype classes presenting a larger proportion of non-Tibetan haplotypes rather than Tibetan ones." There is clearly structure in EGLN1. There is a group of non-Tibetan haplotypes that are closer to Denisovan and a group of Tibetan haplotypes that are distant from Denisovan...How do the authors interpret this?

In the original signet paper (Guoy and Excoffier 2017), they apply signet to data from Tibetans. Zhang et al. PNAS (2021) also applied it to Tibetans. It would be helpful to highlight how the approach here is different.

Reviewer #2 (Public Review):

In the manuscript by Rajan et al., the authors have highlighted the direct interaction between Dbp5 and tRNA, wherein Dbp5 serves as a mediator for tRNA export. This export process is subject to spatial regulation, as Dbp5 ATPase activation occurs specifically at nuclear pore complexes. Notably, this regulation is independent of the Los1-mediated pre-tRNA export route and instead relies on Gle1. The manuscript is well constructed and nicely written.

Reviewer #2 (Public Review):

In their study, Stajich and co-authors present a new 1.03 Gb genome assembly for an isolate of the fungal insect parasite Entomophthora muscae (Entomophthoromycota phylum, isolated from Drosophila hydei). Many species of the Entomophthoromycota phylum are specialised insect pathogens with relatively large genomes for fungi, with interesting yet largely unexplored biology. The authors compare their new E. muscae assembly to those of other species in the Entomophthorales order and also more generally to other fungi. For that, they first focus on repetitive DNA (transposons) and show that Ty3 LTRs are highly abundant in the E. muscae genome and contribute to ~40% of the species' genome, a feature that is shared by closely related species in the Entomophthorales. Next, the authors describe the major differences in protein content between species in the genus, focusing on functional domains, namely protein families (pfam), carbohydrate-active enzymes, and peptidases. They highlight several protein families that are overrepresented/underrepresented in the E. muscae genome and other Entomophthorales genomes. The authors also highlight differences in components of the circadian rhythm, which might be relevant to the biology of these insect-infecting fungi. To gain further insights into E. muscae specificities, the authors identify orthologous proteins among four Entomophthorales species. Consistently with a larger genome and protein set in E. muscae, they find that 21% of the 17,111 orthogroups are specific to the species. To finish, the authors examine the consistency between methods for species delineation in the genus using molecular (ITS + 28S) or morphological data (# of nuclei per conidia + conidia size) and highlight major incongruences between the two.

Although most of the methods applied in the frame of this study are appropriate with the scripts made available, I believe there are some major discrepancies in the datasets that are compared which could undermine most of the results/conclusions. More precisely, most of the results are based on the comparison of protein family content between four Entomophthorales species. As the authors mention on page 5, genome (transcriptome) assembly and further annotation procedures can strongly influence gene discovery. Here, the authors re-annotated two assemblies using their own methods and recovered between 30 and 60% more genes than in the original dataset, but if I understand it correctly, they perform all downstream comparative analyses using the original annotations. Given the focus on E. muscae and the small sample size (four genomes compared), I believe performing the comparisons on the newly annotated assemblies would be more rigorous for making any claim on gene family variation.

The authors also investigate the putative impact of repeat-induced point mutation on the architecture of the large Entomophthorales genomes (for three of the eight species in Figure 1) and report low RIP-like dinucleotide signatures despite the presence of RID1 (a gene involved in the RIP process in Neurospora crassa) and RNAi machinery. They base their analysis on the presence of specific PFAM domains across the proteome of the three Entomophthorales species. In the case of RID1, the authors searched for a DNA methyltransferase domain (PF00145), however other proteins than RID1 bear such functional domain (DNMT family) so that in the current analysis it is impossible to say if the authors are actually looking at RID1 homologs (probably not, RID1 is monophyletic to the Ascomycota I believe). Similar comments apply to the analysis of components of the RNAi machinery. A more reliable alternative to the PFAM analysis would be to work with full protein sequences in addition to the functional domains.

Reviewer #2 (Public Review):

Summary:<br /> The study by Toth et al. investigates the role of RIN4, a key immune regulator, in the symbiotic nitrogen fixation process between soybean and rhizobium. The authors found that SymRK can interact with and phosphorylate GmRIN4. This phosphorylation occurs within a 15 amino acid motif that is highly conserved in N-fixation clades. Genetic studies indicate that GmRIN4a/b play a role in root nodule symbiosis. Based on their data, the authors suggest that RIN4 may function as a key regulator connecting symbiotic and immune signaling pathways.

Overall, the conclusions of this paper are well supported by the data, although there are a few areas that need clarification.

Strengths:<br /> • This study provides important insights by demonstrating that RIN4, a key immune regulator, is also required for symbiotic nitrogen fixation.<br /> • The findings suggest that GmRIN4a/b could mediate appropriate responses during infection, whether it is by friendly or hostile organisms.

Weaknesses:<br /> • The study did not explore the immune response in the rin4 mutant. Therefore, it remains unknown how GmRIN4a/b distinguishes between friend and foe.

Reviewer #2 (Public Review):

This manuscript proposed a new link between the formation of chloroplast budding vesicles (Rubisco-containing bodies [RCBs]) and the development of chloroplast-associated autophagosomes. The authors' previous work demonstrated two types of autophagy pathways involved in chloroplast degradation, including piecemeal degradation of partial chloroplast and whole chloroplast degradation. However, the mechanisms underlying piecemeal degradation are largely unknown, particularly regarding the initiation and release of the budding structures. Here, the authors investigated the progression of piecemeal-type chloroplast trafficking by visualizing it with a high-resolution time-lapse microscope. They provide evidence that autophagosome formation is required for the initiation of chloroplast budding, and that stromule formation is not correlated with this process. In addition, the authors also demonstrated that the release of chloroplast-associated autophagosome is independent of a chloroplast division factor, DRP5b.

Overall, the findings are interesting, and in general, the experiments are very well executed. Although the mechanism of how Rubisco-containing bodies are processed is still unclear, this study suggests that a novel chloroplast division machinery exists to facilitate chloroplast autophagy, which will be valuable to investigate in the future.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Knights et al set out to identify the specific immune cells and their contribution to the development of osteoarthritis. They performed a comprehensive analysis of scRNA-seq and flow cytometry using different stages of the PTOA model and sought to identify specific synovial macrophages in OA. Computational analysis revealed that M-CSF signaling in synovium plays an important role in stromal-immune crosstalk in OA. They also found that four transcription factors including Pu.1, Cebp-alpha, Cebp-beta, and Jun regulate the differentiation of monocytes into pro-inflammatory synovial macrophages in OA.

Strengths:<br /> The main strength of this study is the profiling of immune cells which will be a valuable resource for better understanding the pathogenesis of OA. The work is technically sound, and the level of analysis of gene expression, clustering, cell-cell communication, and dynamic changes in gene modules over time is state-of-the-art.

The reviewer appreciates that the authors uncovered the transcriptional network that regulates the differentiation of synovial macrophages in OA. In addition, the identification of M-CSF signaling as a major crosstalk axis in OA development is also intriguing.