Reviewer #2 (Public Review):

The authors used single cell transcriptome analysis of zebrafish skin cells and characterized various types of cells that are involved in scale formation and stripe patterning. The methods employed in this study is highly powerful to provide mechanistic explanation of these fundamental biological issues and will be a good example for many researchers studying other biological issues. Furthermore, the results characterizing differences in gene expression patterns among various types of cells will be informative for other researchers in the field.

For scale formation, it is known that mineralized tissues may significantly differ in rayfins and lobefins since sox9, col2a1, and col10a1 are all expressed in osteoblasts, in addition to chondrocytes, in zebrafish and gar (Eames et al., 2012, BMC Evol. Biol.). Furthermore, in mammals, Col10 is expressed in chondrocytes in mature cartilage that undergoes ossification. Thus, unlike the authors argue, col10a1 expression is not apparently relevant to the elasticity of scales.

The authors also state that the expression of dlx4a, msx2a, and runx2b characterize cells homologous to mammalian ameloblasts. However, dlx4, runx2, and msx2 are all duplicated in zebrafish, and the function of duplicated genes in teleost fishes may differ from that of single ancestral gene. Moreover, none of Dlx4, Msx2, and Runx2 is expressed specifically by ameloblasts in mammals. Indeed, both Msx2 and Runx2 are expressed in osteoblasts, while the expression of Dlx4 in ameloblasts is not reported. These results, together with the expression of an enamel gene, enam, in dermal cells (SFC), do not appear to support the homology of the surface tissue of mammalian teeth and zebrafish scales.

Reviewer #2 (Public Review):

In a neonatal model of bacterial meningitis induced by s.c. injection of E. coli, transcriptional changes were found across all major cell types including endothelial cells, fibroblasts and macrophages. Among macrophages, they describe 2 resident subsets and 2 inflammatory subsets. By immunohistochemistry of arachnoid and dura flatmounts, they show vascular changes upon infection, including clustering of CLDN5 and PECAM1, and disorganized capillary morphology, which was dependent on Tlr4 signaling but independent of arachnoid macrophages.

The manuscript would benefit from rewriting, it is not written in a concise manner and the rationale for experiments, time points for analyses and their conclusions are not clear. The model of s.c. bacterial infection is not well introduced and overall changes in the periphery, survival curves or bacterial counts (in the KO models) in the meninges/brain are not mentioned.

Reviewer #2 (Public Review):

This study aimed to classify colorectal cancer (CRC) samples based on the expression of genes in selected gene lists, where the gene lists were chosen to represent aspects of the tumour microenvironment, tumour-associated immune cells, and tumour cells. The resulting clusters were then used to define a classifier, followed by a detailed description of molecular features of the tumours and tumour microenvironments assigned to each cluster. The authors claim this study is more "holistic" than previous work on CRC clustering/classifiers because they aimed to explicitly include additional components of the tumour microenvironment in both the clustering/classifier definition and in the subsequent description of molecular characteristics.

The CCCRC clustering and the resulting classifier presented in this paper are derived from published RNAseq studies. The multi-omics aspect of the work is restricted to smaller sample numbers for which both transcriptomic and another omics dataset were available in public resources and comprises a description or correlative analysis of each omics data type within each of the assigned CCCRC subtypes.

By applying solid computational methods to a compendium of published RNAseq datasets (n~1500 tumours), they found that tumour samples from colorectal cancers clustered into 4 subtypes ("CCCRC" subtypes) on the basis of 61 pre-defined gene expression signatures. These subtypes correlated with but did not correspond to, the previously described Consensus Molecular Subtypes (CMS) of colorectal tumours.

Other types of molecular data were available for some tumours, obtained from the same published resources: whole-slide images, mutations, tumour proteomics, and/or scRNAseq. The authors reanalysed these datasets using standard methods and drew correlations with the CCCRC subtypes they had assigned in this work. To (semi-)quantify immune infiltration characteristics from whole-slide images (WSI), they additionally performed automated segmentation in addition to review by pathologists, which in combination produced a convincing WSI-derived dataset.

In combination with existing CRC classifications, this study could facilitate future biomarker discoveries. This appears to be the authors' main claim, and the data and methods broadly support this claim.

Some aspects of the work need to be clarified:

This work relies on the definition of 4 clusters of CRC tumours based on consensus clustering of the 61 gene lists, which in turn depends on the choice of clustering method and the choice of gene lists. Sufficient detail is provided about the gene lists and resulting clusters, but this paper does not show how robust the 4 clusters are to these choices; for example, the "Energy" gene list appears to be a relatively strong component of clusters C2 and C3.

The authors examined whether their CCCRC classification showed differential disease progression in available retrospective cohorts of people treated with anti-PDL1 therapy. The authors presented this work as "significance of CCCRC in guiding the clinical treatment of colorectal cancer", but the data presented in this section cannot support clinical treatment decisions, which would require prospective studies and clinical trial designs. However, this section is potentially useful for generating hypotheses about potential biomarkers related to the CCCRC subtypes, and might, in the future with additional evidence, contribute to the design of a trial. The authors point out that additional experimental evidence would be required.

Other prognostic or predictive clinicopathological variables for colorectal cancer are not discussed in detail in the present work but are important for further work on the prognostic and predictive value of CRC molecular subtypes and biomarker derivation. Discrepancies in treatment response have previously been observed in separate CRC trials of biologically targeted agents with different chemotherapy backbones and other authors have suggested that treatment interactions with the tumour microenvironment might in part explain these discrepancies (e.g. Aderka (2019) PMID:31044725, and others).

Reviewer #2 (Public Review):

This paper provides an important and insightful investigation into patterns of activations that emerge in external task states. The authors use state-of-the-art methods and novel analytic approaches to establish that deactivations in the default mode network during external tasks are driven by activity in brain regions that are important in the current tasks (such as the visual or dorsal attention networks). It will be important in the future to understand whether this is a symmetrical phenomenon by studying this behaviour in states that maximize activity within the default mode network and also drive reductions in networks that are not relevant to these situations.

Reviewer #2 (Public Review):

Since this study is a long-term cohort study in children and adolescents, it is advisable to decide whether to highlight differences by age group or to show consistent effect after exposure. In particular, obesity and related diseases are closely related to socio-economic environmental factors, and its impact might be different according to age (group) at exposure.

The part described in comparison with previous studies is a good attempt. However, some results are consistent with those of previous studies and some are not. This may be related to the time difference in socio-economic environmental factors rather than simply the difference between the West and China (Hong Kong). According to modernization/urbanization, changes in living environment, changes in family relationships, and changes in the care environment can also be factors especially in children.

In studying the effect of environment on gene expression, it can be thought that the influence of genes and the degree of expression might be different depending on the age of the subject (newborn, infant, infant, adolescent, adult) duration of exposure and these still need to be elucidated.

Reviewer #2 (Public Review):

In the manuscript, Chen and colleagues reconstituted the minimal system that indicates the coupling of PSD condensates with actin polymerization. While the functional connection between the assembly and dynamics of PSD and actin was known, the molecular mechanism remained elusive. Using a series of elegant biochemical reconstitutions and in-vitro assays complemented with analysis in living cells and primary neurons, the authors characterized whether PSD condensates of Homer-1, Shank-3 and SAPAP/GKAP are sufficient to induce F-actin bundling. Furthermore, they dissected the positively-charged Arg patch within EVH1 domain of Homer to be crucial for the F-actin bundling. Postsynaptic CaMKII and a short isoform of Homer, Homer1a, can both attenuate this process, suggesting various mechanisms neurons can regulate this process. Overall, the topic is timely, the study is well-designed, and the assays are clearly executed. However, several aspects need to be experimentally addressed, including some important controls:

1. It is well established that molecular crowding plays a crucial role in F-actin bundling. For example, in the reconstitution assays in Fig.1, the authors use 10 µM of each component of PSD (total of 60 µM), to which 5 µM actin is added. Yet, in their control assays (Supp. Fig. 1), only 10 µM of each protein was checked with the same amount of actin. A control is missing where the total protein crowding would be preserved, for example, by adding BSA or protein to mimic non-specific protein crowding.<br /> 2. Is the F-bunding observed under these physiological ratios of PSD proteins and actin? For instance, a recent quantitative study (PMID: 34168338) suggests actin:Homer-1 is 200:1 or 100:1, which is in stark difference from the 1:2 molar ratio used in the study. The protein concentrations (molar ratios) need to match the physiological.<br /> 3. In the cell migration assays, it is somewhat unclear to what extent the interaction is direct. For instance, co-sedimentation at ultra-speed (100,000 g) was used to suggest a direct binding of EVH1-GNC4 fusions (Homer1, Enah) with F-actin. The control that needs to be included is a protein known not to bind to F-actin incubated under the same conditions (salt concentration, duration of incubation) and spun down at 100,000xg. This is important to exclude that the tested proteins non-specifically entangle into F-actin without specifically binding to it, particularly at such high speed.<br /> 4. The imaging assay in hippocampal neurons uses an increased spine head size as a proxy for F-actin bundling. However, one needs to be careful as the baseline includes soluble mCherry, which is both much smaller in size and does not specifically enrich the spines. The image of Homer 1 R3E shows overall lower localization at the spines. Thus, one cannot exclude that the spine enlargement upon overexpression of Homer 1 wt and R3E+EN is not primarily driven by their overall enrichment in the PSD phase. A suitable control for this assay would be mCherry-tagged PSD95, which would localize to the spines yet is not directly involved in F-actin bundling.

Reviewer #2 (Public Review):

This work is a cross-validation of an x-ray tomography technique (SAXS) and an optical microscopy technique (SLI) for imaging axonal orientations ex vivo. These innovative methods were introduced in recent papers by the authors, who have teamed up here to compare them side-by-side on the same tissue samples for the first time. The two methods are both label-free (do not require staining) and they are quite complementary. SAXS can provide full 3D orientation measurements on intact tissue, but it operates at a mesoscopic resolution and requires access to a synchrotron. SLI can measure the orientations of multiple fascicles per voxel at a microscopic resolution and relies on more widely accessible equipment, but its accuracy suffers for fiber orientations perpendicular to the imaging plane and it requires tissue to be sectioned before it is imaged. Therefore it makes a lot of sense to explore the complementary strengths of these two techniques, and to use one to "fill in the blanks" of the other. The paper also compares the orientation measurements obtained with SAXS and SLI to those obtained with diffusion MRI. The latter provides only indirect measurements based on water diffusion, at a mesoscopic resolution somewhat lower than that of SAXS, but has the benefit of being feasible in vivo.

A limitation of this study is that conclusions on the comparison between SAXS and SLI are drawn from only 2 sections of a partial monkey brain sample and 2 sections of a partial human brain sample. Conclusions on diffusion MRI are drawn only on the 2 human sample sections. This is particularly an issue for the comparison to diffusion MRI, as the diffusion MRI voxels are wider than the section thickness, hence one cannot preclude that any orientations detected with diffusion MRI but not with SAXS and SLI come from the portion of the voxel that is missing from the corresponding SAXS/SLI section.

The stated aim of the paper is to provide a framework for combining the complementary benefits of SAXS and SLI, rather than simply presenting the results of a cross-validation study. This is a significant and ambitious aim. However, in order for this to serve as a framework, there would have to be clear prescriptions for how researchers interested in obtaining ground-truth measurements of axonal orientations would do so by using these two methods in tandem. This is not adequately developed in the paper in its present form. For example, the results show reasonable agreement between SAXS and SLI orientations when fibers lie within the SLI imaging plane and decreasing agreement for fibers with increasing through-plane inclination. How would the two methods be combined in voxels where they disagree? Would one use SLI orientations in voxels with fewer through-plane fibers and SAXS orientations in voxels with more through-plane fibers? How would voxels be assigned to each category? How would the orientation vectors from the two modalities be composed and how would the resolution difference between the two be handled? When the through-plane measurement of SLI is unreliable, is its in-plane measurement still reliable? That is if there were one mainly in-plane and one mainly through-plane fiber population, would the orientation of the former still be measured correctly by SLI? There is also considerable agreement reported here between through-plane orientations obtained with SAXS and diffusion MRI. Would this mean that diffusion MRI itself could be used to supplement SLI with through-plane orientations? Any clear set of prescriptions along these lines would represent a framework for imaging orientations by combining modalities. This, however, would require detailed steps for how to perform the combination and use the multi- vs. uni-modal framework to reconstruct connectional anatomy.

A key advantage of SAXS is that it can be performed on intact samples, i.e., before any nonlinear distortions of the tissue are introduced by sectioning. Thus it can provide an undistorted reference, with contrast on axonal orientations that would be absent in, say, a structural MRI of comparable resolution. This contrast could be used to drive registration of the distorted SLI sections to an undistorted SAXS volume, and therefore is a key way in which the two techniques can complement each other. Here, however, this is not explored, as SAXS is performed after sectioning. It is not clear if this is the authors' prescription for how a combined SAXS/SLI framework would be implemented, or if it was done specifically for this study. First, it would seem that SAXS on the intact sample would be lower maintenance, requiring less setup time and hence potentially less overall beamtime than performing SAXS on each section separately. This would make it more practical for routine deployment beyond a few sections. Second, because the SAXS data are now nonlinearly distorted, they cannot be affinely aligned to the MRI volumes. While, in principle, performing both SAXS and SLI on the sections may facilitate the comparison between the two, having to unmount, rehydrate, and remount the sections in between may negate this advantage, as now there is no guarantee that SAXS and SLI can be affinely registered to each other. Here all these registration steps are performed affinely, so it is unclear to which extent the computed errors between modalities are characterizing the inherent limitations of the respective contrasts, or limitations of the registration technique. Some of the alignment is performed manually, for example, specific regions of the images are realigned by hand, and the slice of the diffusion MRI volume that is aligned to the SAXS/SLI sections is chosen by hand. Again, for this to serve as a framework that can be deployed on whole samples, there would have to be clear prescriptions for how to perform these steps robustly, how to ensure that the MRI can be acquired in a coordinate frame parallel to the sections, etc.

Finally, the paper puts forth a general conclusion that diffusion MRI overestimates the number of fiber populations per voxel, on the basis of small ODF peaks appearing perpendicular to the main ODF peaks. Of all conclusions in the paper, this is the least convincingly supported by evidence. First, these small perpendicular peaks are a known artifact, which would be typically eliminated by ignoring ODF peaks below a certain amplitude, a common practice in diffusion tractography algorithms. The authors refrain from using an amplitude threshold, with the rationale that it may also remove true diffusion orientations. However, they apply a threshold when they detect SLI peaks (a rather stringent 8% of the maximum). Second, the explanation that these artifactual peaks may appear due to vessel walls is not convincing. Vasculature is sparse. A single vessel wall will not impact the diffusion signal in the same way as a bundle of parallel axons. In an axon bundle, water molecule displacements are restricted in all directions except parallel to the axons. A single vessel wall in a voxel will not have the same effect on displacements (which are much smaller than the size of the voxel). From Figure 5, it looks like there would be at most 1-2 of these vessels in a diffusion MRI voxel, and they would not be in all voxels. This cannot explain the widespread appearance of these small artifactual peaks. Third, many ODF reconstruction methods have parameters that can be adjusted to make these artifactual peaks more or less prominent. The default parameters may be optimal for in vivo but not ex vivo data, due to the effects of fixation. In light of these concerns, I would caution against making such a general statement about all diffusion MRI in the human brain, especially on the basis of a single diffusion reconstruction method applied to a single location in one brain.

Reviewer #2 (Public Review):

Londoño-Nieto et al. investigated the influence of temperature on the form and intensity of sexual conflict in Drosophila melanogaster. They aimed to test the effect of naturally occurring temperature fluctuations on a wild population of Drosophila while disentangling pre- and post-copulatory episodes of sexual conflict. To this end, they exposed females to males under monogamy or polyandry, hence manipulating the degree of male harm experienced by females. The effect of temperature was explored by exposing these groups to 20, 24, or 28{degree sign}C. They found that female fitness suffered from male harm most at 24{degree sign}C and less at the other two temperatures. Interestingly, pre- and postcopulatory episodes of sexual conflict were affected differently by temperature. Overall, these data suggest that the relationship between sexual conflict and temperature can be strong and complex. Hence, these results can have important implications for the impact of sexual conflict on population viability, especially in light of the climate crisis.

This paper tackles a highly relevant question using an established model organism for sexual conflict and contains a rich dataset obtained using a series of carefully planned experiments and analysed in an appropriate way. Importantly, the authors used biologically meaningful temperatures and mating treatments, which increases the relevance of the data. The main conclusions are well supported by the data. Nevertheless, the devil is in the detail, and given the way the authors frame their study (i.e. testing a natural population under naturally occurring temperature fluctuations) and their results (i.e. sexual conflict is buffered by temperature effects in the wild) there are some limitations to be considered:

1) The authors frame their study as addressing the question of how sexual conflict reacts to naturally occurring temperature fluctuations in the wild. Nevertheless, the population used in this experiment had been kept for nearly 3 years in the laboratory prior to the experiment. Importantly, the authors ensured that the laboratory population maintained genetic diversity, by regularly crossing wild lines into it. Nevertheless, this population remained for some time in the laboratory under standardized conditions. The applied temperature fluctuations are in a biologically meaningful range (though only during the reproductive season), but it remains unclear if the applied fluctuations were in a standardized way (i.e. pre-programmed) or included random fluctuations (i.e. a more natural setting). This laboratory setup has certainly clear advantages, for example, it enables the exclusion of any effects other than the temperature on sexual conflict. Nevertheless, how these will then ultimately play out in the wild could be a different story.

2) The authors highlight clearly that temperature fluctuations in the wild might play an important part in how sexual conflict plays out in natural populations. This very interesting and highly relevant point might lead the reader to assume that this is what was actually tested in the experiment. Nevertheless, in the experiments, different constant temperatures were applied to the flies, while only the stock population was kept at a fluctuating temperature regime. Hence, the influence of fluctuations during episodes of sexual conflict remains untested. While the present data show that sexual conflict can be modulated by temperature, the effect of naturally occurring fluctuations on the net cost of sexual conflict to a population remains unclear.

3) The authors conclude that the effect of sexual conflict can be buffered by temperature in the wild. In general, I agree with this, although a more conservative way of framing this would be to say that temperature modulates or moderates sexual conflict instead of buffers it. If there really is a buffering effect of temperature in the wild remains to be tested, I believe. This will depend on how actual changes in temperature affect this dynamic (see point 2). In addition, I think another interesting open question is what the mechanism behind the observed differences might be. Are male and female interests really more aligned at different temperatures (i.e. males plastically reduce harm)? This would really buffer the harm of sexual conflict at those temperatures. Nevertheless, alternatively, males might not be perfectly adapted to manipulate the female optimally at lower or higher temperatures. This would mean that if the temperatures change, males might evolve to increase the manipulation of females, and hence the scope for sexual conflict might not change in the end under this scenario. Nevertheless, as the authors themselves state: 'An intriguing possibility is thus that SFPs are more effective at lowering female re-mating rates at warm temperatures, thereby buffering these costs.' Therefore, a temperature-dependent increase in the effectiveness of male manipulation might counterintuitively reduce sexual conflict in this species.

4) In the end the authors argue that the climate crisis might have 'unexpected positive consequences via its effect on male harm'. Sexual conflict is indeed widespread, but it takes many different forms (as has been nicely described in the introduction of this paper). Because the studied system seems to be quite a specific example, it is questionable how far spread this phenomenon is in nature. In addition, it remains unclear how male harm will evolve in response to the climate crisis (see point 3). Finally, the relative fitness of females increased in the present experiment, as the tested range was within the reproductive optimum of the species. Nevertheless, the relative importance of the positive effect of sexual conflict on fitness outside of optimal temperatures seems questionable.

Nonetheless, I believe these results to be of exceeding interest to the scientific community and of importance to the field. It opens up many potential research directions and adds further data to the fascinating field of sexual conflict, SFPs, and male harm in Drosophila.

Reviewer #2 (Public Review):

The paper starts with the premise that given the broad immature connectivity between the retina and thalamus during development, locally homogeneous synaptic currents should generate precise spike correlations (on a millisecond timescale) which are not seen in development and could be bad for developmental refinements and "network diversity". Rather, the correlations during development are over much longer timescales. The authors propose that two main factors, the dominance of NMDA (over AMPA) currents and the absence of recurrent connections prevents these precise correlations and preserves diversity.

The paper consists of three parts: (I) develop a biophysical model for a thalamic neuron, (II) use the model to determine which factors govern precise correlations, and then (III) simulate a cortical network and demonstrate loss of network diversity when precise correlations are used. While all parts are interesting, there are several claims in each (and the links between them) that are not fully justified.

What is commending about the paper is that it is one of few theoretical/modeling papers that focuses on neural circuit development and it manages to link experimental results to principles of circuit function. The authors apply quite a few modeling approaches ranging from single-neuron models (including building a database of thalamic neurons based on experimental data) and network models. Some of the claims regarding the timescales of correlations (long in development) are unjustified because the authors use a fixed-timescales kernel to compute these correlations and mainly investigate their amplitude or level, not their timescales. It is also not clear how important is the heterogeneity among thalamic neurons. Are the effects on the correlations the result of NMDA currents or the neuronal diversity from their database? Are precise correlations generated because of the diverse/heterogeneous neurons, or because of the levels of convergence? What happens to homogeneous neurons? The authors also propose that precise correlations impair network diversity but never show this impairment directly beyond a diversity of excitatory-to-excitatory connection strengths. If the authors were to clarify these issues then the paper could be a valuable contribution to the field of developmental systems and computational neuroscience.

Reviewer #2 (Public Review):

The overall objective of this paper is to characterize the cells that are responsible for producing the secretions of the parasitic larvae, Brugia malayi. This parasite is a human pathogen that is one of three responsible for lymphatic filariasis/elephantiasis a disease that threatens half of the world's population. The specific focus of this work is protein secretions made by the parasites. In general, it is well-known that parasitic worms can manipulate and evade host immune immunity via secreted products. Studies have focused on the activities of these secretions and specific molecules. What is lacking is a detailed description of the identity and anatomical location of the cells that produce them. This is especially important as these cells are the target of different classes of anthelminthic drugs. This knowledge could allow new strategies to target these pathogens and to better understand the mechanism of actions.

To better understand this important topic, this manuscript describes a method to dissociate cells of the pre-larval stage (microfilariae) of the human parasitic filarial nematode, Brugia malayi. This method is then used to create an atlas of cells based on the expression profiles of individual dissociated cells. Cells are grouped into clusters with similar patterns of expression using single-cell mRNA sequencing analysis pipelines and the clusters are defined by using a combination of known functionalities based on the well-established, free living, soil nematode, C. elegans, and different functional classifications based on genes of interest. These include known antigens as well as targets of 3 classes of anthelmintic molecules. Using the scRNA-seq data, clear hypotheses can be made about ion channel and structural protein composition, the putative targets of the anthelminthics. Finally, it is proposed that the dissociated cells can be cultured which can facilitate future studies since cell lines or primary cultures of cells from filarial worms are not available.

This paper represents a huge undertaking on an important and understudied area. The authors have taken on a major challenge to gain novel insights, and to provide data and protocols for the field to use. The data are well-presented and support the conclusions of the work. The authors have broadly achieved their goals and the data generated and methodology will be important for the community.

Reviewer #2 (Public Review):

The manuscript is rigorous and clearly written; the experiments are well described, and the conclusions are consistent with the experimental results. Particularly interesting are the data demonstrating the role of cytoneme-like structures. The microscopy images supporting the experimental data are clear and fascinating.<br /> The work is, in my opinion, well conducted.

Reviewer #2 (Public Review):

In this manuscripts, the authors investigated differential role of two closely related proteins, S27 and S27L , which are one of the subunits of ribosome. Ribosomes containing each protein associate with a distinct set of mRNAs, suggesting that ribosomes in the cells play distinct roles depending on which subtype of S27 subunits they contain. The authors also performed functional analyses using mutant mice, and demonstrated that functions of S27-containing ribosome can be rescued by S27L-containing ribosome and vice versa. These findings provide new experimental insights into the origin of family genes fixed during the course of evolution.

Reviewer #2 (Public Review):

In this manuscript, the authors characterize the extent of RNA transfer between cells in culture, with an emphasis on trying to identify RNAs that are transferred through tunneling nanotubes (TNTs). They use an in vitro human-mouse cell co-culture model, consisting of mouse embryonic fibroblasts and human MCF7 breast cancer cells. They take advantage of the CD326 cell surface molecule, which is specifically expressed on MCF7 cells, to separate the two cell populations using magnetic beads conjugated to anti-CD326 antibodies, followed by deep sequencing to identify human RNAs present in mouse cells. They identify many 'transferred' RNAs. Further analysis of sequencing data together with experiments using synthetic reporters indicate that RNA transfer is non-selective, that the amount of transfer strongly correlates with the level of expression in donor cells, and does not appear to require specific RNA motifs. The authors also note that co-culture with MCF7 cells leads to significant changes in the MEF transcriptome.

The experiments are overall carefully designed, and the data are clearly and quite carefully presented to point out limitations in interpretation and to distinguish speculations from experimental conclusions. It should however be kept in mind that it is unclear to what extent these limitations influence the conclusions reached. For example, the identification of transferred RNAs relies on the purity of the isolated cell populations and, while the authors provide some supporting evidence for this, nevertheless potential caveats remain. For instance, the isolated MEF samples used for analysis appear to lack single MCF7 cells, but still contain components, labeled as 'double stained' and 'unstained' cells, which are uncharacterized. The authors present some arguments as to why these would not contribute to 'transferred' reads, but given the low level of detectable transferred RNAs, and the unclear origin of these components, whether they influence the results could be debatable. Furthermore, the small number of replicates (2 replicates for the genome-wide studies and 1 replicate for most of the subsequent experiments) minimizes the confidence in the conclusions. In this context, it is also notable that the profile of transferred RNAs between the two replicates of co-cultured samples appears quite different by PCA analysis. It is thus conceivable that there might be specificity in the RNA 'transferome', influenced by unknown experimental variables, which is though masked when averaging those samples in subsequent analyses.

While the manuscript emphasizes the role of TNTs in RNA transfer, the actual involvement of TNTs relies solely on the observation that potential TNTs form between co-cultured cells. Other means of transfer, such as through engulfment or phagocytosis of cell fragments, could still possibly contribute. Furthermore, the dependence of mRNA transfer on direct cell-to-cell contact is demonstrated for 5 RNAs and extrapolated to transcriptome-wide RNA transfer, an assumption which might, or might not, be valid.

Finally, the results on gene expression changes induced by co-culture (Figures 7, 8) are of unclear relevance. As the authors point out, it is uncertain whether RNA transfer or other paracrine or adhesion-mediated signaling events, underlie these changes. It is therefore not easy to see how these results relate to the rest of the presented work. Furthermore, while the authors expand on the potential significance of changes observed in genes related to cancer-associated fibroblasts or to immunity-related genes, these remain speculative and untested.

Overall, the manuscript presents evidence indicating that RNA is transferred non-selectively in co-cultured cells, under specific conditions and between the cell types tested. The impact of the work is reduced by the lack of mechanistic understanding underlying this transfer and the uncertainty of whether this phenomenon has any subsequent physiological relevance.

Reviewer #2 (Public Review):

This manuscript by Laturney et al. has found a previously uncharacterized neural link between female mating status and upregulation of sugar intake in the common fruit fly, Drosophila melanogaster. Although mated female flies have been known to increase both yeast and salt intake compared to virgin females, changes in sugar intake have not been previously described. Using quantitative monitoring of food intake, functional calcium imaging, connectome tracing, and neuronal manipulations, authors convincingly demonstrated that the Sex Peptide sensory neurons (SPSN) and their downstream neural circuit control the activity of female-specific Lgr3 neurons in a mating-dependent manner. In virgin females, the SPSN circuit (including its output pCd-2 neurons) is active, which is predicted to inhibit hunger-promoting Lgr3 neurons. After mating, the SPSN circuit becomes silent, which should disinhibit Lgr3 neurons. Indeed, they found that optogenetic silencing of pC2-d neurons promoted sucrose consumption. The newly characterized pCd-2 neurons are sexually dimorphic, consistent with their role in female-specific post-mating modulation of sucrose consumption.

Aside from the novelty of the mating-dependent changes in sugar intake, an exciting discovery of the current study is that separate circuits control different aspects of post-mating behavioral changes (increased egg-laying, mating rejection, increased sugar consumption). This finding illustrates a general neural mechanism by which a single "internal state" exerts its influences on multiple behaviors via branches of circuits from a hub for the given state (pC1 for the female mating status), which is a powerful mechanistic model for other internal states.

The high-quality data based on elegant yet rigorous experiments deserve praise as a textbook example. They presented multiple independent lines of evidence to demonstrate the function of each component of the SPSN circuit over the sucrose consumption Lgr3 neurons, which convincingly proves that the pCd-2a/b neurons transmit information of mating status to a hunger-controlling hub. Experiments have been exceptionally rigorous. Genetic manipulations were performed with multiple controls. They used multiple split GAL4 lines to target specific classes of neurons to eliminate the neuronal off-target effect. They also used multiple types of feeding assays to clarify the feeding phenotype induced by mating. Overall, the scientific rigor of this work sets a standard for researchers in the field to follow.

That the activity levels of pCd-2 neurons and their downstream Lgr3 neurons are indeed influenced by mating has not been directly tested. Since multiple previous publications consistently demonstrated that the SPSN-SAG-pC1 axis is suppressed by the Sex Peptide, the authors' conclusion that pCd-2 neurons are suppressed after mating (for example, see line 319) is very likely correct. However, what the authors showed was that silencing of the SPSN circuit "can" increase sucrose consumption in virgin females. To what extent mating suppresses pCd-2 neurons (and disinhibits Lgr3 neurons) remains uncharacterized. The inhibition exerted by the Sex Peptide is likely partial, which might not be precisely recapitulated by the optogenetic silencing. Mated female flies show an increased preference for protein and salt. The authors' finding that they also increase sugar consumption after mating indicates that mating causes a substantial change in female feeding patterns. The current work elevates the value of Drosophila as a neurogenetic model to understand how the nervous system achieves the complex tasks of nutritional homeostasis after mating, which dramatically alters the energy allocation in many species (including mammals). Data presented in this work will advance our understanding of how females coordinate feeding priorities in a face of changing nutritional demands after mating, which is one of the fundamental questions in neuroscience.

Reviewer #2 (Public Review):

The large genetic association studies conducted in East Africa have shown that the Dantu blood group provides substantial protection against severe malaria. The proposed mechanism of protection is reduced red cell invasion resulting in reduced parasite multiplication. This hypothesis was tested in adult Kenyan volunteers infected with P. falciparum under careful monitoring. The strength of the study is that the CHMI model using a single parasite strain has few confounders and it provides a very clear answer. The data reported on the other "protective" genetic polymorphisms is also fascinating. The hypothesis that Dantu reduces merozoite invasion has some support from previous laboratory studies, but it would be useful to confirm, once invasion is successful, that intraerythrocytic growth is unimpaired (e.g. count merozoites per schizont, measure asexual cycle length etc).

Reviewer #2 (Public Review):

In this manuscript, Seelbinder et al, introduce a novel, elegant approach to study the organization of cell nuclei, which complements currently existing technology. The authors employ localized temperature gradients to move chromatin inside the nucleus noninvasively, and they study flow fields and deformations of different nuclear compartments in different experimental settings. The study is timely and should be of broad interest to a wide readership, in particular since the method can also be applied to study mechanical relationships of subcellular compartments in other cellular and extracellular systems.

The non-invasive manipulation of cell organelles in intact cells has been a challenge for decades. The new technique introduced in this study contributes to closing this important gap, enabling experiments to better understand spatial and mechanical relationships between different cell compartments. This study is a very nice example of how concepts and approaches from physics can be exploited to better understand biology.

Reviewer #2 (Public Review):

Ramesh, Liu et al. investigated the dynamics of the histone H3 lysine 27 trimethyl mark (H3K27me3) in the cerebellum during postnatal development. They profile the mark and measure gene expression at three time points (P7, P14, P60) to show that there is a global increase in the amounts of H3K27me3 genome-wide, but a generalized loss of the mark at promoters. This loss is associated with neuronal genes that become expressed in the mature cerebellum. Through conditional knockout and transcription factor analysis, they implicate the autism-associated lysine demethylase gene KDM6B in the removal of H3K27me3 at genes that become active postnatally and show that the ZIC transcription factors are candidates to mediate some of these effects. They then use pharmacologic inhibition of KDM6B and the PRC2 component, EZH2, in a granule neuron culture system to further dissect the function of these enzymes in H3K27me3 dynamics.

The authors employ multiple genomic methods to carry out rigorously controlled experiments and their conclusions are well supported by the data. The study provides fundamental insights into the dynamics of H3K27me3 during the postnatal development of circuits in the brain. In particular, the findings that substantial changes in the H3K27me3 mark continue through the later steps of cerebellar maturation (P14 to P60) and that the autism-associated gene KDM6B is involved in this process, will be of significant interest to the field.

The study has some limitations with regard to scope and mechanism. For example, given the importance of enhancers in the regulation of gene expression, the omission of any analysis of H3K27me3 at defined enhancer elements is a limitation of the study. In addition, while the observations supporting the role of ZIC proteins in the removal of H3K27me3 during gene activation are interesting, the lack of direct mechanistic analysis investigating this biology limits the strength of the conclusions that can be made about the direct function of these factors in H3K27me3 dynamics.

Reviewer #2 (Public Review):

In this work Ushio et al. combine environmental DNA metabarcoding with novel statistical approaches to demonstrate how fish communities respond to changing sea temperatures over a seasonal cycle. These findings are important due to the need for new techniques that can better measure community stability under climate change. The eDNA metabarcoding dataset of 550 water samples over two years is, I feel, of sufficient scale to provide power to detect fine-scale ecological interactions, the experiments are well controlled, and the statistical analysis is thorough.

The major strengths of the manuscript are: (1) the magnitude of the dataset, which provides densely replicated sampling that can overcome some of the noise associated with eDNA metabarcoding data and scale up the number of data points to make unique inferences; (2) the novel method of transforming the metabarcode reads using endogenous qPCR "spike-in" data from a common reference species to obtain estimates of DNA concentration across other species; and (3) the statistical analysis of time-series and network data and translating it into interaction strengths between species provides a cross-disciplinary dimension to the work.

I feel like this kind of study showcases the power of eDNA metabarcoding to answer some really interesting questions that were previously unobtainable due to the complexities and cost of such an exercise. Notwithstanding the problems associated with PCR primer bias and PCR stochasticity, the qPCR "spike-in" method is easy to implement and will likely become a standardised technique in the field. Further studies will examine and improve on it.

Overall I found the manuscript to be clear and easy to follow for the most part. I did not identify any serious weaknesses or concerns with the study, although I am not able to comment on the more complex statistical procedures such as the "unified information-theoretic causality" method devised by the authors. The section on limitations of the study is important and acknowledges some issues with interpretation that need to be explained. The methods, while brief in parts, are clear. The code used to generate the results has been made available via a GitHub repository. The figures are clear and attractive.

Reviewer #2 (Public Review):

The authors convincingly show multiple inner and outer leaflet non-protein (lipid) densities in a cryo-EM closed state structure of GLIC, a prokaryotic homologue of canonical pentameric ligand-gated ion channels, and observe lipids in similar sites during extensive simulations at both resting and activating pH. The simulations not only corroborate structural observations, but also suggest the existence of a state-dependent lipid intersubunit site only occupied in the open state. These important findings will be of considerable interest to the ion channel community and provide new hypotheses about lipid interactions in conjunction with channel gating.

Reviewer #2 (Public Review):

This is an interesting study from Admin Peng's laboratory that builds on previous work by the PI implicating Greatwall Kinase (the mammalian gene is called MASTL) in checkpoint recovery.

The main claims of this study are:

1) Greatwall stability is regulated by the E6-AP ubiquitin ligase and this is inhibited following DNA damage in an ATM dependent manner.<br /> 2) Greatwall directly interacts with E6-AP and this interaction is suppressed by ATM dependent phosphorylation of E6-AP on S218<br /> 3) E6-AP mediates Greatwall stability directly via ubiqitylation<br /> 4) E6-AP knock out cells show reduced ATM/ATR activation and quicker checkpoint recovery following ETO and HU treatment<br /> 5) Greatwall mediated checkpoint recovery via increased phosphorylation of Cdk substrates

In my opinion, there are several interesting findings presented here but the overall model for a role of the E6-AP -Greatwall axis is not fully supported by the current data and will require further work. Moreover, there are a number of technical issues making it difficult to assess and interpret the presented data.

Major points:

1) The notion that Greatwall is indeed required for checkpoint recovery hinges on two experiments shown in Figures 5A and B where Greatwall depletion blocks the accumulation of HELA cells in mitosis following recovery from ETO treatment and in G2/M following release from HU. An alternative possibility to the direct involvement of Greatwall in checkpoint recovery could be that Greatwall in HeLA cells is required for S-phase progression (as for example Charrasse et al. suggested). A simple control would be to monitor the accumulation of mitotic cells by microscopy or FACS following Greatwall depletion without any further checkpoint activation.

2) The changes in protein levels of Greatwall and the effects of E6-AP on Greatwall stability are rather subtle and depend mostly on a qualitative assessment of western blots. Where quantifications have been made (Figures 2D and 4F) the loading control and the starting conditions for Greatwall (0 timepoints in the right panel) appear saturated making precise quantification impossible. I would argue that the authors should at least quantify the immuno-blots that led them to conclude on changes in Greatwall levels and make sure that the exposure times used are in the dynamic range of the camera (or film). A more precise experiment would be to use the exogenously expressed CFP-Greatwall that is described in Figure 6 and measure the acute changes in protein levels using quantitative fluorescence microscopy in live cells. This is, in my opinion, a lot more trustworthy than quantitative immuno-blots.<br /> I also note here that most experiments linking Greatwall levels to E6-AP were done using siRNA, while the E6-AP ko cells would be a more reliable background for these experiments, especially with reconstituted controls.

3) This study has no data linking the effects of Greatwall to its canonical target PP2A:B55. The model shown in Figure 9 is therefore highly speculative. The possibility that Greatwall could act independently of PP2A:B55 should at least be considered in the discussion given the lack of experimental evidence.

4) The major effect of E6-AP depletion on the checkpoint appears to be a striking reduction in ATM/ATR activation, suggesting that this ubiquitin ligase is involved in checkpoint activation rather than recovery. It is not clear if this phenotype is dependent on Greatwall. If so it would be hard to reconcile with the default model that E6-AP acts via the destabilisation of Greatwall. In the permanent absence of E6-AP, increased Greatwall levels should inactivate B55:PP2A. How would this lead to a decrease in ATM/ATR activation? This is unlikely, and indeed Figure 5E shows that the reduction of MASTL in parallel to E6-AP does not result in elevated levels of ATR/ATM activation. Conversely, the S215A E6-AP mutant does have a strong rescue impact on ATR/ATM (Figure 8D).

5) In summary, I do not think that the presented experiments clearly dissect the involvement of E6-AP and Greatwall in checkpoint activation and recovery. E6-AP depletion has a strong effect on checkpoint activation while Greatwall depletion is likely to have various checkpoint-independent effects on cell cycle progression.

Reviewer #2 (Public Review):

This paper describes a novel synthetic lethal interaction between BRCA2 loss and the cytokinesis regulators, including ROCK. The SL effects are restricted to short-term in vitro assays, and the underlying mechanisms remain largely elusive. The impact of the work in its current form is limited.

Strengths:<br /> - A novel synthetic lethal (SL) interaction, which appears independent from the -BRCA2 SL interaction that depends on replication fork stalling and DNA damage induction.<br /> - The SL interaction is validated in a panel of genetic models of BRCA2 deficiency.<br /> - The SL interaction can be induced using clinically approved agents.

Weaknesses<br /> - The evidence that this SL interaction is independent of replication defects is not solid.<br /> - The SL interaction is based on chemical inhibitors only, with 6 out of 9 ROCK inhibitors not demonstrating the SL interaction.<br /> - The mechanisms by which ROCKi specifically affects BRCA2-defective cells are elusive.<br /> - It remains unclear what the cause of the multiple mitotic defects is.

Combined, it remains unclear if the identified SL interaction is therapeutically meaningful. Clinical stage inhibitors are available, and various BRCA2-deficient cancer models have been described, allowing the authors to address this in long-term in vitro assays and in vivo assays. Also, the authors describe multiple phenotypic consequences, but the order of events and the reason why the effects are specific to BRCA2 remain largely unclear. Furthermore, the notion that the observed effects are independent of replication defects requires further substantiation.

Reviewer #2 (Public Review):

Zheng et al., investigated the molecular and functional mechanisms of two homeodomain missense mutations causing human retinal photoreceptor degeneration diseases in photoreceptor development regulated by the CRX transcription factor. They analyzed the E80A mutation associated with dominant cone-rod dystrophy (CRD) and the K88N mutation associated with dominant Leber Congenital Amaurosis (LCA). The authors found that E80A CRX binds to the same target DNA sites as WT CRX, but the binding specificity of K88N CRX is altered from that of WT in an in vitro assay. They generated Crx(E80A) and Crx(K88N) KI mice and performed ChIP assay and observed that K88N CRX binds to novel genomic regions from the WT-binding sites, while E80A binds to the WT sites. In addition, using the KI mice, they found that E80A and K88N differently affect the expression of Crx target genes. This study is well executed with proper and solid methodologies, and the manuscript is clearly written. This study gives us the insights how single missense CRX mutations lead to different types of human retinal photoreceptor degeneration diseases.

While the study has strengths in principle, it has a couple of weaknesses. One is how well E80A KI mice function as a pathological model of dominant CRD, in which cones are mainly first affected, is not clearly shown in this study. More data investigating how cones are affected by performing histological, molecular, and physiological analyses will be helpful and useful. For example, in the Discussion, the authors describe that E80A associates with S-cone opsin promoter results is "data now shown". This data must be presented for the readers. In addition, more molecular insights as to how E80A affects cones will strengthen this study. Another point is that it will be very valuable if the authors could show how E80A and K88N differently affect the 3D structure of the CRX homeodomain. Even a simulation model would be valuable.

Reviewer #2 (Public Review):

In this work from Zhou et al., the authors address mechanisms of mitotic chromosome size scaling during development. Their approach, which employs complementary use of in vivo (Xenopus embryos) and in vitro systems (Xenopus extracts), rendered investigation of this relationship experimentally tractable and allowed the authors to convincingly demonstrate that mitotic chromosome scaling is mediated by differential loading of maternal chromatin remodeling factors during interphase. The authors show that this scaling is dependent on an increasing nucleo-cytoplasmic (N/C) and that condensin I is titrated away from chromosomes as the N/C ratio is increased. Interestingly, the authors found that spindle and nuclei did not scale with changes in N/C ratio, suggesting that although mitotic chromosome scaling correlates with spindle and nuclei scaling, it is mechanistically distinct. Complementary Hi-C analyses of chromatin architectures of both larger condensin I-rich chromosomes and smaller condensin I-poor chromosomes support a condensin-based looping model to explain the inverse relationship between chromosome-associated condensin and chromosome length, however, this model seems somewhat contrived due to inherent limitations of the approach. A characterization of an independent importin-α-dependent mitotic chromosome scaling mechanism, though potentially interesting, is too premature to be included and a bit of a non sequitur in terms of the overarching narrative and major findings of the work. Though there is some room for improvement in terms of image analysis and measurements, the work is well-written, comprehensive in scope, and addresses a fundamental biological question. Furthermore, the authors' major conclusions and substantive claims are well-supported by the experimental results.

Reviewer #2 (Public Review):

The Covid-19 pandemic has had major adverse impacts on cancer screening globally. Despite this, most prior reports have not included observations from LMICs. This paper aims to address this important gap.

Because comparable data were not available across the countries reported here, comparisons would not be appropriate, so the authors chose a case study design, which was a prudent decision and a strength of the work.

The authors make use of data from IARC's CanScreen5 reporting system, which is completely appropriate. In addition, this aspect serves to demonstrate the usefulness of the CanScreen5 system, as it can be used to support this type of study. National data were not available in all countries.

The main findings in the paper describe the early impact of the Covid-19 pandemic on cancer screening participation for the screening programs reported on in the 6 countries that were selected.

I would anticipate that, having demonstrated that this type of case study focusing on cancer screening in LMICs is feasible, this would encourage others to conduct further studies among LMICs, which would be welcomed by the field.

Reviewer #2 (Public Review):

The abstract and introduction framework asserts that ketamine's enhancement of excitatory synaptic drive in the hippocampus is presumed to underlie its rapid antidepressant effects. This is not the only, and perhaps not the primary effect mechanism suggested by prior experiments, also strongly implicating disinhibitory effects in the prefrontal cortex as necessary and sufficient to mediate antidepressant effects. Nevertheless, it is valuable to seek mechanistic motifs that provide multiple paths for explaining the seemingly counterintuitive effects where NMDAR blocker enhances excitatory transmission. These need not be conserved across brain regions and cell classes. The primary result of this study demonstrates that 1 hr-long ketamine application to cultured cells reduces calcineurin and GCaMP activity to elevate AMPA receptor subunit GluA1 phosphorylation and enhance the expression of Ca2+-permeable, GluA2-lacking (CP-)AMPARs. These observations are then evaluated in vivo, where calcineurin shows a similar response to ketamine and CP-AMPAR antagonist-abolished ketamine effects on behavior in the open field and tail suspension tests. One significant uncertainty this study helps resolve is whether GluA2-containing AMPARs are removed from synapses or whether GluA2-lacking AMPARs are inserted following ketamine administration. GCaMP imaging, FRET and glutamate uncaging assays provide a strong complement to biochemistry and in vivo data. There are several significant technical and conceptual limitations in this work, which substantially limit the extent of conclusions that can be drawn at this point.

1. The age of neurons in cell culture experiments was 14 days in vitro (DIV), representing developing cultures that are just starting to form synapses. How these effects carry over to more mature cultures or adult animals is unclear.

2. Phosphorylation analyses, forming the foundation of this work, are carried out 1 hr after ketamine treatment. This is prior to the observed clinical effects of ketamine and this point should be acknowledged. Whether and how long this effect lasts remains to be examined. If the goal is to highlight the earliest likely effects of ketamine that should precede potential clinical effects, this should be acknowledged, and in that case, the onset of effects should be clarified. At this point, the temporal features remain undersampled, with a single time-point.

3. A lower dose (50%) treatment was used to evaluate potential sex differences in ketamine effects, which is not sufficiently justified, except post hoc based on behavioral data. The discussion section does consider potential factors that can account for observed differences.

4. The 1-hr timeline to behavioral testing is fast, relative to clinical effects on behavior as well as behavioral effects measured in most studies using mouse models.

5. Tail suspension test is broadly acknowledged as an inadequate model of antidepressant effects.

6. There is no evidence from the in vivo experiments that effects in the hippocampus are due to direct actions of ketamine, as those reported for the cell culture studies. Intraperitoneal injections cannot be used to localize primary effects in vivo to the hippocampus, which would require local delivery.

7. If (MNI)-caged L-glutamate was used at 1 μM concentration, as stated in methods, this is considerably below typical concentrations reported in the literature.

Reviewer #2 (Public Review):

In this paper by Banerjee et al., the authors described the potential role of two universal stress proteins in M. smegmatis and M. tuberculosis in regulating intracellular free cAMP concentration, which was a unique observation. The experiments were logically designed to prove the expression and interactions; it would have been worthwhile to explore beyond to gain an insight into how the changing levels of free cAMP could modulate any key phenotypes in the bacteria such as virulence, antibiotic resistance, etc. in the content of knockout/knockdown and overexpression of MSMEG_3811 and Rv1636 in individual organisms. The preliminary data of natural inhibitor STOCKIN43384 impacting the survival of M. smegmatis was interesting, but authors need to prove the MOA by using knockdown and overexpression strains of Rv1636.

Reviewer #2 (Public Review):

The evolution and control of the three-part life history of holometabolous insects have been controversial issues for over a century. While the functioning of broad as a master gene controlling the pupal stage and of E93 as a master gene for the adult stage has been known for about a decade or more, chinmo has only recently been proposed as being the master gene responsible for maintaining the larval stage (Truman & Riddiford, 2022). While the former paper focused on the embryonic and early larval function of Chinmo, this paper explores its metamorphic effects and defines the roles of Broad and E93 in the phenotypes produced by manipulations of Chinmo expression.

Overall, the paper is well presented but in places, readers would be helped if the authors were more explicit about the logic and details of their manipulations. There are a couple of conceptual issues that the authors should address.

The role of Broad in larval tissues:<br /> One intriguing issue relates to the relationship of Chinmo to Broad and E93 in larval versus imaginal tissues prior to metamorphosis. The knock-down of chinmo in imaginal discs results in severe suppression of growth and the lack of metamorphic patterning genes such as cut and wingless. Normal growth and patterning are reestablished though, if broad is also knocked-down, supporting the notion that the effects of the lack of Chinmo are mediated through the premature expression of Broad.<br /> In the salivary glands, by contrast, chinmo knock-down suppresses growth, and this growth suppression is not reversed by simultaneous broad knockdown. They properly conclude that the role of Chinmo in supporting the growth of larval tissues does not involve Broad, but their data on the expression of salivary gland proteins suggest that Broad still plays some role in Chinmo function in salivary glands. Fig. 5E shows the levels of various salivary glue proteins in the glands of Chinmo knock-down larvae. The levels are reduced, as expected by the lack of salivary gland growth, but a significant finding is that they are there at all! The Costantino et al. (2008) paper shows that these genes are only induced in the mid-L3. Ecdysone, acting through Broad isoforms, is necessary for their appearance and these SGS genes can be induced in the L1 and L2 stages by ectopic expression of some Broad isoforms. Their low levels in Fig 5, would be due to the small size of the gland, but the gland's premature expression of Broad likely causes their induction. In larval cells, then, Chinmo may feed into two parallel pathways, one that does not involve broad and regulates growth and the other, utilizing Broad, regulating premetamorphic changes.<br /> It would be useful to look at early larval salivary gland proteins such as ng-1 to -3 that are expressed in salivary glands before the critical weight. Also, it would be interesting if the appearance of the SGS proteins after chinmo knock-down (Fig 5E) is abolished by simultaneous knock-down of broad.

Role of Chinmo and Broad in Hemimetabolous insects:<br /> In the conclusion of their comparative studies on the cockroach (line 342), the authors state that Broad exerts no role in the development of hemimetabolous insects. However, this conclusion is not consistent with the literature. The first study of broad knockdown in a hemimetabolous insect was in the milkweed bug Oncopeltus fasciatus by Erezyilmaz et al. (2006). Surprisingly to Erezyilmaz et al., broad knock-down in early-stage nymphs did not cause premature metamorphosis. However, Broad expression was essential for tissues of the wing pads and dorsal thorax to undergo morphogenetic growth (rather than simple isomorphic growth), and for stage-specific changes in coloration through the nymphal series (but not for the nymph to adult color change). A similar function for Broad on wing growth during the later nymphal stages was later shown in Blattella (Fernandez-Nicolas et al., 2022; Huang et al., 2013). The wing- and genital pads represent "imaginal" tissues in the nymph and the need for Broad in these tissues are the same as seen in imaginal discs as the latter shift from isomorphic growth to morphogenesis at the critical weight checkpoint in the L3.<br /> This would suggest that important roles for Broad and E93 are already established in the hemimetabolous insects with E93 controlling the shift from immature (nymphal) to adult phenotypes and Broad controlling the premetamorphic growth of imaginal tissues in early-stage nymphs. Chinmo might then be needed to keep both in check.

Reviewer #2 (Public Review):

Like humans, Bengalese finches rely on auditory feedback to maintain the acoustic stability of their learned vocalizations, and deafening causes acoustic degradation of their songs. How disruptions to sensory input alter gene expression in brain regions important for singing and song learning remains relatively unexplored. The authors develop an innovative serial laser capture RNA-sequencing method, which allows them to conduct large-scale analyses of gene expression in spatially defined singing-related regions, as well as in surrounding non-singing-related regions. These methods are used to demonstrate that deafening preferentially alters gene expression in song-related regions relative to surrounding song-related areas, and that deafening reduces correlations in gene expression between connected song-related regions. The authors then compare their findings to a previous single-cell RNA sequencing dataset to determine the cell types whose gene expression is likely to be most strongly affected by deafening and song degradation. Finally, the authors repeat their measurements of gene expression changes in RA following unilateral lesion of LMAN and find that LMAN lesions have the largest effect on groups of genes whose expression was also strongly affected by deafening. The study is elegant and rigorous, and its conclusions are well-supported. This work reveals candidate genes that may play a role in stable vocal performance and whose changes in expression may contribute to the acoustic degradation of vocal performance following deafening.

Reviewer #2 (Public Review):

Neurons of the inferior olive exhibit strong subthreshold oscillations, and drive complex spiking through climbing fiber synapses onto Purkinje cells in the cerebellar cortex. This activity plays an essential role in coordinating motor control and the induction of cerebellar plasticity. In this study, the authors make use of optogenetic and electrophysiological approaches to examine the interplay between intrinsic oscillations and two important excitatory and inhibitory input populations to the inferior olive. The authors show that excitation is enhanced when it occurs in the rebound phase of the preceding inhibition. Using a computer model, the authors also show that enhanced excitation can effectively recruit larger populations of neurons, presumably through gap junctional coupling. The strengths of the study include the authors' ability to independently control both excitatory and inhibitory pathways, as well as the rigorous and systematic examination of input timing and amplitude and their effects on spike output. There were some weaknesses; high variability in cell resting potentials raised questions about how cell health impacted the findings, and there needed to be better documentation of recording conditions and parameters. There also needed to be a more extensive discussion about the nature of input timing and frequency under behaviorally relevant conditions. Given these relatively minor issues, the study provides new insight and depth into synaptic integration in the inferior olive and adds to our understanding of how input timing is translated into climbing fiber signals.

Reviewer #2 (Public Review):

The authors report a study comparing self-reported stable and unstable knees with total knee arthroplasty. Advanced imaging methods (dynamic fluoroscopy with model-image registration) and analysis of muscle activities were used to characterize the study subjects during three ambulatory activities (level gait, downhill walking, and stair descent).

The subject cohort all received one design of TKA in a similar time period. The unstable subgroup was >60% female, while the stable knee cohort was 70% male, which is a notable limitation. The measurement methods are state-of-the-art and expertly applied.

The results suggest there may be measurable differences in knee kinematics in subjects with unstable knees, but this was not strongly supported across groups. Rather it was highlighted in 3 individuals who self-reported instability during the test session. The muscle activity analysis supports there being differences between the stable and unstable knee groups.

Despite the limitations of a small subject cohort with only a single TKA design, the study highlights important methods that appear suitable to further study the factors contributing to clinical dissatisfaction with TKA as it relates to joint stability and function during ambulatory tasks.

Reviewer #2 (Public Review):

In this manuscript, Villalobos-Cantor et. al. described a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. PhAc-OPP contains a blocking group that prevents it from incorporating into the growing peptide, and the blocking group can be cleaved off by the enzyme PGA, which is expressed in the cell type of interest.

The authors validated POPPi in different cell types in the Drosophila brain and showed that this method could be used to image general translation or to biochemically enrich nascent peptides in a cell-type-specific manner. They also showed that with an optimized labeling protocol, it is possible to achieve efficient labeling with minimum effect on animal viability and health. The authors further used POPPi to provide independent support for a previously known phenomenon: age-dependent decline in general translation in the neurons. The results of this work are solid, and the main conclusions are well supported by the data presented. The manuscript is very well written with a clear logic flow and is very easy to read.

What is less clear is how generally useful POPPi will be to the community. The authors pointed out two major cell-type specific applications of POPPi, 1) imaging general translation and 2) biochemically purifying nascent peptides. For application #1, although POPPi might be a more desirable method in some cases, a combination of non-cell-type specific labeling using OPP, and marking the cell type of interest by a fluorescent protein might be simpler. Because labeling with OPP eliminates the enzymatic step that converts non-reactive PhAc-OPP to reactive OPP, the labeling kinetics can be improved, and the toxicity associated with PGA expression can be avoided. For application #2, a currently widely used strategy for a similar purpose is various types of ribosome profiling techniques. Ribosome profiling may be easier to perform than POPPi, and because proteins cannot be amplified, a very large quantity of starting materials will be needed if one wants to use POPPi to characterize cell-type specific nascent proteome. In fact, in this manuscript, the authors used western blots to detect candidate proteins and did not use mass spec to characterize the nascent proteome.

Reviewer #2 (Public Review):

The work from Nakajima-Takagi et al describes the phenotypes and study of a PCGF1 mutant mouse model. PCGF1 is a core component of the non-canonical PRC1.1 complex and specific functions of this complex in hematopoiesis. Using somatic inactivation models, the authors demonstrate that the acute deletion of PCGF1 from adult hematopoiesis leads to a progressive myeloid bias in the bone marrow and peripheral blood. This occurs at the expense of the HSPC compartment, with a reduction in all populations and of the lymphoid committed populations. The myeloid bias is cell intrinsic, as competitive transplant of the PGCF1 deficient bone marrow recapitulates the phenotype. The effect is not due to exhaustion or loss of self-renewal of the HSCs.<br /> To understand the basis for the myeloid bias, the authors first assessed transcriptome signatures and see a shift in gene expression programs related to myeloid development and targets of the key myeloid transcription factor C/EBPa. Further analysis demonstrated an increased expression of Cebp1 in the PCGF1-deficient LSK cells. Reducing the expression of Cebpa could modify the myeloid skewing of Pcgf1 deficient cells in culture. This de-repression of Cebpa correlates with changed local H2AK119ub1 levels in the HPSC populations.

Additional studies assessed how the loss of Pcgf1 changed the response to hemoablation, in this instance with a single dose of 5-FU. This study coupled with scRNA-seq suggested that PRC1.1 was important in regulating the GMP populations, potentially through a self-renewal program. This led to a focussed analysis of the GMPs, with evidence for altered Hoxa9 and b-catenin levels contributing to the altered GMP behaviours. Both have been implicated and demonstrated to have functional roles in these programs in other studies.

Finally, ageing of Pcgf1 deficient mice demonstrated that these mice were predisposed to developing T-ALL and MPN. The authors provide a characterisation of these moribund states and their phenotypes are consistent with the diagnosis.

Overall the work demonstrates a specific requirement for Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. I think the authors largely achieved the aims and the results are supportive of the conclusions. The work shows myeloid bias, experimental evidence that this is due to a derepression of a myeloid lineage program in the HPSC and associated chromatin changes, and functions for Pcgf1 in both hematopoietic regeneration and malignancy. This suggests a unique role for non-canonical PRC1.1 compared to canonical PRC 1.

Strengths:<br /> - in vivo experiments and evidence;<br /> - multiple lines of evidence supporting the conclusion;<br /> - mechanistic studies provide direct evidence of the proposed mechanism.

Weaknesses:<br /> - can the authors demonstrate normal maturation of the myeloid lineages as this would be important to differentiate between myeloid bias and a block in myeloid differentiation? This is important to distinguish between.<br /> - include analysis of mature myeloid cells and FACS plots to allow assessment of maturation.

Reviewer #2 (Public Review):

In this manuscript, Niethamer et al. investigate the role of the transcription factor ATF3 in lung regeneration after H1N1 influenza. They focus on endothelial ATF3 which is present in a subset of lung capillaries in the adult mouse lung. Interestingly, they found that influenza infection upregulates endothelial ATF3 and that endothelial deletion of Atf3 results in impaired regeneration, leading to enlarged airspaces after viral infection. They further show that this effect may be due to an increase in apoptosis and a decrease in proliferation, suggesting that endothelial ATF3 is necessary for pulmonary vascular regeneration, as well as recovery of the alveolar architecture.<br /> Given the recent publications in the field describing lung endothelial heterogeneity, as well as its possible role in injury repair, this work is relevant to the community. It also supports the idea that epithelial-endothelial crosstalk is important for lung regeneration and proposes a potential candidate for this process.

Strengths:<br /> The authors identified and tested the role of endothelial Atf3 in lung regeneration using well-established techniques. They identified this transcription factor as a candidate using state-of-the-art scRNA-seq. They also carefully lineage traced ATF3 expressing cells using an inducible reporter before and after infection and then used a pan-endothelial driver Cdh5 to delete Atf3 specifically in the endothelium. Thus, the authors successfully show significant changes in the alveolar structure after infection in their mutant model.

Weaknesses:<br /> Although there is evidence that the author's claims have biological relevance, this paper would benefit from strengthening and/or clarifying some things:

• The scRNA-seq analysis is performed in two separate objects ("control lung" and "H1N1 infected lung 14dpi"). For these two sets of data to be comparable, the authors should have integrated the objects and analyzed them together. This is not only important for deciding the clusters' identities and making sure that the same clusters are compared between control and infected, but also necessary to compare gene expression.<br /> • ATF3 is not only present in Cap1_B, in the infected lung there seems like Cap1_A express less ATF3. The authors should comment on this difference.<br /> • It is unclear how the clusters Cap1_A and Cap1_B were decided. The manuscript would benefit from clarification.<br /> • It would be beneficial to see via immunofluorescence the morphological and spatial differences between ATF3-expressing and non-expressing endothelial cells since this transcription factor is expressed in multiple endothelial cell types.<br /> • The authors mention ATF3 is not endothelial-specific. Expression of ATF3 in other cell types should be evaluated via immunofluorescence.<br /> • The authors should have shown evidence of the deletion in their Atf3EC-KO mouse and addressed whether they had residual ATF3. If there is no antibody available, RNAscope could be used, or Western Blot or RT-PCR on sorted endothelial cells.<br /> • The authors only show the epithelium as evidence that the alveolar region is altered in their mutant after infection. The endothelium should have also been investigated, especially since their mutant is an endothelial-specific deletion. Within this, the different endothelial cells should have been assessed by a method other than RNAscope such as immunofluorescence, given that this method is unable to show morphology and there are antibodies available.<br /> • Bulk RNA-seq from endothelial cells is used in the manuscript. However, because ATF3 is not specific to Cap1_B cells or even capillaries alone, the downstream gene expression analysis of bulk RNA should be placed into the context of lung endothelial heterogeneity.<br /> • Although the authors mentioned that the infection with H1N1 influenza can have regional differences, they do not show how they picked regions for their analysis and quantification, and whether ATF3 upregulation was found in more severely affected regions. Furthermore, since they quantified via FACS, this heterogeneity in the infection itself could have affected their observations.

Reviewer #2 (Public Review):

1) A detailed step-by-step approach to validation of some previously known outcomes.<br /> 2) Useful for more focus to be placed on data from the second half of the paper.<br /> 3) Some reflection on the media used to study paracrine effects is needed - more experiments here would be beneficial.<br /> 4) Path clamp experiments - how does bath solution alter the effect of any limited paracrine effect - we are removing cells from the treatment media and putting them in physiological solutions - an opportunity to recover?

Reviewer #2 (Public Review):

This study assessed the inflammatory and metabolic profiles of a healthy sub-Saharan Africa (Tanzania) population versus a healthy population outside Africa (Dutch). Using plasma samples from these cohorts, an O-Link proteomics inflammatory panel and targeted metabolomics platforms were utilised. The study shows that 'healthy' Tanzanians display an enhanced pro-inflammatory phenotype versus Dutch volunteers. Specific pathways and metabolites identified included - increase activation of the Wnt/Beta catenin pathway, and the metabolites 4E-BP1 and FGF21. The study highlights some interesting findings regarding the impact of diet on inflammatory pathway activation.

Major Strengths & Weaknesses - This is an interesting study and approach that aims to address some challenging questions in underrepresented populations. The findings demonstrate the importance of diet and dietary interventions on metabolic health, as well as key inflammatory proteins. It does raise the question whether anti-inflammatory therapies need to be targeted to specific at-risk populations, more so than other populations.

Impact - The study demonstrates the importance of considering differences between populations and the inclusion of underrepresented populations in such studies. The data suggests that lifestyle changes in sub-Saharan Africa are potentially contributing to altered inflammatory and metabolic profiles. Thus, health initiatives advocating traditional diets may alleviate the NCD epidemic in sub-Saharan Africa.

Reviewer #2 (Public Review):

The authors combine NMR experiments, cell experiments, and molecular simulations to address the question of how lipid interactions of the prolactin receptor contribute to signalling. They assess the interactions of the disordered cytoplasmic tail of the receptor with phosphoinositides among others by chemical shift perturbations from NMR for different PIP2-containing membranes, by coarse-grained simulations, as well as site-directed mutagenesis and subsequent cell signalling experiments to monitor the activation of the mutants. A major result is that PIP2 interactions are functionally important, which so far has not been known for this receptor. Their results are likely relevant for other non-receptor tyrosine kinases.

The hypothesis that the protein complex is regulated by IDR-membrane interactions is very novel. A major strength is the close connection of and feedback between state-of-the-art experiments and simulations.

This is where I see weaknesses:<br /> 1. The motivation of focusing on LID1 is limited.<br /> 2. The data and analysis for the JAK2-PRLR complex appear somewhat superficial, and a connection between conformational states to their functional relevance is lacking. In fact, the majority of the simulation part of the paper is about suggesting different states of the PRLR-JAK2 complex but the states and their hypothesized functional relevance are not further taken up, e.g. by experiments, and yet presented as major results, e.g. in the abstract.<br /> 3. The connection between simulations and mutational study is not very direct.<br /> An open question is if the mutants can distinguish between the effects of PRLR-PIP2 interaction or PRLR-JAK2 interaction, even though this conclusion is still drawn from the data.<br /> 4. The conclusions drawn from the mutagenesis study (lines 547-555) are not directly supported by data. Only a partial correlation between PRLR membrane localisation and STAT5 activation is no reason to attribute the unexplained part of the STAT5 activation to PRLR-JAK2 interactions without further studies.<br /> 5. PIP2 is identified as an important regulator, with very solid support from the presented data. PIP3 is part of the model but not discussed before or as part of the results. The analysis could be similarly applied or the data directly relevant to the understanding of PIP3 plays a similar role, as interactions are likely primarily electrostatically driven.

Reviewer #2 (Public Review):

In A. Ruppel, et al, the authors study the mechanics of one cell, two cells, and cell monolayers upon a transient local activation of contractility. First, the authors characterize the tractions and stress maps (measured via Traction Force Microscopy and Monolayer Stress Microscopy, resp.) for one and two cells in the absence of contractility activation, and found a correlation between the principal stress direction and actin fiber orientation. Next, the authors use the theory of foams to infer, combining traction force data and cell geometry data, the mechanical parameters of cells like the line tension or the force of adherent fibers. Next, the authors activate contractility by means of optogenetic tools on one half of the system and quantify the response on both halves, concluding that the receiver half response is driven by active processes, increasing contractility for two cells, while fluidizing for one cell. Next, the authors estimate the level of active response in cell doublets by comparing the stress maps to numerical simulations of a thin elastic medium with anisotropic contractility. By varying aspect ratios of the H pattern, the authors find a correlation between the principal stress direction and the orientation of stress fibers and find that the previous active response is in general enhanced when the principal stress direction is perpendicular to the orientation of the fibers. Finally, these features are also found in a cell monolayer for a fixed confinement aspect ratio.

Overall, the manuscript contains a broad characterization of the steady state mechanics and the dynamical response to the activation of contractility for one cell, two cells, and cell monolayers.

Reviewer #2 (Public Review):

The authors hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. Using a number of methods, they have established a regulatory feedback mechanism of this transcription factor and the PTH receptor in osteoblastic precursors as well as showing that PrrxCre deletion of Zfp467 causes an increase in trabecular bone mass, while AdipoCre does not. Nevertheless, they have not established the actual mechanism of action of the transcription factor nor which gene it acts on in the osteoblast. They have mostly achieved their aims and the results partially support their conclusions. However, the work is descriptive and does not address the central issue of how ZFP467 acts. At present, its impact on the field is limited.

Reviewer #2 (Public Review):

This manuscript introduces a novel assay in a 'phenomics' approach to address an important aspect of S. aureus pathogenesis. The authors set out to identify mutations that arise during clinical S. aureus infections that cause a decrease in intracellular host-cell toxicity and increase intracellular persistence. To do this, they use a 'phenomics' approach. For phenotype, they quantify HeLa cell toxicity for each strain in a panel of 387 clinical S. aureus isolates. This is done by measuring HeLa cell death induced by intracellular S. aureus via propidium-iodide uptake. The whole genomes of each of these 387 isolates had previously been sequences. They use the genomic data and phenotype data to carry out a genome-wide association study (GWAS) looking for genetic signatures that correlate with reduced HeLa cell cytotoxicity. As expected, mutations in agr were the strongest locus-level signal, but the study did identify one agr-independent mutation in ausA, which was able to be independently validated, showing that the assay is robust enough to find causal mutations. The analysis is thoughtful, the assay appears robust, and I think the discussion of conclusions and limitations is mostly valid. Thus, my concerns are focused on further understanding the practical utility of the approach and whether or not the HeLa cell model recapitulates what happens in professional phagocytes. For example, it is not clear to me that this system has the statistical power to find novel, biologically relevant rare mutations without first being very mindful in selecting strains that are extremely genetically similar. It is also not clear to me that the toxicity assay captures the important features of the intracellular persistence that occurs in vivo within professional phagocytic cells. Thus, given these practical limitations and a somewhat artificial model system, the impact on the field is likely to be moderate in nature. However, the analysis and approach taken could be re-purposed to any robust quantitative phenotype, and this will certainly be of great interest to others that study bacterial evolution in clinical contexts.

Reviewer #2 (Public Review):

Krishnan, et al describe a unique and powerful approach to assessing the role of genetic variation on mitochondrial and cardiac function and health. Utilizing a panel of inbred mouse strains, on which they performed proteomics on heart samples, they measured 840 mitochondrial proteins and correlated these data to heart function using two heart stress models. This resulted in a number of correlative observations, three of which were explored in more detail to connect three specific genes to cardiac hypertrophy. This is an interesting dataset and there is clearly value in what is presented. The data were largely correlative, however, and there are only a couple of causation-oriented experiments. It's hard to adjudicate between these strengths and weaknesses in determining the overall impact of the manuscript.

Reviewer #2 (Public Review):

This manuscript reports on mermithid nematode fossils from amber which dates from the Cretaceous period. The specimens described in the manuscript consist of insects and associated nematodes which have been trapped in amber and fossilised. The nematodes have been identified as belonging to the Mermithidae family, a family of nematode worm that infect insects.

The findings of this manuscript provide an insight into the evolution history of nematodes and parasitism. Despite the ubiquity of both nematodes and parasites in extant ecosystems, fossil records of both are very rare. This is because nematodes and many parasites are soft bodied, and many are located inside their hosts' bodies, thus they rarely become fossilised. Thus, most of what is known about the evolutionary history of nematodes, and evolution of parasitism are based on what could be inferred from extant examples.

The specimens described in this manuscript provides a valuable contribution to our understanding of parasitism in the geological past. These amber specimens are a snapshot of parasite-host interactions - interactions which are commonly found in nature but are rarely captured in fossils. The identification of the specimens as mermithid nematodes are based on sound scientific reasoning. The worms' morphology and position in relation to the insects are consistent with what have been observed with extant mermithid nematodes.

Additionally, one of the values of such parasite fossils is that they provide us with insight into parasite-host combinations or interactions which may have existed throughout the geological past, but no longer exist today or cannot be inferred from extant taxa. It helps fill in major gaps in our understanding of parasitism. This was the case with the amber fossil that contained a bristletail with its nematode parasite.

Reviewer #2 (Public Review):

This very interesting study uses a combination of high channel count neural recordings and machine learning to characterize neural representations of complex natural and synthetic sounds in the inferior colliculus. The authors use deep neural networks to model sound evoked activity in a large number of IC multiunits with high accuracy in gerbils with normal hearing and hearing loss. They then use the DNNs to simulate activity evoked by a wide range of stimuli and demonstrate systematic differences in latent population representations between normal hearing and hearing-impaired animals. Models for hearing impaired animals show activity consistent with impaired representations of speech in noise. These results lay the groundwork for a potentially valuable approach to improving signal processing in hearing aids and prosthetics.

The large speech dataset and clean hearing loss effects are particularly impressive. While the approach and associated data are novel and likely to be of broad interest, there are some substantial concerns about the study. First, the authors fail to acknowledge substantial previous work on super-threshold activity in cortex of animals with hearing loss, making it appear that they overstate the novelty of the current results. There are also many cases where they fail to clearly report the details of statistics used to support their claims. Finally, while the accuracy of the DNN models is compelling for the speech stimuli in the data set, it is not clear that the comparisons of simulated activity reflect actual neural activity in the stimulus conditions tested.

Reviewer #2 (Public Review):

In the study by Li et al., the authors hypothesize that RELMa, a macrophage-derived protein, plays a sex-dimorphic role as a protective factor in obesity in females vs males. The authors perform largely in vivo studies utilizing male and female WT and RELMa KO mice on a high-fat diet and perform an in-depth analysis of immune cell composition, gene expression, and single-cell RNA Sequencing. The authors find that WT females are protected from obesity and inflammation vs males, and this protection is lost in female RELMa KO mice. Further analysis by the authors including flow cytometry of the visceral fat SVF in female WT mice showed reduced macrophage infiltration, higher levels of eosinophils, and Th2 cytokine expression compared to WT male mice and female KO mice. The authors show that protection from obesity and inflammation in female RELMa KO mice can be rescued with an injection of eosinophils and recombinant RELMa. Lastly, the authors use single-cell RNA-Sequencing to further analyze SVF cells in WT and KO male and female mice on a high-fat diet.

Overall, we find that the study represents an important finding in the immunometabolism field showing that RELMa is a key myeloid-derived factor that helps influence the macrophage-eosinophil function in female mice and protects from diet-induced obesity and inflammation in a sexually dimorphic manner. Overall, the study provides strong and convincing data supporting the authors' hypothesis and conclusion.

Reviewer #2 (Public Review):

Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

Gui et al. (2021) already reported on a cryo-EM observation in bovine tracheal cilia, with the conclusion similar to this paper in the structure of OAD/DC on DMT. Using knockout zebrafish strain, the authors present detailed interaction of calaxin with other DC components. They show that the binding of calaxin induces the changes of conformation in N-terminal region of CCDC151/114. The conformation further changes in the presence of Ca2+; specific conformation of N-terminal region of CCDC151/114 becomes undetectable, instead additional structure appears in the vicinity of calaxin.

1) The authors conclude that the Ca2+-dependent conformational change of DC is subtle and not dynamic. This result is eventually valuable information but may be somewhat unexpected from the point of view that calaxin plays an important role in the regulation of flagellar motility in Ciona sperm. The authors found that calaxin changes the conformation of N-terminal CCDC151/114 region but the core dynein structure shows no dynamic change. What about the changes in the interaction between calaxin, core dynein, and DMT? Is this beyond the resolution of cryo-ET analysis?

2) It would be very helpful if the authors could add the cryo-ET images of calaxin-/- axoneme in the presence of 1 mM EGTA in Figure 7. Although these images are thought to be similar or identical to Figure 4F, it would help to confirm that the conformational changes in CCDC151/114 and additional part of DC are induced in a Ca2+-dependent manner.

3) To clarify the molecular interaction of calaxin with other components, it would also be helpful if the authors add the images rotated 80 degree to Figure 4F and G, in similar way in Figure 7,

4) Despite the molecular phylogenetic difference, there are several similarities between calaxin and Chlamydomonas DC3, not only in the in situ structure and configuration but in the phenotype of mutants; Chlamydomonas mutant lacking DC3 shows OAD loss in the distal part of a flagellum (Casey et al, MBC, 2003). It may be a good reference if the authors add the position of DC3 in Figure 4. A', B', and C.

5) There is a significant difference in sperm motility between WT and calaxin-/- or WT and armc4-/- (Figure 2E). However, it is not clear whether immotile sperm were included in the data for VAP (Figure 2F) or BCF (Figure 2G). For example, WT and calaxin-/- show similar VAP, although both are significantly different in the percent of motile sperm.

6) In calaxin-/- mouse, OAD was clearly detected from the base to two-thirds of a flagellum with unclear border (Figure 2A). Typical distribution of OAD+class and OAD-class are shown in Figure 5 in the ~3 micrometer tomograms. Were these taken from around this unclear border? Are proximal most region of a flagellum occupied with OAD+class only? The authors should clearly indicate the region of a flagellum where the tomograms in Figure 5C and D were selected.

7) Line 229~: It is not clear what the authors meant by "probably reflecting the different distance from the sperm head". In relation to this and the comment 6, does the "proximal" in the sentence "OAD loss occurred even in the proximal part of the flagella" (line 232) indicate the region near the base of a flagellum?

8) In conjugation with comment 7, it would be appreciated to show an authors' idea on why distal region of flagella tends to lack calaxin, if they do not discuss anywhere in the text,

9) Immunofluorescence in twister-/- epithelial cilia showed that the localization of calaxin is independent of OAD (line 271-274). Based on the authors' finding, the localization of calaxin requires Armc4, which is preassembled with calaxin in the cytoplasm. If this is true and the localization of calaxin is NOT resulting from diffusion, Armc4 must be localized with calaxin along the entire length of cilia in twister-/- epithelial cilia (Figure 6D). Although Armc4 is shown localized in cryo-ET images (e.g. Figure 1, Figure 7), authors may provide the immunofluorescence of Armc4 along the entire length of sperm flagella and epithelial cilia.

Reviewer #2 (Public Review):

In this study, Rmus and colleagues contribute to the important open question of whether reinforcement learning deficits observed in older adults are due to impairments in basic learning processes, or can be attributed to a decline in working memory function. The authors present cross-sectional behavioral data from a task designed to assess the role of working memory in reinforcement learning. And they use computational modeling in conjunction with MR spectroscopy to demonstrate a relationship between prefrontal glutamate and age-related impairments in learning specific to working memory decay. I found the overall story compelling, the data novel, and the analysis carefully executed. Below I outline some areas in which the claims of the manuscript could be strengthened.

1. I may have missed this, but does glutamate correlate with other model parameters? Or did the authors only focus on the WM parameters because of the age difference? In support of the specificity argument, it would be important to show that glutamate only predicts WM related parameters regardless of whether there was an age difference or not.<br /> 2. As it is somewhat common with these tasks, it seems like the model does not fully capture the performance deficit in OA (Fig. 2B), even when all the individual difference parameters in WM are allowed to vary. Can the authors say more about the discrepancy? This is an interesting datapoint which may give clues to mechanism.<br /> 3. Relatedly, it may not be possible with these data alone, but can authors discuss what the WM decay parameter captures? In particular for OA, the distinction between generating and maintaining a "task set" has been extensively written about. Older adults tend to have difficulty internally generating and flexibly deploying task sets, but somewhat paradoxically can perform better than YA in certain decision situations (e.g. when reward is dependent on previous choices, see Worthy et. Al. 2011). The task in this study necessarily pushes OA in a regime in which relying on familiar decision strategies is sub-optimal, and task sets must be continuously generated. Is there a type of intervention do authors expect would reverse the observed deficit in WM?<br /> 4. There is a wealth of evidence suggesting striatal DA loss in older adults, which served as the basis for many of the original investigations and hypotheses regarding a simple RL deficit in OA (e.g. work by Shu-Chen Li and others). While the authors do not directly measure DA in this study, it would be helpful to place the results in the context of that literature.<br /> 5. Finally, the main argument of the paper as I read it is that PFC glutamate mediates the performance deficits observed in RL because it reflects a compromised WM system. Sample size permitting, it would be helpful to see a formal test of this mediation relationship.

Reviewer #2 (Public Review):

The manuscript by Aderounmu presents an interesting attempt to reconstruct evolution of the function of the helicase domain in ancestral Dicers, RNase III enzymes producing siRNAs from long double-stranded RNA and microRNAs from small hairpin precursors. The helicase has a role in long dsRNA recognition and processing and this function could have an antiviral role. Authors show on reconstructed ancestral Dicer variants that the helicase was losing dsRNA binding affinity and ATPase activity during evolution of the lineage leading to vertebrates while an early divergent Dicer-2 variant in Arthropods retained high activity and seemed better adapted for blunt ended long dsRNA, which would be consistent with antiviral function.

The work is consistent with apparent adaptation of vertebrate Dicers for miRNA biogenesis and two known modes of substrate loading: "bottom up" dsRNA threading through the helicase domain where the helicase domain recognizes the end of dsRNA and feeds it into the enzyme and "top-down" where the substrate is first anchored in the PAZ domain before it locks into the enzyme. Some extant Dicer variants are known to be adapted for just one of these two modes while Dicer in C. elegans exemplifies an "ambidextrous" variant. The reconstruction of the helicase domain complex enabled authors to test how well would be ancestral helicases supporting the "bottom up" feeding of long dsRNA and whether the helicase would be distinguishing blunt-end dsRNA and 3' 2 nucleotide overhang. Although the reconstruction of an ancestral protein from highly divergent extant sequences yields just a hypothetical ancestor, which cannot be validated, the work provides remarkable data for interpreting evolutionary history of the helicase domain and RNA silencing in more general. While it is not surprising that the ancestral helicase was a functional ATPase stimulated by dsRNA, particularly new and interesting are data that the decline of the helicase function started already at the level of the common deuterostome ancestor and the helicase was essentially dead in the vertebrate ancestor. It has been reported two decades ago that human Dicer carries a helicase, which has highly conserved critical residues in the ATPase domain but it is non-functional (10.1093/emboj/cdf582). Recently published mouse mutants showed that these highly conserved residues are not important in vivo (10.1016/j.molcel.2022.10.010). Aderounmu et al. now suggest that Dicer carried this dead ATPase with conserved residues for over 500 million years of vertebrate evolution.

I do not have any major comments to the biochemical analyses and while I think that the ancestral protein reconstruction could yield hypothetical sequences, which did not exist, I think they represent reasonable reconstructions, which yielded data worth of interpretations. My major criticism of the work concerns clarity for the readership and interpretations of some results where I wish authors would clarify/revise the text. The following three examples are particularly significant:

1) It should be explained to which common ancestor during metazoan evolution belongs the ancestral helicase AncD1D2 or at least what that sequence might represent in terms of common ancestry during metazoan evolution.

2) This is linked to the first point - authors work with phylogenetic trees reconstructed from a single protein sequence, which are not well aligned with predicted early metazoan divergence (https://doi.org/10.1098/rstb.2015.0036). While their sequence-based trees show early branching of Dicer-2 as if the two Dicers existed in the common ancestor of almost all animals (except of Placozoa), I do not think there is sufficient support for such a statement, especially since antiviral RNAi-dedicated Dicers evolve faster and Dicer-2 is restricted to a few distant taxonomic group, which might be better explained by independent duplications of ambidextrous ancestral Dicers. I would appreciate if authors would discuss this issue in more detail and make readers more aware of the complexity of the problem.

3) Authors should take more into the account existing literature and data when hypothesizing about sequences of events. Some decline of the helicase activity is apparent in AncD1DEUT suggesting that it initiated between AncD1D2 and AncD1DEUT. This implies that a) antiviral role of Dicer was becoming redundant with other cellular protein sensors by then and b) Dicer was already becoming adapted for miRNA biogenesis, which further progressed in the lineage leading to vertebrates to the unique top-down loading with the distinct pre-dicing state where the helicase forms a rigid arm. Authors even cite Qiao et al. (https://doi.org/10.1016/j.dci.2021.103997) who report primitive interferon-like system in molluscs - this places the ancestry of the interferon response upstream of AncD1DEUT and suggests that this ancestral protein-based system was taking over antiviral role of Dicer much earlier. In fact, a bit weaker performance of AncD1LOPH/DEUT combined with the aforementioned interferon-like system and massive miRNA expansion in extant molluscs (10.1126/sciadv.add9938) suggests that molluscs possibly followed a convergent path like mammals. While I am missing this kind of discussion in the manuscript, I think that the model where "interferon appears ..." in AncD1VERT (Fig. 6) is incorrect and misleading.

Reviewer #2 (Public Review):

In this work, Cunha et al provide an insightful and exhaustive analysis of the role of hypoxia and HIF-1a for T cell activation and function. The work contributes to the field by showing that transient hypoxia occurring simultaneously with T cell stimulation (antigen recognition) induces an effector program in T cells that results in increased cytotoxicity in vivo and in mouse models. Importantly, the induction of this effector phenotype is not necessarily linked with an increase in proliferation in vitro, and in vitro differences are mostly observed upon antigen re-challenging.

The major strengths of the work are the use of different complementary methods to modulate HIF-1a (low oxygen conditions, inhibition of PDH by FG-4592, and deletion of VHL) and the combination of mouse and human models, especially addressing how to implement the findings to the production of CAR-T cells. Besides, the authors not only evaluate T cell function but also dive into the pathways driving the responses observed, which provides mechanistic insight.

While activation of HIF-1a through the different means mentioned before results in similar signatures in terms of T cell effector phenotype and animal response, there are some aspects that differ between the models. This is probably indicating that low levels of oxygen have other effects beyond the regulation of HIF, and that pharmacological modulation of HIF-1a might not be exactly equivalent to HIF-1a stabilization by real hypoxia.

The work is useful to better understand the discrepancies in the field, where it has been previously shown that hypoxia can have both a pro-inflammatory effect and an immunosuppressive effect on T cells. The answer proposed by the authors is that it´s a matter of timing, and not so much the magnitude of the HIF-1a response. Despite this being relatively easy to control ex vivo, the challenge occurs when considering the role of hypoxia in vivo, which probably lasts longer than the transient hypoxia needed for beneficial effects on T cells, causing T cell exhaustion.

From the translational perspective, the study suggests strategies to improve CAR-T cell therapy but also has some limitations. Despite an improvement of cytotoxicity and survival observed in mouse models upon adoptive cell transfer or injection of CAR-T cells with previously increased HIF1a levels, these approaches do not result in curation and survival is still quite low in all groups. Interestingly, improved survival with HER2 CARs exposed ex vivo to low oxygen conditions for 1 day is clear and more promising.

Reviewer #2 (Public Review):

This work formulates a detailed theoretical polymer physics model intended to explain the observed morphology of chromatin in the Drosophila cell nucleus. The model is examined in detail by both analytical calculation and computer simulation. The central premise of the suggested theory is that it is based on equilibrium statistical mechanics. Within this paradigm, authors explore the model that views chromatin fiber as a block copolymer and, most importantly, describes the role of RNA polymerase as it interacts with one of the copolymer blocks and regulates its effective solvent quality. Blocks are assumed to be fixed on the time scale of interest by, e.g., different levels of acetylation or methylation. RNA polymerase is supposed to interact only with one of the chromatin blocks, called active, and assumed interaction is quite peculiar. Namely, RNA polymerase complex may absorb on chromatin fiber and, the model assumes, the fiber decorated with absorbed RNA polymerase molecules is less sticky to itself, or more repulsive than the fiber itself. This peculiar assumption allows authors to make interesting predictions about how proteins can regulate the genome folding architecture.

STRENGTH

The work includes a rather detailed theoretical description of the model and its equilibrium statistical mechanics. As both analytical theory and accompanying simulation indicate, the assumptions put forward in formulating the model do indeed produce the desired morphology, with isolated regions ("micells") of core inactive chromatin surrounded by the less dense shell region in which RNA polymerization may potentially take place. Having such a detailed theory is potentially beneficial for the field and opens up avenues for further exploration.

WEAKNESS

The underlying assumption about the interaction of RNA polymerase complex with the fiber, although important and organic for the model, does not seem easy to justify from a molecular standpoint, especially thinking of the charges and electrostatic interactions.

Reviewer #2 (Public Review):

The paper from Marchal-Duval et al reports for the first time the important role played by the transcription factor PRRX1, expressed specifically in the mesenchyme of the lung, in the context of fibrosis. The authors used a combination of human (Donor and IPF) and mouse lungs (saline and bleomycin treated) as well as associated fibroblasts and PCLS to test the functional role of PRRX1 in the context of proliferation and differentiation induced by TGFb1. The work is supported by an impressive amount of data (7 main figures and 14 supplementary figures).

A main weakness in this work is the counterintuitive result that PRRX1 is downregulated in human lung fibroblasts (from both IPF and Donor) treated with TGFb1. Another smaller weakness is the inactivation of Prrx1 in vivo using ASO starting at d7 post bleomycin treatment.

The strengths of this work are the multiple approaches used by the authors to test the role of PRRX1 in lung fibrosis. The results are statistically solid and informative. The results presented are extremely convincing to support their conclusion that PRRX1, downstream of TGFb1 signaling is important for fibrosis.

Reviewer #2 (Public Review):

The work presented by Jordan and Keller aims at understanding the role of noradrenergic neuromodulation in the cortex of mice exploring a visual virtual environment. The authors hypothesized that norepinephrine released by Locus Coeruleus (LC) neurons in cortical circuits gates the plasticity of internal models following visuomotor prediction errors. To test this hypothesis, they devised clever experiments that allowed them to manipulate visual flow with respect to locomotion to create prediction errors in visuomotor coupling and measure the related signals in LC axons innervating the cortex using two-photon calcium imaging. They observed calcium responses proportional to absolute prediction errors that were non-specifically broadcast across the dorsal cortex. To understand how these signals contribute to computations performed by V1 neurons in layers 2/3, the authors activated LC noradrenergic inputs using optogenetic stimulations while imaging calcium responses in cortical neurons. Although LC activation had little impact on evoked activity related to visuomotor prediction errors, the authors observed changes in the effect of locomotion on visually evoked activity after repeated LC axons activation that were absent in control mice. Using a clever paradigm where the locomotion modulation index was measured in the same neurons before and after optogenetic manipulations, they confirmed that this plasticity depended on the density of LC axons activated, the visual flow associated with running, and the concurrent visuomotor coupling during LC activation. Based on similar locomotion modulation index dependency on speed observed in mice that develop only with visuomotor experience in the virtual environment, the authors concluded that changes in locomotion modulation index are the result of experience-dependent plasticity occurring at a much faster rate during LC axons optogenetic stimulations.

The study provides very compelling data on a timely and fascinating topic in neuroscience. The authors carefully designed experiments and corresponding controls to exclude any confounding factors in the interpretation of neuronal activity in LC axons and cortical neurons. The quality of the data and the rigor of the analysis are important strengths of the study. I believe this study will have an important contribution to the field of system neuroscience by shedding new light on the role of a key neuromodulator. The results provide strong support for the claims of the study.

Reviewer #2 (Public Review):

This work presents an exhaustive study of inferred functional networks from in vitro neuronal cultures across several modalities: primary rat cultures (with varying densities and longitudinal points), and human iPSC monolayers from different cell types and organoids. The authors first estimated the functional connectivity of these networks from their spontaneous activity (recorded with high-density MEAs) and then tried to find which wiring principle could better explain the observations. By deploying generative network models (with 13 different wiring principles) they observed that models with homophilic wiring principles were systematically outperforming the other ones. This proposes a universal rule for how neurons connect, which is that they tend to connect with neurons that have many common neighbors.

One of the major strengths of this study is its scope. They analyzed sparse and dense primary rat cultures at 4 different time points during development (from 7 to 28 DIV in total) as well as with the pharmacological application of GABAA blockers; 3 different cell lines: spinal-cord motor neurons, dopaminergic neurons, and glutamatergic neurons, and organoid slices. However, the big scope of this study is also one of its weaknesses, the techniques presented here to analyze the data are used inconsistently; for some preparations, there's much more detail than in others, and the constant jump between preparations and methodologies makes the findings hard to follow.

Similarly, the number of samples used in some preparations (ranging from 6 to 12) appears to be insufficient, since the study relies on multiple comparisons across the results from 13 different generative models. In many cases, it is not possible to identify which results are significant and which aren't. Most of the methodology used in this study has been used before in the context of the human connectome project (Betzel et al, Neuroimage 2016); in there they used data from 380 total participants, which made the comparisons across all the different models much more robust.

Most previous research with generative models for neural connectivity has focused on structural connectivity. In there, the link between wiring principles, energetic costs, and network topology can be made. This study, however, focuses on functional connectivity (measured by the spike time tiling coefficient), where the link between these quantities is unclear. Although the authors highlight this point in the manuscript, the constant comparisons to structural connectivity concepts and studies often lead to confusion. A clear example of this is the section where the authors explore the effect of chronic GABAA receptor blockade. It is unclear whether the authors are trying to claim that this protocol alters the development of the structural network or only the dynamics. The former could have needed additional controls.

The authors have been diligent and thorough with their statistical testing and their claims are commensurate with that. However, given the large number of different types of results and tests being presented, it is often difficult to find the corresponding explanation in the methods.

This is valuable work for experimental and computational neuroscientists studying the development of neuronal networks and the link between structural and functional connectivity. It would greatly benefit from homogenizing the results, methods, and statistics across the different experimental preparations. The conceptual similarities and differences between structural and functional wiring principles also need to be emphasized.

Reviewer #2 (Public Review):

In the present manuscript, Perrodin et al. investigated which properties of ultrasonic vocalizations determine their attractiveness for female mice. They collected a set of male courtship vocalizations and compared their attractiveness for female mice against a number of conditions, including silence, and a number of modified sequences.

The study has a clear design and used insightful modifications on the vocalization sequences, which allow the present results to be linked to previous results. The most interesting outcome of the study is that female mice prefer regularly timed sequences of vocalizations over less regularly timed sequences. This result is novel and adds to our understanding of the determinants of social communication between mice. Overall the study is likely underpowered, which was, however, hard to assess as animal numbers were largely not reported for the individual tests, and statistical analysis was carried out on the level of sessions only.

The study has a very good discussion embedding the current results with the previous literature, although the discussion steps beyond the results in a few respects, in particular when trying to determine the underlying reasons for the preference for regularly spaced sequences.

Methodologically the study is carried out at the appropriate level, although some improvements could be made to the experimental apparatus to avoid reflections.

The study will likely have a substantial impact on the field of mouse communication because the regularity of spacing has not been a focus of previous research. In addition, the confirmation that a lot of other modifications are less determining for the attractiveness of the vocalizations provides solid data on which to base future work.

Reviewer #2 (Public Review):

The eleven paralogs of SLC26 proteins in humans exhibit a remarkable range of functional diversity, spanning from slow anion exchangers and fast anion transporters with channel-like properties, to motor proteins found in the cochlear outer hair cells. In this study, the authors investigate human SLC26A6, which functions as a bicarbonate (HCO3-)/chloride (Cl-) and oxalate (C2O42-)/Cl- exchanger, combining cryo-electron microscopy, electrophysiology, and in vitro transport assays. The authors provide compelling evidence to support the idea that SLC26A6's exchange anions at equimolar stoichiometry, leading to the electroneutral and electrogenic transport of HCO3-/ Cl- and C2O42-/Cl-, respectively. Furthermore, the structure of SLC26A6 reveals a close resemblance to the fast, uncoupled Cl- transporter SLC26A9, with the major structural differences observed within the anion binding site. By characterizing an amino acid substitution within the SLC26A6 anion binding site (R404V), the authors also show that the size and charge variance of the binding pocket between the two paralogs could, in part, contribute to the differences in their transport mechanisms.

The strength of this work lies in the reductionist, in vitro approach that the authors took to characterize the transport process of SLC26A6. The authors used and developed an array of functional experiments, including two electrogenic transport assays - a fast kinetic (electrophysiology) and a slow-kinetic (fluorescent-based ACMA) - and two electroneutral transport assays, probing for Cl- (lucigenin) and HCO3- (europium), which are well executed and characterized. The structural data is also of high quality and is the first structure of an SLC26 coupled anion exchanger, providing essential information for clarifying our understanding of the functional diversity between the SLC26 family of proteins.

To my knowledge, the outward-facing conformational state has not been determined for any mammalian SLC26 paralog, which limits the mechanistic interpretation of transport and is a weaker point of this manuscript. However, this is a very minor point.

Reviewer #2 (Public Review):

In this work, the authors investigate the role of CRB3 in the formation of the primary cilium both in a mouse model and in human cells. They confirm in a conditional knock-out (KO) mouse model that Crb3 is necessary for the formation of the primary cilium in mammary and renal epithelial tissues and the new-born mice exhibit classical traits of ciliopathies. In the mouse mammary gland, the absence of Crb3 induces hyperplasia and tumorigenesis and in the human mammary tumor cells MCF10A the knock-down (KD) of CBR3 impairs ciliogenesis and the formation of a lumen in 3D-cultures with less apoptosis and spindle orientation defects during cell division.

To determine the subcellular localization of CRB3 the authors have expressed exogenously a GFP-CRB3 in MCF10A and found that this tagged protein localizes in cell-cell junctions and around pericentrin, a centrosome marker, while endogenous CRB3 localizes at the basal body. To dissect the molecular role of CRB3 the authors have performed proteomic analyses after a pull-down assay with the exogenous tagged-CRB3 and found that CRB3 interacts with Rab11 and is present in the endosomal recycling pathway. CRB3 KD also decreases the interactions between components of the γTuRC complex. In addition, the authors showed that CRB3 interacts with a tagged-Rab11 by its extracellular domain and that CRB3 promotes the interaction between Rab11 and CEP290 while CRB3 KD decreased the co-localization of GCP6 with Rab11 and γTub. Finally, the authors showed that CRB3 depletion cannot activate the Hh pathway as opposed to the Wnt pathway.

Reviewer #2 (Public Review):

The manuscript by Minkowicz et al., investigates the presence of neuronal ensembles in the striatum that may encode grooming (as a model of a naturalistic behavior). They implemented a semi-automated detection of grooming, and by recording populations of striatal cells they show that individual neurons in the striatum contain activity modulations around the start, end, or during grooming. Then using this activity they identify ensembles of cells in individual sessions/animals at the start, end or during grooming.

The behavioral tracking and recordings are remarkable, the manuscript is clearly written and the finding mostly sound with the proposed conclusions, providing original findings in the field. Nonetheless some points are raised that need further clarification

1. When claiming that the findings show encoding of transitions into or out of grooming (and duration of grooming) one could expect to see specific regressions between the neuronal activity (of individual cells or ensembles) and the parameters mentioned besides the analysis shown in figure 3 and 5.<br /> 2. Was the detection of ensembles presented in figure 4 sensible to use less than 5 seconds before/after grooming. I am thinking that 5 seconds are times that could contain behaviors that may have their own ensembles. Why 5 seconds?<br /> 3. According to Figure 2-figure supplement 1. The recordings were performed covering the lateral and in some cases the central part of the striatum. Shall it be specified along the text where the specific recordings come from?

Reviewer #2 (Public Review):

The authors embarked on a study to identify SNPs in clinical isolates of S. aureus that influence sensitivity to serum killing. Through a phenotypic screen of 300 previously sequenced S. aureus bacteremia (SAB) isolates, they identified ~40 SNPs causing altered serum survival. The remainder of the study focuses of tcaA, a gene with unknown function. They show that when tcaA is disrupted, it results in increased resistance to glycopeptides and antimicrobial components of human serum.

They perform an elegant series of experiments demonstrating how a tcaA knockout is more resistant to killing by whole serum. arachadonic acid, LL-37 and HNP-1. They provide compelling evidence that in the absence of tcaA resistance to arachidonic acid is mediated through release of wall teichoic acids from the cell wall, which acts as a decoy and sequesters the fatty acid.

Similarly, they suggest that resistance to cationic antimicrobial peptides is through alteration of the net charge of the cell wall due to loss of negatively charged WTAs based on reduced cytochrome C binding.

They continue to show that tcaA is induced in the presence of human serum, which causes increased resistance to the glycopeptide teichplanin.

They propose that tcaA disruption causes altered cell wall structure based on morphologic changes on TEM and increased sensitivity to lysostaphin and increased autolysis via triton x-100 assay.

5, Finally, they propose that tcaA influences mortality in SAB based on raw differences in 30-day morality. Interestingly they do decreased fitness during murine bacteremia model compared to wild-type.

Strengths:

1. The manuscript is well-written and easy to follow<br /> 2. The identification of SNPs leading to altered serum killing is convincing and valuable data<br /> 3. The mechanism for tcaA-mediated resistance to arachadonic acid and AMPs is compelling and novel<br /> 4. The murine infection data demonstrating that tcaA mutants exhibit reduced virulence is important data

Weaknesses:

1. Some of the conclusions are not supported by the data shown (either missing or incomplete)<br /> 2. The authors conclude that tcaA mutants show reduced peptidoglycan crosslinking. This conclusion is based on qualitative TEM images and increased sensitivity to lysostaphyin/autolysis. While these data are suggestive. it is difficult to draw such a conclusion without analysis of the cell wall by LC-MS (such as http://doi.org/10.1371/journal.ppat.1009468).<br /> 3. The authors conclude "TcaA contributes to increased disease severity in mice and humans". While it seems biologically plausible that a polymorphism known to increase glycopeptide MIC affects mortality, the human data presented is based on raw 30-day mortality numbers. It is misleading to make the association with mortality without adjusting for confounding variables known to influence mortality in SAB (e.g. age, comorbidities, presence of sepsis, endocarditis, duration of bacteremia). Also, with just 12 patients in the SNP group, this is likely underpowered to detect any difference.

Overall, I think this is a good submission and the majority of their conclusions are supported by the data. The mechanism behind the clinically relevant tcaA mutation is important, given its known role in glycopeptide resistance and therefore likely clinical outcomes. This manuscript would benefit with the inclusion of some additional experiments to help support their finding.

Reviewer #2 (Public Review):

In this work, the authors reported cryo-EM structures of four types of zinc-binding site mutants of a bacterial Zn2+/H+ antiporter YiiP, and proposed distinct structural/functional roles of each of the binding sites in the intramolecular Zn2+ relay and the integrity of the homodimeric structure of YiiP. MST analysis using the mutants with a single Zn2+-binding site at different pH further clarified the pH dependence of Zn2+ binding affinity of each site. Moreover, the inverse Multibind approach refined the CpHMD pKa values of the key Zn2+-binding residues so that they agreed with the MST data. Consequently, energetic coupling of Zn2+ export to the proton-motive force has been suggested. These findings definitely provide new mechanistic insight into this Zn2+/H+ antiporter.

Regrettably, the resolutions of the cryo-EM structures presented in this work are, overall, not high enough to describe detailed structure of some specific regions including the Zn2+-binding sites, and the density is missing for some important regions including the kinked segment of TM5. Further attempts toward higher resolution cryo-EM maps would be beneficial to corroborate their conclusions. Additionally, it may, in a sense, appear that the MD simulations have been carried out forcibly so that the outcomes are compatible with or nicely explain the experimental data. Although this is not unusual or unacceptable, I am concerned that the determined pKa values of some residues, especially of Asp residues at Site A, are unusually high. These outcomes seem to need careful interpretation and discussion.

Reviewer #2 (Public Review):

Zacharopoulos et al. investigated the relationship between MR spectroscopy-detected neurotransmitter concentrations (GABA/glutamate) in the intra-parietal sulcus (IPS) and middle frontal gyrus (MFG), behaviourally measured indices of sensory and cognitive processing, and fMRI measured functional connectivity within the frontoparietal network. They find that increased IPS glutamate concentration is related to poorer visuomotor processing in younger participants and better performance in older participants, while IPS GABA predicts the opposite pattern. They further show that these relationships are mediated by frontoparietal functional connectivity. Finally, they show that IPS GABA and glutamate concentration are related to fluid intelligence and that this relationship is mediated by visuomotor processing and moderated by the developmental stage. These data add to our understanding of the dynamic role of excitatory and inhibitory neurotransmitter systems in cognitive processes throughout development.

Strengths:

The study employs an impressively large cross-sectional, multimodal, dataset, with almost 300 participants ranging from 6 to 18+ years old.

The main finding (i.e., the interaction between GABA/Glu, visuomotor processing, and age) is found across three behavioural tasks and replicated in a second dataset collected 1.5 years after the first.

The authors extensively report the results of the numerous analyses performed in the supplementary material.

Weaknesses:

Pre-registration of experimental and analytical plans should be the norm, e.g., to reduce so-called 'p-hacking'. I am by no means asserting that this behaviour has occurred in the current study; however, it is disappointing that there is no reported pre-registration for such a large-scale study, where the selection and order of analyses (and the subsequent corrections applied) can meaningfully influence the pattern of results.

Many tests were performed in the study using frequentist statistics, and the way the results are reported makes it difficult to discern how distinguishable those that were reported as meaningful are from those that were disregarded.

Insufficient analyses were conducted to describe the relationships between a) GABA and glutamate, b) repeated behavioural measures, and c) test-retest reliability. This reduces the strength of the claims, some of which could be accounted for by simpler, potentially less interesting, explanations.

Reviewer #2 (Public Review):

The mechanisms of action potential firing were studied by whole-cell patch-clamp recordings in acute brain slices of the zebra finch. The study builds on the initial finding by Zemel et al. (2021) that the action potentials of robustus arcopallialis projection neurons (RAPNs) have an exceptional small half-duration of about 0.2 ms at 40C. The authors, therefore, set out to investigate the mechanisms of action potential repolarization. They use an impressive set of complementary techniques including voltage clamp and current clamp recordings, pharmacological interventions with classical and novel subunit-specific blockers, in situ hybridization, and comparative genomics of the KCNC/Kv3 potassium channel genes. The data convincingly demonstrate that the Kv3.1 but not Kv3.2-Kv3.4 nor Kv1.1/1.2/1.6, Kv7, or BK channels mediate the rapid repolarization. The manuscript is clearly written and the data and the presentation of the data are of the highest scientific quality. The study is of interest to a broad readership because the zebra finch is a fascinating and novel model to investigate the mechanisms of rapid motor control. The similarities of these neurons of the zebra finch with the specialized Betz cells in the motor cortex of humans and other primates demonstrates the exciting advantages of this animal model in comparison with well-established rodent models to investigate the mechanisms of complex sensory-motor control in vertebrates.

Reviewer #2 (Public Review):

This paper is an interesting and novel addition to our understanding of the link between ER stress and lipid homeostasis. Utilizing a genetic screen to determine modulators of the UPRER, Garcia, G., et al., determine C. elegans cannot activate the UPRER as strongly with knockdown of the putative hydroxysteroid dehydrogenase let-767. Additionally, let-767 knockdown results in smaller lipid droplets and changes to ER morphology. Both lipid droplet size and ER morphology size can be restored with supplementation of lipids, while the defect to UPRER activation persists. The authors elegantly show that one impact of let-767 knockdown on UPR is downstream of XBP1 splicing. The authors then go on to show that in mammalian cells, the lipid precursor 3-oxoacyl-CoA can cause a similar reduction to UPRER activation to that seen in C. elegans with let-767 knockdown. Some limitations of this study are that let-767 exact role in lipid metabolism is not well understood and it is unclear what the impact of let-767 knockdown in C. elegans has on lipid composition. It is also unclear mechanistically how let-767 is able to effect UPRER, as the authors show one potential mechanism is by blocking activation of the UPR downstream of XBP1 splicing. While the authors demonstrate that high levels of 3-oxoacyl-CoA can cause a reduction in the UPR response in mammalian cells, this finding is not recapitulated in C. elegans, nor does the study determine whether this compound accumulates in a let-767 knockdown.

Reviewer #2 (Public Review):

Aimon et al. used fast whole-brain imaging to investigate the relationship between walking and neural activity in adult fruit flies. They find that increases in brain-wide activity are tightly correlated with walking behavior, and not with grooming or flailing, and are independent of visual input. They reveal that excitatory, inhibitory, and neuromodulatory neurons all contribute to brain-wide increases in neural activity during walk. Aimon et al. extend their observations of brain-wide activity to reveal that activity in some inferior brain regions is more correlated with walk than in other brain regions. The authors further analyzed their imaging dataset to identify candidate brain regions and cell types that may be important for walking behavior, which will be useful in hypothesis generation in future studies. Finally, the authors show that brain-wide activity is similar between spontaneous and forced walk and that severing the connection between the ventral nerve cord and central brain abolishes walk-related increases in brain activity. These results suggest that increases in brain-wide activity during walking may be largely attributed to sensory and proprioceptive feedback ascending to the central brain from the ventral nerve cord rather than to top-down executive and motor control programs. The observations presented in this study suggest hypotheses that may be tested in future studies.

Strengths: This paper presents a rich imaging dataset that is well-analyzed and cataloged, which will be valuable for researchers who use this paper for future hypothesis generation. The comparison of many different reagents, imaging speeds, and behavioral conditions suggests that the observed increases in brain-wide activity during walking are quite robust to imaging methods in adult fruit flies.

Weaknesses: This study is largely observational, and the few experimental manipulations presented are insufficient to support the author's broad claims about the generation of brain-wide neural activity.

Notably, the authors suggest that their image analysis can reveal individual cell types that are important for walking by matching their morphologies to registered components from whole-brain imaging experiments. While these predictions are a useful starting point for future experiments, they have not convincingly shown that their method can identify individual cell types in genetic reagents with more restricted expression patterns. Adding further validation to show that genetically subtracting the candidate neurons from the overall expression pattern of the calcium indicator abolishes that component from the response would strengthen this claim. Furthermore, imaging the matched candidate neuronal cell type to show that it recapitulates the activity dynamics of the proposed component would add additional evidence.

In addition, increases in neural activity prior to walk onset in specific brain regions are intriguing but insufficient to demonstrate the neurons in these regions trigger walking. This claim should await further studies that employ targeted and acute manipulation of neural activity, as noted by the authors. Furthermore, that activity in these brain regions is significantly increased prior to walk onset awaits more rigorous statistical testing, as do the authors' claims that spontaneous versus forced walking alters these dynamics. The suggestion that walking increases brain-wide activity via feedback from the ventral nerve cord is an interesting possibility and would also benefit from additional experimental validation. Activating and silencing neurons that provide proprioceptive feedback from the legs and determining the effect of this manipulation on brain-wide neural activity would be a good starting point.

Reviewer #2 (Public Review):

In this study, Tomasi et al identify a series of tRNA modifying enzymes from Mtb, show their function in the relevant tRNA modifications and by using at least one deleted strain for MnmA, they show the relevance of tRNA modification in intra-host survival and postulate their potential role in pathogenesis.

Conceptually it is a wonderful study, given that tRNA modifications are so fundamental to all life forms, showing their role in Mtb growth in the host is significant. However, the authors have not thoroughly analyzed the phenotype. The growth defect aspect or impact on pathogenesis needs to be adequately addressed.

- The authors show that ΔmnmA grows equally well in the in vitro cultures as the WT. However, they show attenuated growth in the macrophages. Is it because Glu1_TTC and Gln1-TTG tRNAs are not the preferred tRNAs for incorporation of Glu and Gln, respectively? And for some reason, they get preferred over the alternate tRNAs during infection? What dictates this selectivity?

- As such the growth defect shown in macrophages would be more convincing if the authors also show the phenotype of complementation with WT mnmA.

An important consideration here is the universal nature of these modifications across the life forms. Any strategy to utilize these enzymes as the potential therapeutic candidate would have to factor in this important aspect.

Reviewer #2 (Public Review):

In this publication, the authors provide a comprehensive trajectory of transcriptional changes in Müller glia cells (MG) in the regenerating retina of zebrafish. These resident glia cells of the retina can differentiate into all neural cell classes following injury, providing full regenerative capabilities of the zebrafish retina. The authors achieved this by using single-cell RNA sequencing of Müller glia, progenitors, and regenerated progeny, comparing uninjured and light-lesioned retinae.

The isolation strategy involves using two transgenic strains, one labelling dividing cells and their immediate progeny, and the other Müller glia cells. This allowed them to separate injury-induced proliferating and non-reactive Müller glia cells. Subsequent single-cell transcriptomics showed that MG could be non-reactive under both uninjured and lesioned conditions and reactive MG give rise to a cell population that both replenishes the pool of MG and replenish neurogenic retinal precursor cells. These precursor cells produce regenerated neurons in a developmental time series with ganglion cells being born first and bipolar cells being born last. Interestingly hybrid populations have been detected that co-share characteristics of photoreceptor precursors and reactive glia.

This is the first study of its kind following the dynamic changes of transcriptional changes during retinal regeneration, providing a rich data source of genes involved in regeneration. Their finding of transcriptionally separable MG populations is intriguing.

This study focuses on the light-lesioned retina and leaves open the question if the observed transcriptional trajectories of regenerating neurons are generalizable to other lesion models (e.g. chemical or mutational lesions) or are specific to the light-damaged retina.

Reviewer #2 (Public Review):

The authors sought to characterize normal placental aging to better understand how the molecular and cellular events that trigger the labor process. An understanding of these mechanisms would not only provide insight into term labor, but also potential triggers of preterm labor, a common pregnancy complication with no effective intervention. Using bulk transcriptomic analysis of mouse and human placenta at different gestational timepoints, the authors determined that stabilization of HIF-1 signaling accompanied by mitochondrial dysfunction and cellular senescence are molecular signatures of term placenta. They also used in vitro trophoblast (choriocarcinoma) and a uterine myocyte culture system to further validate their findings. Lastly, using chemically induced HIF-1 induction in vivo in mice, the authors showed that stabilization of HIF-1 protein in the placenta reduced the gestational length significantly.

The major strength of this study is the use of multiple model systems to address the question at hand. The consistency of findings between mouse and human placenta, and the validation of mechanisms in vitro and in vivo modeling are strong support for their conclusions. The rationale for studying the term placentas to understand the abnormal process of preterm birth is clearly explained. Although the idea that hypoxic stress and placental senescence are triggers for labor is not novel, the comprehensiveness of the approach and idea to study the normal aging process are appreciated.

There are some areas of the manuscript that lack clarity and weaknesses in the methodology worth noting. The rationale for focusing on senescence and HIF-1 is not clearly given that other pathways were more significantly altered in the WGCNA analysis. The placental gene expression data were from bulk transcriptomic analyses, yet the authors do not explicitly discuss the limitations of this approach. Although the reader can assume that the authors attribute the mRNA signature of aging to trophoblasts - of which, there are different types - clarification regarding their interpretation of the data and the relevant cell types would strengthen the paper. Additionally, while the inclusion of human placenta data is a major strength, the differences between mouse and human placental structure and cell types make highlighting the specific cells of interest even more important; although there are correlations between mouse and human placenta, there are also many differences, and the comparison is further limited when considering the whole placenta rather than specific cell populations.

Additional details regarding methods and the reasons for choosing certain readouts are needed. Trophoblasts are sensitive to oxygen tension which varies according to gestational age, and it is unclear if this variable was taken into consideration in this study. Many of the cellular processes examined are well characterized in the literature yet the rationale for choosing certain markers is unclear (e.g., Glb1 for senescence; the transcripts selected as representative of the senescence-associated secretory phenotype; mtDNA lesion rate).

Overall, the findings presented are a valuable contribution to the field. The authors provide a thoughtful discussion that places their findings in the context of current literature and poses interesting questions for future pursuit. Their efforts to be comprehensive in the characterization of placental aging is a major strength; few placental studies attempt to integrate mouse and human data to this extent, and the validation and presentation of a potential mechanism by which fetal trophoblasts signal to maternal uterine myocytes are exciting. Nevertheless, a clear discussion of the methodologic limitations of the study would strengthen the manuscript.

Reviewer #2 (Public Review):

This preprint presents a compelling study examining the relationship between genotypic changes and phenotypic traits in bacteria over an extended period using the Long-Term Evolution Experiment (LTEE) as a model. The primary advances in methodology include employing high-resolution mass spectrometry for comprehensive metabolic profiling and combining it with previous gene expression and DNA sequencing datasets. This approach provides insight into how specific genetic mutations can alter metabolic pathways over 50,000 generations, enabling a deeper understanding of how genetic changes lead to observed differences in evolved bacterial strains. The findings reveal that evolved bacteria possess more diverse metabolic profiles compared to their ancestors, suggesting that these populations have uniquely adapted to their environment. The work also attempts to uncover the molecular basis for this adaptive evolution, demonstrating how specific genetic changes have influenced the bacteria's metabolic pathways.

Overall, this is a significant and well-executed research study. It offers new insights into the complex relationship between genetic changes and observable traits in evolving populations and utilizes metabolomics in the LTEE, a novel approach in combination with RNA-seq and mutation datasets.

However, the paper's overall clarity is lacking. It is spread too thin and covers many topics without a clear focus. I strongly recommend a substantial rewrite of the manuscript, emphasizing structure and readability. The science is well executed, but the current writing does not do it justice.

Reviewer #2 (Public Review):

Hersperger et al. investigated the importance of Drosophila immune cells, called hemocytes, in the response to oxidative stress in adult flies. They found that hemocytes are essential in this response, and using state-of-the-art single-cell transcriptomics, they identified expression changes at the level of individual hemocytes. This allowed them to cluster hemocytes into subgroups with different responses, which certainly represents very valuable work. One of the clusters appears to respond directly to oxidative stress and shows a very specific expression response that could be related to the observed systemic metabolic changes and energy mobilization. However, the association of these transcriptional changes in hemocytes with metabolic changes is not well established in this work. Using hemocyte-specific genetic manipulation, the authors convincingly show that the DNA damage response in hemocytes regulates JNK activity and subsequent expression of the JAK/STAT ligand Upd3. Silencing of the DNA damage response or excessive activation of JNK and Upd3 leads to increased susceptibility to oxidative stress. This nicely demonstrates the importance of tight control of JNK-Upd3 signaling in hemocytes during oxidative stress. However, it would have been nice to show here a link to systemic metabolic changes, as the authors conclude that it is tissue wasting caused by excessive Upd3 activation that leads to increased susceptibility, but metabolic changes were not analyzed in the manipulated flies. The overall conclusion of this work, as presented by the authors, is that Upd3 expression in hemocytes under oxidative stress leads to tissue wasting, whereas in fact it has been shown that excessive hemocyte-specific Upd3 activation leads to increased susceptibility to oxidative stress (whether due to increased tissue wasting remains a question). The DNA damage response ensures tight control of JNK-Upd3, which is important. However, what role naturally occurring Upd3 expression plays in a single hemocyte cluster during oxidative stress has not been tested. What if the energy mobilization induced by this naturally occurring Upd3 expression during oxidative stress is actually beneficial, as the authors themselves state in the abstract - for potential tissue repair? It would have been useful to clarify in the manuscript that the observed pathological effects are due to overactivation of Upd3 (an important finding), but this does not necessarily mean that the observed expression of Upd3 in one cluster of hemocytes causes the pathology.

Reviewer #2 (Public Review):

In whole exome sequencing of two patients suffering from MMAF syndrome, mutations of CCDC146 gene that result in premature stop codons were identified. The position of mutations could result in a truncated form of protein, thus whether these patients do indeed lack CCDC146 protein or if present, whether the truncated protein is functional, is unanswered by showing the CCDC146 protein localization only in the sperm from healthy donors. The main claim that CCDC146 protein is microtubule associated protein in the axoneme is well supported imaging expanded sperm flagellum to increase spatial resolution. However, the author's claim that the signal in the mid-piece is not specific is less supported by experimental evidence. The detection of CCDC146 in the sperm head is not further explored while TEM images show spermatogenesis defects in the manchette and acrosome formation. Increased detection of the CCDC146 protein in mouse sperm with sarkosyl supports its association with microtubules but does not exclude its potential role in the formation of sperm head. Overall, this study provides valuable information on CCDC146 function in male germ cells during spermatogenesis.

Reviewer #2 (Public Review):

This study visualizes a specific localized form of cell-to-cell communication and conveys very well with what dynamics and sensitivity this biological phenomenon occurs.

Using a FRET-based PKA biosensor, the authors observed that radial localized kinase activity changes spontaneously occur in adjacent cells of certain cell density. This phenomenon of radial propagation of PKA activity changes in groups of cells was further mechanistically elucidated and characterized. Interestingly, the authors found that individual cells in the cell groups form spontaneous Ca2+ transients, which at a certain strength can trigger the biosynthesis and release of prostaglandin E2 (PGE2). PGE2 then acts on the neighboring cells and triggers the increase of cAMP levels and the associated activation of the PKA via G-protein-coupled receptors (EP2 and EP4). In systematic, well-structured experiments, it was then found that the frequency of occurrence of such radial activations depends not only on the cell density but also on the activation state of the ERK MAP kinase pathway. The authors skillfully used various modern genetically encoded biosensors and other tools such as optogenetic tools to visualize and characterize an interesting biological phenomenon of cell-to-cell communication. The insights gained with these investigations produce a better understanding of the dynamics, sensitivity, and spatial extent with which such communications can occur in a cell network. It is also worth noting that the authors have not limited the studies to 2D cell culture in vitro, but were also able to confirm the findings in an animal model.

Reviewer #2 (Public Review):

In this study, Huang et al. investigated Bacillus velezensis, a species that colonizes plant roots as part of the rhizosphere. They showed that clone of B. velezensis SQR9 retains a division of labor of motile, planktonic subpopulation that do not produce extracellular matrix (ECM) and biofilm-forming sessile subpopulation that do produce ECM. Specifically, the sessile subpopulation secret toxins named bacillunoic acids (BAs) to kill some, but not all, of the planktonic subpopulation. The killing mechanism is mediated by a global regulator Spo0A, which co-activates BAs production and immunity, as well as ECM production. A strain that has a disrupted policing system revealed reduced biofilm formation, lower resistance to environmental stresses and alleviated ability to colonize plant roots. Overall, the toxin-mediated policing system is important for B. velezensis to mediate division of labor for enhancing population stability and ecological fitness when required (e.g., cell transition from a planktonic style to a multicellular style).

Reviewer #2 (Public Review):

Raykov et al. reported that TrafE, a member of the E3 ubiquitin ligase family similar to the TRAF proteins in mammalian cells, is essential for Dictyostelium discoideum to effectively respond to endolysosomal damage and defend itself against Mycobacterium marinum infection. First, the authors demonstrate that TrafE is recruited to the site of Mycobacterium-Containing Vacuole (MCV) damage along with ubiquitin molecules. This recruitment is necessary for the effective suppression of M. marinum growth in the cells. They also found that this response was not limited to the damage caused by M. marinum, but was also triggered by sterile damage caused by chemical compounds. Furthermore, the authors revealed that TrafE plays a role in the recruitment of Vps4 to sites of membrane damage and regulates the disassembly of ESCRT subunits. While TRAF6 has been previously implicated in ubiquitination in response to invaded bacteria in mammalian cells, this study provides solid data that furthers our understanding of the mechanism behind xenophagy. The authors conducted a thorough analysis to contribute to this field of research.

Reviewer #2 (Public Review):

The manuscript "Rapid and precise genome engineering in a naturally short-lived vertebrate" describes the development of a CRISPR- based knock-in technology in Nothobranchius furzeri, or the African turquoise killifish, an innovative model species for studying aging and age-related disorders. While Tol2 systems had been demonstrated to be successful in generating reporter killifish lines, endogenous reporters via knock-in had not been reported so far. The major strength of the paper is that the authors show that they have been successful in developing 5 different knock-in fish lines with large inserts (up to 1.8kb) with high efficiency. They have inserted single or dual fluorescent reporters and demonstrated expression in line with the expected pattern. This is a breakthrough in the field and this method can be instrumental for many researchers working with unusual model species, and in particular, will expand the killifish community toolbox.

While this is very promising, the paper would benefit from a more rigorous validation of the KI lines that were generated. The authors did not show a co-localisation of the target gene expression with the reporter to prove bona fide reporting. In addition, it was not clear whether the KI affects the endogenous expression level of the target genes. The targeting efficiency of the method is high, but the quantifications are based on rather limited numbers of animals, which might not yet be very robust. A larger number of animals would have strengthened the efficiency conclusion.

The figures of the manuscript are well designed and support the conclusions, but several contain information that is not discussed in the main text, such as (un)expected bands on gels, reporter staining in WT animals, and unusual staining patterns. The body text seems to ignore these and only discusses findings that are in line with the story. A key point to the efficiency of the method seems to be a chemical modification of the repair template, which was not disclosed in the method section which at the moment hampers replication.

Finally, the discussion is brief and does not benchmark the method to other CRISPR-based KI methods in Xenopus or more typical model species such as mouse.

In conclusion, this paper describes a breakthrough method for a rising animal model that would benefit from a more thorough validation. Full disclosure of the methodology will boost the generation of genetically edited killifish lines and aid in the establishment of this promising animal model.

Reviewer #2 (Public Review):

The goal of this study was to determine whether heartbeat-evoked responses measured at the scalp level with EEG, which followed regularity violations, could potencial help inform the diagnosis of patients with altered states of consciousness.

The authors use high density EEG and an oddball paradigm that probes violations of both local and global regularities. Four groups were considered including unresponsive wakefulness syndrome patents, minimally consciousness patients, emerging minimally consciousness patients and healthy controls. A difference was found between unresponsive and minimally conscious patients in the amplitude of the heartbeat evoked responses measure with EEG following a sound that violated a global regularity. Similarly, differences were found between the variance of these responses between the two above mentioned groups (N=58 and N=59), but no differences were found in relation to the healthy control group, which appear to be "in between" the two other groups (at least for global effect of HER). I thought this was a little counterintuitive and raises some questions about what this neural signature can tell us about the state of consciousness. Having said that, the healthy control sample was very small, more than 5 times smaller (only N=11).

In general, I thought the Discussion section was a little light on the implications of the findings, what they tell us about the brain mechanisms of consciousness and their different levels/states. A question is raised about whether it is necessary to lock EEG to heartbeats to find differences between patients. The data appeared to say that this is not the case but the discussion does not appear to reflect that very clearly.

Reviewer #2 (Public Review):

A distinguishing feature of live cells is that intracellular organelles move powered by molecular motors. However, the arsenal of molecular motors is limited relative to the vast variety of cargoes and processes involving long-distance movement. Cells cope with this mismatch by using adaptors that "bridge" a given molecular motor with a specific cargo, whose identity is dictated by peripheral membrane proteins, such RABs, or identity-determining lipids, such as PtdIns3P. Cytoplasmic dynein walks towards the minus end of the microtubules. A score of cellular processes is dependent on dynein, such that deficient regulation of the motor has deep consequences in cellular homeostasis, and the identification of new adapters is of broad interest, both basic and, potentially, clinical.

Dynein adaptors usually stabilize the binding of dynactin to dynein using coiled-coil regions to longitudinally embrace dynactin, holding it to the elongated dynein cap of the super-complex. Not only do they adapt cargo but additionally increase the processivity and speed of the motor. In this manuscript, Julie and collaborators present evidence that a protein denoted kazrin, which is involved in a variety of processes, is actually an adaptor connecting endosome domains specialized in recycling cargo back to the surface of the cell by way of the RAB11 perinuclear recycling endosome. The topic is important, experiments have been carefully conducted and well controlled and display items faithfully guide readers through the main findings. However, I feel that the evidence that kazrin is a dynein adaptor is somewhat thin and that it could be improved with relatively little additional work. The manuscript would also benefit from better integration of the conclusions in the current state of the art in the dynein field.

Reviewer #2 (Public Review):

In the current study, Mondoloni and colleagues investigate the neural correlates contributing to nicotine aversion and its alteration following chronic nicotine exposure. The question asked is important to the field of individual vulnerability to drug addiction and has translational significance. First, the authors identify individual nicotine consumption profiles across isogenic mice. Further, they employed in vivo and ex vivo physiological approaches to defining how antiparticle nuclei (IPn) neuronal response to nicotine is associated with nicotine avoidance. Additionally, the authors determine that chronic nicotine exposure impairs IPn neuronal normal response to nicotine, thus contributing to higher amounts of nicotine consumption. Finally, they used transgenic and viral-mediated gene expression approaches to establish a causal link between b4 nicotine receptor function and nicotine avoidance processes.

The manuscript and experimental strategy are well designed and executed; the current dataset requires supplemental analyses and details to exclude possible alternatives. Overall, the results are exciting and provide helpful information to the field of drug addiction research, individual vulnerability to drug addiction, and neuronal physiology. Below are some comments aiming to help the authors improve this interesting study.

1. The authors used a two-bottle choice behavioral paradigm to investigate the neurophysiological substrate contributing to nicotine avoidance behaviors. While the data set supporting the author's interpretation is compelling and the experiments are well-conducted, a few supplemental control analyses will strengthen the current manuscript.<br /> a. The bitter taste of nicotine might generate confounds in the data interpretation: are the mice avoiding the bitterness or the nicotine-induced physiological effect? To address this question, the authors mixed nicotine with saccharine, thus covering the bitterness of nicotine. Additionally, the authors show that all the mice exposed to quinine avoid it, and in comparison, the N-Av don't avoid the bitterness of the nicotine-saccharine solution. Yet it is unclear if Av and N-Av have different taste discrimination capacities and if such taste discrimination capacities drive the N-Av to consume less nicotine. Would Av and N-Av mice avoid quinine differently after the 20-day nicotine paradigm? Would the authors observe individual nicotine drinking behaviors if nicotine/quinine vs. quinine were offered to the mice?<br /> b. Metabolic variabilities amongst isogenic mice have been observed. Thus, while the mice consume different amounts of nicotine, changes in metabolic processes, thus blood nicotine concentrations, could explain differences in nicotine consumption and neurophysiology across individuals. The authors should control if the blood concentration of nicotine metabolites between N-Av and Av are similar when consuming identical amounts of nicotine (50ug/ml), different amounts (200ug/ml), and in response to an acute injection of a fixed nicotine quantity.

2. Av mice exposed to nicotine_200ug/ml display minimal nicotine_50ug/ml consumption, yet would Av mice restore a percent nicotine consumption >20 when exposed to a more extended session at 50ug/kg? Such a data set will help identify and isolate learned avoidance processes from dose-dependent avoidance behaviors.

3. The author should further investigate the basal properties of IPn neuron in vivo firing rate activity recorded and establish if their spontaneous activity determines their nicotine responses in vivo, such as firing rate, ISI, tonic, or phasic patterns. These analyses will provide helpful information to the neurophysiologist investigating the function of IPn neurons and will also inform how chronic nicotine exposure shapes the IPn neurophysiological properties.

Reviewer #2 (Public Review):

The authors successfully show that how EPAC and PKCε work together to recruit presynaptic proteins for neurotransmitter release instead of synaptic vesicle formation since the absence of EPAC and PKCε does not affect the number of synaptic vesicles. In addition, the data clearly demonstrate that EPAC and PKCε function specifically at the presynaptic terminals and thus is required for induction of presynaptic LTP. Their suggested EPAC- PKCε module is also essential for proper cerebellar motor performance and motor learning.

Furthermore, the order of data analysis perfectly matches the logical explanation of the entire story. The authors first prove that EPAC and PKCε, together with RIM1a, are necessary for neurotransmitter release at the presynaptic terminal. Then, by using specific knockdown mice of presynaptic granule cells, both proteins contribute to the release of synaptic vesicles in that only the frequencies of EPSC have changed. In addition, presynaptic LTP is only induced with the presence of EPAC and PKCε, highlighting the important role of the EPAC- PKCε module. Ultimately, the impact of EPAC and PKCε is shown by conducting the behavior tasks including OKR, VOR, and VVOR.

The authors suggest the missing link between EPAC and RIM1 is PKCε. Phosphorylation of RIM1 by PKCε is a novel signaling cascade found in this paper. The authors' data from the heterologous expression system and cerebellar granule cell-specific PKCε KO mice indicate that PKCε can regulate RIM1Threonine phosphorylation.<br /> The EPAC-PKCε unit is essential to both presynaptic neurotransmitter release and presynaptic LTP in parallel fiber-Purkinje cell synapse. Future work is necessary to dissect which is responsible for cerebellar motor performance and motor learning.

The study provides the necessity of exploring the new part of the motor learning circuit since the significant focus of cerebellar motor learning has been only confined to postsynaptic plasticity. Generally, postsynaptic plasticity is affected by the presynaptic properties, such as presynaptic vesicle release and recycling of neurotransmitters at the synapse. Also, the presynaptic terminal, which can be referred to as an inducing force of the postsynaptic plasticity, does not merely release the neurotransmitters at a constant rate; they also change as a result of incoming stimuli. Such change is called presynaptic plasticity. Therefore, it should be further scrutinized how presynaptic plasticity is conducted and determined.

operator norm is actually a type of norm. Linear operators as homomorphism between the vector spaces are indeed a vector space itself. See here.

The properties of the operator norm basically interits the properties from the normed vector space. This is direct from the definition. However, these results are only for the bounded linear operators.

Reviewer #2 (Public Review):

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

In some places the absence of crosslinks is reported as a lack of interaction, however it could also be that there are no residues that can be crosslinked in this region. The distance is also not reported in the figures so we do not know how valid these model are. For example for TRAP binding to KHC, there are not many crosslinks but it is not clear if there was an issue with the complex assembly or crosslinking reaction-as there is no EM data of this complex. There is also a structural model of KHC and KLC (Fig 4) where the domains are too far apart for the crosslinks to be allowed, raising a question about whether that model is correct or not. The structural data are supported by single molecule motility assays with various mutants of Kinesin-1, which greatly help characterising the domains functionally.

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

Reviewer #2 (Public Review):

To date, only a handful of studies have addressed the importance of AGS3, a paralog of the relatively well-characterized spindle orientation factor LGN. The authors now show that AGS3 acts as a negative regulator of LGN and propose that this activity could work through competition for binding partner(s). Remarkably, regulation is temporally restricted in such a way that the conserved role played by LGN in metaphase spindle orientation is unaffected. Instead, AGS3 regulates a post-metaphase function for LGN, namely Telophase Correction.

The article is well-written, the experiments are performed at a high level, and the claims are generally supported by the data. Two main points of confusion are raised in the current version. 1) The authors show that AGS3 regulates cortical localization of LGN, but would need to clarify how LGN is being affected. 2) The authors propose in the discussion that AGS3 might exert its regulatory effect through competition for NuMA, an important binding partner for LGN, but would need to clarify how and why NuMA would be involved in Telophase Correction.

Reviewer #2 (Public Review):

This manuscript by Walker et al describes an elegant study that synergizes our knowledge of virulence gene regulation of Vibrio cholerae. The work brings a new element of regulation for CRP, notably that CRP and the high density regulator HapR co-occupy the same site on the DNA but modeling predicts they occupy different faces of the DNA. The DNA binding and structural modeling work is nicely conducted and data of co-occupation are convincing. The work could benefit from doing a better job in the manuscript preparation to integrate the findings into our current state of knowledge of HapR and CRP regulated genes and to elevate the impact of the work to address how bacteria are responding to the nutritional environment. Importantly, the focus of the work is heavily based on the impact of use of GlcNAc as a carbon source when bacteria bind to chitin in the environment, but absent the impact during infection when CRP and HapR have known roles. Further, the impact on biological events controlled by HapR integration with the utilization of carbon sources (including biofilm formation) is not explored. The rigor and reproducibility of the work needs to be better conveyed.

Specific comments to address:

1) Abstract. A comment on the impact of this work should be included in the last sentence. Specifically, how the integration of CRP with QS for gene expression under specific environments impacts the lifestyle of Vc is needed. The discussion includes comments regarding the impact of CRP regulation as a sensor of carbon source and nutrition and these could be quickly summarized as part of the abstract.<br /> 2) Line 74. This paper examines the overlap of HapR with CRP, but ignores entirely AphA. HapR is repressed by Qrrs (downstream of LuxO-P) while AphA is activated by Qrrs. WithLuxO activating AphA, it has a significant sized "regulon" of genes turned on at low density. It seems reasonable that there is a possibility of overlap also between CRP and AphA. While doing an AphA CHIP-seq is likely outside the scope of this work, some bioinformatic or simply a visual analysis of the promoters known AphA regulated genes would be interest to comment on with speculation in the discussion and/or supplement.<br /> 3) Line 100. Accordingly with the above statement, the focus here on HapR indicates that the focus is on gene expression via LuxO and HapR, at high density. Thus the sentence should read "we sought to map the binding of LuxO and HapR of V. cholerae genome at high density".<br /> 4) Line 109. The identification of minor LuxO binding site in the intergenic region between VC1142 and VC1143 raises whether there may be a previously unrecognized sRNA here. As another panel in figure S1, can you provide a map of the intergenic region showing the start codons and putative -10 to -35 sites. Is there room here for an sRNA? Is there one known from the many sRNA predictions / identifications previously done? Some additional analysis would be helpful.<br /> 5) Line 117. This sentence states that the CHIP seq analysis in this study includes previously identified HapR regulated genes, but does not reveal that many known HapR regulated genes are absent from Table 1 and thus were missed in this study. Of 24 HapR regulated investigated by Tsou et al, only 1 is found in Table 1 of this study. A few are commented in the discussion and Figure S7. It might be useful to add a Venn Diagram to Figure 1 (and list table in supplement) for results of Tsou et al, Waters et al, Lin et al, and Nielson et al and any others). A major question is whether the trend found here for genes identified by CHIP-seq in this study hold up across the entire HapR regulon. There should also be comments in the discussion on perhaps how different methods (including growth state and carbon sources of media) may have impacted the complexity of the regulon identified by the different authors and different methods.<br /> 6) The transcription data are generally well performed. In all figures, add comments to the figure legends that the experiments are representative gels from n=# (the number of replicate experiments for the gel based assays). Statements to the rigor of the work are currently missing.<br /> 7) Line 357-360. The demonstration of lack of growth on MurNAc is a nice for the impact of the work. However, more detailed comments are needed for M9 plus glucose for the uninformed reader to be reminded that growth in glucose is also impaired due to lack of cAMP in glucose replete conditions and thus minimal CRP is active. But why is this now dependent of hapR? A reminder also that in LB oligopeptides from tryptone are the main carbon source and thus CRP would be active.<br /> 8) A great final experiment to demonstrate the model would have been to show co-localization of the promoter by CRP and HapR from bacteria grown in LB media but not in LB+glucose or in M9+glycerol and M9+MurNAc but not M9+glucose. This would enhance the model by linking more directly to the carbon sources (currently only indirect via growth curves)<br /> 9) Discussion. Comments and model focus heavily on GlcNAc-6P but HapR has a regulator role also during late infection (high density). How does CRP co-operativity impact during the in vivo conditions? Does the Biphasic role of CRP play a role here (PMID: 20862321)?

Reviewer #2 (Public Review):

DNA gyrase is an essential enzyme in bacteria that regulates DNA topology and has the unique property to introduce negative supercoils into DNA. This enzyme contains 2 subunits GyrA and GyrB, which forms an A2B2 heterotetramer that associates with DNA and hydrolyzes ATP. The molecular structure of the A2B2 assembly is composed of 3 dimeric interfaces, called gates, which allow the cleavage and transport of DNA double stranded molecules through the gates, in order to perform DNA topology simplification.<br /> The article by Germe et al. questions the existence and possible mechanism for subunit exchange in the bacterial DNA gyrase complex.

The complexes are purified as a dimer of GyrA and a fusion of GyrB and GyrA (GyrBA), encoded by different plasmids, to allow the introduction of targeted mutations on one side only of the complex. The conclusion drawn by the authors is that subunit exchange does happen, favored by DNA binding and wrapping. They propose that the accumulation of gyrase in higher-order oligomers can favor rapid subunit exchange between two active gyrase complexes brought into proximity.<br /> The authors are also debating the conclusions of a previous article by Gubaev, Weidlich et al 2016 (https://doi.org/10.1093/nar/gkw740). Gubaev et al. originally used this strategy of complex reconstitution to propose a nicking-closing mechanism for the introduction of negative supercoils by DNA gyrase, an alternative mechanism that precludes DNA strand passage, previously established in the field. Germe et al. incriminate in this earlier study the potential subunit swapping of the recombinant protein with the endogenous enzyme, that would be responsible for the detected negative supercoiling activity.

Accordingly, the authors also conclude that they cannot completely exclude the presence of endogenous subunits in their samples as well.

Strengths

The mix of gyrase subunits is plausible, this mechanism has been suggested by Ideka et al, 2004 and also for the human Top2 isoforms with the formation of Top2a/Top2b hybrids being identified in HeLa cells (doi: 10.1073/pnas.93.16.8288).<br /> Germe et al have used extensive and solid biochemical experiments, together with thorough experimental controls, involving :<br /> - the purification of gyrase subunits including mutants with domain deletion, subunit fusion or point mutations.<br /> - DNA relaxation, cleavage and supercoiling assays<br /> - biophysical characterization in solution (size exclusion chromatography, mass photometry, mass spectrometry)

Together the combination of experimental approaches provides solid evidence for subunit swapping in gyrase in vitro, despite the technical limitations of standard biochemistry applied to such a complex macromolecule.

Weaknesses

The conclusions of this study could be strengthened by in vivo data to identify subunit swapping in the bacteria, as proposed by Ideka et al, 2004. Indeed, if shown in vivo, together with this biochemical evidence, this mechanism could have a substantial impact on our understanding of bacterial physiology and resistance to drugs.

Reviewer #2 (Public Review):

I believe the authors succeeded in finding neural evidence of reactivation during REM sleep. This is their main claim, and I applaud them for that. I also applaud their efforts to explore their data beyond this claim, and I think they included appropriate controls in their experimental design. However, I found other aspects of the paper to be unclear or lacking in support. I include major and medium-level comments:

Major comments, grouped by theme with specifics below:<br /> Theta.<br /> Overall assessment: the theta effects are either over-emphasized or unclear. Please either remove the high/low theta effects or provide a better justification for why they are insightful.

Lines ~ 115-121: Please include the statistics for low-theta power trials. Also, without a significant difference between high- and low-theta power trials, it is unclear why this analysis is being featured. Does theta actually matter for classification accuracy?

Lines 123-128: What ARE the important bands for classification? I understand the point about it overlapping in time with the classification window without being discriminative between the conditions, but it still is not clear why theta is being featured given the non-significant differences between high/low theta and the lack of its involvement in classification. REM sleep is high in theta, but other than that, I do not understand the focus given this lack of empirical support for its relevance.

Line 232-233: "8). In our data, trials with higher theta power show greater evidence of memory reactivation." Please do not use this language without a difference between high and low theta trials. You can say there was significance using high theta power and not with low theta power, but without the contrast, you cannot say this.

Physiology / Figure 2.<br /> Overall assessment: It would be helpful to include more physiological data.

It would be nice, either in Figure 2 or in the supplement, to see the raw EEG traces in these conditions. These would be especially instructive because, with NREM TMR, the ERPs seem to take a stereotypical pattern that begins with a clear influence of slow oscillations (e.g., in Cairney et al., 2018), and it would be helpful to show the contrast here in REM. Also, please expand the classification window beyond 1 s for wake and 1.4 s for sleep. It seems the wake axis stops at 1 s and it would be instructive to know how long that lasts beyond 1 s. The sleep signal should also go longer. I suggest plotting it for at least 5 seconds, considering prior investigations (Cairney et al., 2018; Schreiner et al., 2018; Wang et al., 2019) found evidence of reactivation lasting beyond 1.4 s.

Temporal compression/dilation.<br /> Overall assessment: This could be cut from the paper. If the authors disagree, I am curious how they think it adds novel insight.

Line 179 section: In my opinion, this does not show evidence for compression or dilation. If anything, it argues that reactivation unfolds on a similar scale, as the numbers are clustered around 1. I suggest the authors scrap this analysis, as I do not believe it supports any main point of their paper. If they do decide to keep it, they should expand the window of dilation beyond 1.4 in Figure 3B (why cut off the graph at a data point that is still significant?). And they should later emphasize that the main conclusion, if any, is that the scales are similar.

Line 207 section on the temporal structure of reactivation, 1st paragraph: Once again, in my opinion, this whole concept is not worth mentioning here, as there is not really any relevant data in the paper that speaks to this concept.

Behavioral effects.<br /> Overall assessment: Please provide additional analyses and discussion.

Lines 171-178: Nice correlation! Was there any correlation between reactivation evidence and pre-sleep performance? If so, could the authors show those data, and also test whether this relationship holds while covarying our pre-sleep performance? The logic is that intact reactivation may rely on intact pre-sleep performance; conversely, there could be an inverse relationship if sleep reactivation is greater for initially weaker traces, as some have argued (e.g., Schapiro et al., 2018). This analysis will either strengthen their conclusion or change it -- either outcome is good.

Unlike Schönauer et al. (2017), they found a strong correspondence between REM reactivation and memory improvement across sleep; however, there was no benefit of TMR cues overall. These two results in tandem are puzzling. Could the authors discuss this more? What does it mean to have the correlation without the overall effect? Or else, is there anything else that may drive the individual differences they allude to in the Discussion?

Medium-level comments<br /> Lines 63-65: "We used two sequences and replayed only one of them in sleep. For control, we also included an adaptation night in which participants slept in the lab, and the same tones that would later be played during the experimental night were played."

I believe the authors could make a stronger point here: their design allowed them to show that they are not simply decoding SOUNDS but actual memories. The null finding on the adaptation night is definitely helpful in ruling this possibility out.

Lines 129-141: Does reactivation evidence go down (like in their prior study, Belal et al., 2018)? All they report is theta activity rather than classification evidence. Also, I am unclear why the Wilcoxon comparison was performed rather than a simple correlation in theta activity across TMR cues (though again, it makes more sense to me to investigate reactivation evidence across TMR cues instead).

Line 201: It seems unclear whether they should call this "wake-like activity" when the classifier involved training on sleep first and then showing it could decode wake rather than vice versa. I agree with the author's logic that wake signals that are specific to wake will be unhelpful during sleep, but I am not sure "wake-like" fits here. I'm not going to belabor this point, but I do encourage the authors to think deeply about whether this is truly the term that fits.

Reviewer #2 (Public Review):

The authors reexamine the effects of depth on crowding, using a clever display that presents at three depths at once, and find that placing the target or flanker at far depth greatly increases crowding, contrary to what might have been expected by prior work with small depth differences. These stimuli avoid creating conflicting cues to depth and are thus the most relevant to viewing in daily life, indicating more crowding than was expected.

Reviewer #2 (Public Review):

This study was designed to study the cortical response to violations in auditory temporal sequences during wakefulness and sleep. To this end, the study had three levels of temporal sequence, a regular temporal sequence, an auditory tone that was yoked to the cardiac signal, and an irregular tone. The authors show significant EEG differences to an omitted tone when the auditory tone was predictable both during wakefulness and sleep.

The authors analyze the ERP to the omitted tone as well as when aligned to the R-peak of the HEP. The analysis was comprehensive and the effects reported align with the interpretation given. Of particular interest was the fact that a deceleration of the heart rate was present for omissions when the auditory tone was yoked to the R-peak (synch) in all stages of wakefulness and sleep.

However, one weakness was the rationale for the current study and how the results link to current theoretical frameworks for the role of interoception in perception and cognition. This was in contrast to the clear background and explanation to study the response to omissions for a predictable auditory sequence in wakefulness and sleep. It was unclear why the authors selected the cardiac signal to yoke their auditory stimuli. What is the specific motivation for the cardiac signal rather than the respiratory signal? This was not clear.

Reviewer #2 (Public Review):

The authors tackled a longstanding question for brain evolution: if the brain regions change based on functional constraints or developmental constraints.

The strength of this study is that the authors introduced an automated method for brain segmentation based on the zebrafish tool, which is a highly advanced technology. They also performed the volume and landmark-based shape analyses in a surface, cave and their F1 and F2 hybrid, highlighting genetic regulations, and revealed 3 genetically correlating clusters of brain regions, which are brand new as far as I know. This study needs intense effort, fine skills to conduct, and intellectual efforts to summarize the vast dataset. I simply admire how the authors achieve their study at this level.

The weakness of this study is that the method/approach used in this study is difficult to test the functional constraint hypothesis although the authors nicely tested the developmental constraint hypothesis, which was highlighted in their correlation studies (volumetric and shape: Fig 4 and 5). I am also a little concerned with the accuracy of the automated segmentation algorithm shown in Figure 1-figure supplement 2. The original zebrafish paper (CobraZ) showed a similar accuracy (cross-correlation as 80%). If this level of accuracy is accepted in the field, I am OK with it.<br /> Their data support the conclusion 'brain-wide evolution occurs in distinct developmental modules' because of their correlation study. However, I am not so positive at the point that one of two central hypotheses were directly tested in this study: the functional constraint hypothesis - to test it, for example, the authors need to address the functional connectivities (calcium imaging, etc) and then test if the correlation between calcium-transients and the size/shape of each pair of brain regions.

Reviewer #2 (Public Review):

Jangir et al test the hypothesis that resistance to the antimicrobial peptide (AMP) colistin can simultaneously increase resistance to other AMPS with related modes of action. Because AMPS comprise part of innate immunity, their central concern is that colistin resistance may compromise host defenses and thereby increase bacterial virulence. Their results show that MCR-1, whether expressed from naturally circulating or synthetic plasmids, can increase the MIC to AMPS from humans, pigs, and chickens, and impart fitness benefits at sub-MIC concentrations. In addition, they find that MCR-1-containing strains have increased survival in human plasma and are more lethal in an insect infection model.

The conclusions of the paper are generally well supported by the results, but some aspects could be clearer and better defended with a few small additional experiments.

Strengths:<br /> Using both synthetic and natural plasmids makes it possible to cleanly separate the effects of MCR-1 from the effects of other plasmid-borne genes or plasmid copy numbers. This helps confirm the causal role of MCR-1 on altered AMP susceptibility.

Testing the survival of transformed isolates in human serum and in insects points to relevance in the more immunologically complex host environment where cells are exposed to a suite of factors that reduce bacterial survival.

Weaknesses/suggestions:<br /> Although increases in MIC are evident for different AMPS, the effects are generally modest. To address this, it might be helpful to use pairwise competition assays, as in Figure 1, to establish that even small changes to MIC are associated with clear selective benefits. This would be especially helpful in assays with human serum and in Galleria where the concentrations of AMPS or other immune components are unknown.

Assays using human serum are interesting but challenging to interpret given the diverse causes of bacterial killing, including complement. Although this was partly addressed in Supplementary Figure 6, I found the predictions of these experiments unclear. First, I think these experiments are too central to be relegated to the supplemental materials; they belong in the main text. Secondly, it is important to explicitly spell out the expectations of using heat-killed serum (which will degrade any heat-labile components) or complement-deficient serum. It should be clearer under which conditions MCR-1-containing strains are predicted to do better or worse than controls.

Galleria is a useful infection model for virulence, but it is unclear what drives differences between strains. First, bacterial numbers aren't measured in this assay, so it isn't known if increased virulence is due to increased bacterial growth or decreased bacterial clearance. As above, I think these assays would be stronger using the competition-based approach in Figure 1. This would indicate bacterial numbers through time and directly show the selective benefit associated with MCR-1. Second, it would be useful to elaborate on why MCR-1 increases virulence, especially any known similarities between Galleria AMPS and those tested in Figures 1 and 2. Overall, it would help if Galleria were less of a black box.

Reviewer #2 (Public Review):

The study by Yanase et al. investigated the details of the 3D architecture of Leishmania haptomonad promastigote's adhesion to the midgut of the insect vector. The authors generated a dataset of images that reveal intricate details of the formed adhesion plaque and expanded the study with in vitro alternatives for the exploration of how Leishmania promastigotes strong adhesion by hemidesmosomes to surfaces can happen and be maintained. They show with unprecedented detail the ultrastructure of the attachment plaque. The in vitro dataset of the paper adds to the specific literature important details on how to explore micro/nanostructures involved in an important attachment step for this eukaryotic parasite. However, the in vitro data should be reconsidered in its discussion and conclusions as it does not support direct comparison with in vivo Leishmania forms as pictured by the authors. In general, the dataset presented in this manuscript adds valuable data and resources for the study of Leishmania promastigotes to surfaces, especially to the thoracic midgut parts of its insect vector.

The dataset of this paper is well-collected and robust, but some aspects of image analysis need to be clarified and extended. Also, the in vitro data from the manuscript will benefit from an extensive adjustment in its discussion. Points to focus on:

1) The haptomonad promastigote is indeed a possible critical form for transmission, but it lacks formal demonstration still in all literature available. This should not be claimed without proper formal demonstration.

2) Literature available and cited in this manuscript regarding in vitro adhesion of culture Leishmania promastigotes does not provide direct evidence for haptomonad differentiation. Haptomonads are still a largely unknown promastigote form with no defined ontogeny. With that, to propose an in vitro haptomonad differentiation protocol, more detailed direct evidence of in vivo haptomonads will be necessary. The in vitro experiments available show how cultured promastigotes attach to surfaces. Detailed studies in vivo will be needed still to attribute the findings in vitro to haptomonads.

3) This manuscript will benefit by having a detailed description of how to analyze and get to the 3D models presented. This has a strong potential for usage beyond the Leishmania/sand fly field. Statistics should be made available with ease across the manuscript and with a dedicated section on methods.

Reviewer #2 (Public Review):

The manuscript by Sampaio and colleagues utilizes an elegant and delicate approach to manipulate fluid dynamics in zebrafish Kupffer's vesicle (KV) to answer a long-standing question in the field - is it fluid movement or something in the fluid that governs the break in symmetry?

The researchers extract fluid from KV at different times during somitogenesis and find this procedure results in left-right organ defects when fluid is removed from the 3 to 5 somite stage, peaking at 5 somites. The effect on left-right patterning by this manipulation is not significant from the 6 somite stage onward. This technique is non-trivial and the researchers have used it with great effect.

Fluid extraction in this sensitive time window (3-5 somites) did not affect cilia number, length, or distribution within KV suggesting the effect on left-right patterning is due to disruption of the fluid. There is a clear effect of the manipulation on dand5 RNA asymmetry as expected. Manipulated embryos that developed left-right defects also showed a decrease in angular velocity of particle movement in the anterior LRO. Increasing the viscosity of the fluid in KV with methylcellulose also results in left-right patterning defects. Taken together, these results are in strong support of fluid movement and detection being important in breaking symmetry in a ciliated left-right organizer. They also argue against the idea that there are signals in the fluid that are being moved asymmetrically to signal to the "left" to break the symmetry. Importantly, they help set a time window when fluid flow is critical for this process.

Reviewer #2 (Public Review):

The authors conducted a wide-ranging series of experiments which lead to the conclusion that NBR1 is involved in the clearance of photodamaged chloroplasts. It is a novel finding because the role of NBR1 in this process was never documented. Notably, the NBR1-mediated clearance is only one of the several possible mechanisms responsible for chloroplast turnover. It is not surprising, considering that the nbr1 mutants are viable. The work is arranged very well. The rationale of the subsequent experiments is logically justified and the outcomes and followed by clear conclusions. In consequence, the authors managed not only to observe the association of NBR1 with the chloroplasts but they threw some light on the corresponding mechanisms. The manuscript contains numerous high-quality images from a confocal microscope and from a transmission electron microscope. All images are accompanied by statistical analysis of the respective microscopic observations, which greatly improves the credibility of the conclusions. Shortly, the authors demonstrated that NBR1 decorates not only the exterior but also the interior of damaged chloroplasts in an ATG7-independent way. Next, they establish that NBR1 and ATG8 are recruited to different populations of damaged chloroplasts, and they document differences in chloroplasts turnover, differences in chlorophyll abundance and chlorophyll photochemical properties, as well as differences in the total proteome of the nbr1 mutant in comparison to the wild type and atg7 mutant in two light regimes (low light and high light). Finally, they exclude the requirement for the known E3 ligases PUB4 and SP1 for NBR1-mediated degradation and show that the NBR1 internalization relies rather on the chloroplastic membrane rupture than on the TIC-TOC-dependent processes. In summary, the authors postulate that NBR1-mediated chloroplast clearance is a novel, not yet described mechanism and summarize it in a clear diagram.

The work is interesting, the figures are convincing and the conclusions are justified by the results. It provides novel data on the function of selective autophagy receptors NBR1 in plant cells, however, it also leaves the reader with some unanswered questions. The most important is the relative contribution of each of the chloroplast's degradation routes to the turnover of these organelles in different stresses, light regimes, plant growth stages, etc. This is a difficult problem because the mutations in relevant genes have pleiotropic effects and it is difficult to separate the functions of the individual turnover routes. For example, the defects in core autophagy genes (like the atg7 mutant used in this study) result in an increased level of NBR1. These issues are not sufficiently addressed in the discussion.

Reviewer #2 (Public Review):

In this work Xia et al have generated CRISPR resources for genome-wide gain-of-function genetic perturbation in the Drosophila genome and have used them to identify novel genes that cause Rapamycin resistance in Drosophila cells. To do so, they have used the SAM system, already established to work well in flies (Jia et al., PNAS 2018). 3 of these candidate genes they discovered in the screen, were further characterized to study how they affect the mTOR pathway leading to Rapamycin resistance. Since genome-wide libraries for GOF studies do not exist for Drosophila, these resources will be very useful for a wider Drosophila community.

Strengths

1. GOF CRISPR library does not exist currently to be used in Drosophila and hence this is going to be useful for the wider Drosophila community<br /> 2. Authors have used already established and currently the most effective SAM system for gene activation for a genome-wide genetic screen.<br /> 3. From this screen they have found candidate genes overexpression which leads to Rapamycin resistance. They have validated 3 of these genes by multiple methods and have also tried to elucidate the mechanism by which these genes might regulate mTOR signaling and confer resistance to Rapamycin. The authors have shown the strength and usefulness of the resource that they have generated and this resource will be complementary to loss-of-function screens of similar nature.

Weaknesses

1. Authors have taken a number of measures to maintain the integrity of the CRISPRa library, including multiple gRNA targets per gene, 1000 cells per gRNA, and deep sequencing. However, do the authors have an idea of what percentage of the gRNA vectors are functional? Looking at the data they show for the 3 candidate genes, at least half of them are not functional, which could be either because of gRNA location or efficiency. Considering this to be an average situation, there might be a large number of genes for which all gRNAs might not function at all. I understand this might be a caveat for all such studies, but an estimate of some kind in discussion might be useful for anyone who might want to use these resources.<br /> 2. As the authors mention that ~32% of genes in Drosophila have transcription start side <1kb apart, off-targeting (neighbouring genes getting activated in addition to the intended gene) will be an issue. To address this, the authors describe one example of genes where although the genes TSS are within one kb of each other, the sgRNA specifically activated only one gene and not the other. However, since following this, authors have generated genome-wide resources keeping 500bp upstream as their benchmark, a large percentage of these 32% genes might have off-targets. It would be useful to know the estimates of off-targeting for such a resource. In addition, have authors looked at the transcripts of genes close to the specific genes they have studied? CG9932 is in close proximity to (although not within the 1 kb range) a few genes including mTOR.

Reviewer #2 (Public Review):

The authors conduct a structure-function analysis of an uncharacterized gene, DltE, which was found by a genetic screen to be involved in the growth promotion of Drosophila larvae by Lactiplantibacillus plantarum, a bacterium that is consistently associated with Drosophila. They find that DltE is a D-Ala carboxylesterase that removes D-Ala from lipoteichoic acids in the cell envelope and that D-alanylated lipoteichoic acids stimulate Drosophila larval growth. The result that D-Ala LTA stimulates larval growth is compelling, although some minor experimental details to do with biological replicates are not shown and the tracking of bacterial abundances should be addressed to make the conclusions more solid. Additionally, I think the use of the terms "direct" and "symbiotic" is inappropriate in the manuscript, but this can be resolved by removing them or performing additional experiments.

The authors make these claims:<br /> - DltE is not a carboxypeptidase modifying Lp peptidoglycan;<br /> - DltE is a D-Ala esterase acting upon D-Ala-LTA;<br /> - only LTAs but not WTAs are D-alanylated in LpNC8 cell envelopes;<br /> - D-Ala-LTAs, in addition to PG, are direct symbiotic cues supporting<br /> (1) intestinal peptidase expression and<br /> (2) juvenile growth in Drosophila.<br /> I find all of the claims to be well supported by data except the suggestion that these are "direct symbiotic" cues. I think the authors provide the support that D-Ala LTAs are nutritional cues, not symbiotic ones.

Overall, I find the work compelling.

Reviewer #2 (Public Review):<br /> <br /> MAPKs are key fundamental enzymes and out of the 14 MAPKs, ERK3 and ERK4 remain less studied. The authors have made some interesting discoveries on ERK3, especially in the context of chemotaxis and tumourigenesis previously (Bogucka et al eLife 2020). Here they investigated the role of ERK3 in the control of cell architecture. Loss of ERK3 led to a reduction in the formation of actin-rich protrusions which led the authors logically to look for the activation of RhoGTPases. Intriguingly, they found that ERK3 functioned as a GEF for Cdc42 but not for Rac1. Further, they identified that Rac-WAVE and Arp2/3 were present at endogenous levels in a heteromeric complex in cells. As ERK3-deficient breast epithelial cells exhibit less F-actin content, this has led the authors to check for Arp2/3-dependent events here. By employing a variety of knockdown and complementation approaches, the authors convincingly demonstrate that the kinase activity of ERK3 is not required for the total F-actin content but for the formation of actin-rich protrusions. Finally, loss of ERK3 reduced random cell motility in vitro and in vivo, which was accomplished by intravital imaging of breast cancer cells in mice. Many protein kinases have catalysis-dependent and -independent functions (catalytic activity versus allosteric activity) and here is another example that deserves further investigation and opens new lines of investigation.

Reviewer #2 (Public Review):

De Filippo et al. investigated the spatiotemporal dynamics of the ripples propagation in the hippocampus of head-fixed mice. By leveraging the LFP and the isolated units of an open dataset of 49 animals with ~6 Neuropixels probes in the longitudinal axis of the hippocampus, they found: first, that stronger ripples (>ninth decile of power) originated in the most septal pole of the hippocampus (medially, anatomically) tend to travel more (M to L) than more lateral ripples (closer to the temporal pole). Second, while strong ripples were mainly local, the authors found that they are most likely to be generated in the temporal pole of the hippocampus, from where they can travel with relatively small attenuation. Finally, they found that strong/septal ripples elicit high spiking activity along the entire mediolateral axis of the hippocampus. Longer/stronger ripples have been proposed to be important in situations with high memory load, and these analyses increase our understanding of their physiology and mechanisms of generation.

The conclusions of this paper are mostly well supported by data, but some aspects of interpretation and data analysis need to be clarified and extended.

1) High amplitude ripples preferentially occur in distal CA1, and ripples can propagate at a higher degree on the proximo-distal than in the septo-temporal axis of the hippocampus (Kumar and Deshmuckh, 2020). Therefore, a proximo-distal bias in the Neuropixel positioning could explain part of the variance the authors report. Authors should consider (or control for) the proximodistal positioning of the electrodes.

2) In my opinion, the dynamics of the ripple-induced spiking activity for the events generated in the medial or lateral section of the hippocampus are very striking, more even considering that only a minority of the detected ripples are strong/long events (less than 5% in a familiar environment, Fernandez-Ruiz et al, 2019), while, according to the authors, majority of the ripples (grouped as 'common' by the authors) travel on the opposite direction (from the lateral section towards the septal pole, figure 2). Moreover, in the 50-120ms window, the most lateral positions (>3500um) seem to be more influenced by the medial ripples than relatively more central electrodes (~3000um). How can the authors explain this? To understand a little bit more how ripple features relate to the spiking dynamics, authors could try to generate heatmaps of the differential spiking between medial and lateral ripples (as they did in Fig. 4D-E) for 'strong' and 'common' ripples, or for local and propagating ripples.

Reviewer #2 (Public Review):

Lloyd et al examine the relationship between pupil size and fMRI signals in six brain nuclei responsible for providing the four major neuromodulators in the brain: norepinephrine from the locus coeruleus (LC), dopamine from ventral tegmental area (VTA) and substantia nigra, serotonin from the dorsal and median raphe nuclei, and acetylcholine from the cholinergic basal forebrain. Importantly, the authors focus on the relationship between these nuclei in the ascending arousal system (AAS) and the pupil at rest, outside of the context of any task, to determine the extent that small changes in pupil size are predictive of AAS activity.

Very few previous studies have examined this relationship at rest, perhaps in part because of the increased sensitivity required in the absence of event-based averaging. These nuclei are small (especially the LC), and thus are difficult to measure with standard fMRI.

The authors use a number of data collection and processing techniques to increase the sensitivity and precision of their recordings targeted to small ROIs. They find robust correlations between multiple AAS nuclei and pupil size with a time course that is not well captured by a standard hemodynamic response function (HRF).

The latter methodological finding is likely to be useful to the field for future studies focused on extracting useful signals from these nuclei, and the observed relationship between multiple AAS nuclei and the pupil support an emerging consensus from animal research that pupil fluctuations are correlated with neuromodulators besides norepinephrine.

Reviewer #2 (Public Review):

Mansur et al highlight interesting aspects of KLHL40-mediated proteostatic mechanisms in secretion and skeletal muscle development in zebrafish. They propose that KLHL40-mediated ubiquitylation of functional modules in the muscle proteome, particularly membrane traffic components, regulates protein abundance to control development. The authors present solid evidence for the role of KLHL40-mediated ubiquitylation and degradation of the cellular proteome but would benefit from further supporting evidence for their direct consequences on protein secretion.

Reviewer #2 (Public Review):

The availability of large collections of Mycobacterium tuberculosis (Mtb) isolates has enabled many important studies looking to identify mycobacterial genetic polymorphisms associated with anti-tuberculosis (TB) drug resistance, including both classical "resistance-conferring" mutations and novel "resistance-enabling" mutations. Importantly, these studies have expanded our understanding of mycobacterial genetic adaptations undermining chemotherapy, in many cases allowing for improved diagnostic tests and predictions of treatment failure. In this submission, Gao and colleagues adopt a different approach to the problem: although also applying a GWAS-type analysis, they instead attempt to elucidate polymorphisms implicated in poor outcomes of TB patients undergoing treatment for the drug-susceptible disease. Starting with a large dataset comprising 3496 samples with corresponding clinical (host) metadata, the authors generate Mtb whole-genome sequence data for 91 samples obtained from patients with "poor" outcomes and 3105 patients with "good" outcomes. These are used to identify 14 fixed and >230 unfixed mutations that might be associated with "poor" treatment outcomes, a conclusion which they argue is plausible given transcriptional evidence implicating many of the identified genes in the mycobacterial response in vitro to first-line drug exposure and/or hypoxia, both of which are considered relevant to clinical disease. Notably, they also identify a tendency for a greater proportion of "ROS mutational signatures" in unfixed mutations from "poor" outcome samples. Finally, incorporating these observations in a prediction model, the authors observe that the mycobacterial factors aren't adequate on their own but, when combined with key host factors - including patient age, sex, and duration of diagnostic delay (which have stronger predictive value) - they enhance predictive capacity. In summary, this paper reports a novel approach yielding observations that offer tantalizing insight into the mycobacterial factors which might influence TB treatment outcomes independent of drug resistance, however, the following must be considered:

(i) The manuscript provides little to no detail about how the samples were obtained, other than the fact that they comprise "pre-treatment" samples: are they all sputum samples? Were they induced? Similarly, no information is provided about sample propagation: were the samples cultured to achieve sufficient biomass for whole-genome sequencing? If so, in what growth media, for how long, and how many passages? Were all samples treated identically? And were they plated to single colonies - or are the "isolates" referred to throughout the manuscript actually heterogenous populations of potentially different Mtb clones obtained - and propagated - as a mixed sample? This information is critical given the potential that the identified polymorphisms - both fixed and (perhaps even more so) unfixed - might have arisen as a consequence of in vitro (laboratory) manipulation under standard aerobic conditions.

(ii) A key question that arises from this study (and others like it) is whether causation has been adequately established. Ideally, the Mtb genotypes contained within samples obtained pre-treatment should be compared with samples obtained from the same patients following treatment - that is, when the "poor" outcome was manifest. The expectation is that the polymorphisms identified prior to initiation of therapy - especially the 14 fixed mutations - should be evident (even dominant) at the later stage when therapy failed (or at the subsequent presentation in cases of relapse). Recognizing that this is not easily accomplished, though, it seems fair to suggest that the perceived relevance of the identified mutations would be strengthened if the authors were able to provide any other evidence - perhaps from studies of drug-resistant Mtb isolates - supporting their inferred role in undermining frontline treatment.

(iii) Related to the above, the authors make the valid point that their intention here was different from other studies which have deliberately utilized drug-resistant Mtb isolates to identify resistance-conferring and resistance-enabling mutations (such as in the study they cite by Hicks et al). It would be interesting to know, however, if any of the mutations identified in those other studies were also picked up in this work - and, if not, why that might be the case.

(iv) Finally, the analyses presented in this study are heavily dependent on the use of appropriate statistical methods to identify potentially rare genetic polymorphisms. However, as noted for sample processing (see my earlier comment above), there is very little detail provided about the methodology applied. This omission detracts from the interpretation, especially given that the predominance of lineage 2 (which contributes >75% of the isolates, with sublineage 2.3 constituting >50%) risks a lineage-specific association, rather than a more generalizable pathogenicity phenotype. Similarly, the heavy skew in the numbers of "good" (3105 samples) versus "poor" (91 samples) collections (approximately 34x difference in sample size) raises the possibility that mutations identified in the "poor" category might be artificially over-represented. More clarity in detailing the statistical methods is required to allay any concerns about the identification of candidate polymorphisms.

Reviewer #2 (Public Review):

Acute lung injury (ALI) and ARDS are major causes of morbidity and mortality in critically ill patients and patients infected with Sars-Cov-2. There are no effective therapies for ALI/ARDS, and the 28-day mortality rate is ~40%. One of the main pathological features of ALI/ARDS is a vascular injury characterized by endothelial dysfunction, inflammation, and in situ thrombosis. Using a murine model of ALI/ARDS triggered by diphtheria toxin (DT) mediated endothelial specific ablation, the authors apply sc-RNA-seq analysis to study how lung cell populations respond to injury and identify two main endothelial subpopulations responsible for regenerating lung vasculature over seven days. The study's implications are exciting as they provide evidence of intrinsic repair mechanisms that could be targeted for vascular regeneration and recovery of lung function in the context of ALI/ARDS. In particular, the apelin pathway rises as a prime therapeutic candidate given its role in coordinating the behavior of general and aerocyte capillary cells in lung vascular repair.

While the results of this study are exciting and novel, it must be recognized that several limitations need to be properly addressed to facilitate the translation of the findings toward medical care. For instance, the animal model used in this study (DT mediated EC ablation) does not fully recapitulate all the pathological hallmarks of ALI/ARDS, the most important of which is that repair proceeds at a very slow pace as a result of multiple factors that are not recapitulated in this made. Since the authors use only one model of ALI/ARDS, it is not entirely clear whether the current findings can be generalized to other models. Since no one model truly recapitulates the complexity of human ALI/ARDS, it is important to use at least two or more models that can narrow genetic and molecular mechanisms fundamental to lung injury and recovery. Another important aspect is the lack of validation in human samples and cells, which could strengthen the conclusions raised by the authors in the discussion. Finally, the authors appropriately emphasize how this study could help efforts to understand Sars-Cov2 mediated ALI/ARDS. Still, no studies explore any overlap with currently available Omics data from COVID lungs.

Despite these weaknesses, this study is the first to apply rigorous scRNA-seq analysis to this unique model of ALI/ARDS. It also provides data to support the importance of the two newly discovered endothelial cell subpopulations (gCap and aCap) in lung repair and regeneration, which hold the potential to offer unique mechanistic insights into the genetic and molecular mechanisms responsible for vascular repair and offers the opportunity to consider apelin based therapeutic approaches to treat ALI/ARDS. In conclusion, this study is expected to contribute to our lung biology understanding greatly. It provides the research community with novel resources and tools that greatly aid efforts to understand ALI/ARDS and identify therapeutics to treat this devastating disease.

Reviewer #2 (Public Review):

The authors utilized a label-free LC-MS/MS analysis in formalin-fixed paraffin-embedded (FFPE) tumors from 143 LNM-negative and 78 LNM-positive patients with T1 CRC to identify protein biomarkers to determine LNM in T1 CRC.

The authors used a fair number of clinical samples for the proteomics investigation. The experimental design is reasonable, and the statistical methods used in this manuscript are solid.

The authors largely achieved their aims and the results supported their conclusion. The method used in this proteomic study can also be used for the proteomics analysis of other cancer types to identify diagnostic and prognostic biomarkers. In addition, the 9 marker panel has a potential clinical diagnosis practice in determining LNM in T1 CRC.

Nevertheless, the authors need to justify their standards in selecting the biomarkers. For example, a p-value cut-off of 0.1 is not a usual criterion in similar proteomic studies. In addition, an identification frequency of 30% in patients seems not preferable for biomarker identification. The authors also need to justify the definition of fold change in the three subtypes with Kruskal-Walli's test. The authors need to describe more details on how they identified the 13 proteins from a 55-protein database. In addition, what is the connection between the final 9 proteins and the 19 proteins? What is the criterion to select 5 proteins for IHC validation from the 9 proteins?

Reviewer #2 (Public Review):

The paper by Laurenz Lammer and colleagues used cohort data to investigate the cross-sectional and longitudinal association between loneliness and brain structure and cognitive function. The main finding was that baseline social isolation and change in social isolation were associated with smaller hippocampus volumes, reduced cortical thickness, and poorer cognitive function. Given that more and more people feel lonely nowadays (e.g., due to the pandemic), the study by Lammer and colleagues addresses a highly relevant health concern of our time.

Significant strengths of the study:

- large cohort;<br /> - the cross-sectional and longitudinal analyses confirmed the findings;<br /> - the study was preregistered;<br /> - the study included men and women;<br /> - analyses were sound and controlled for essential confounders.

The major weaknesses of the study:

- it is unclear whether loneliness causally contributes to brain structure and cognitive function;<br /> - the factors that may cause loneliness are unclear.

Reviewer #2 (Public Review):

Overall, this manuscript provides a thorough characterization of the role of microtubules in the movement of GLUT4 in muscle fibers, and demonstrates the need for an intact microtubule network for GLUT4 responsiveness but only after the initial round of response.<br /> The study poses a very interesting question, rooted in studies in the literature studying the effects of Nocodazole (Noco) and C2-ceramide on GLUT4 traffic in cell systems. It is important to validate or refute predictions from those studies and, largely through this group's work, the quest to examine these questions in isolated muscle fibers and intact muscles as feasible is commendable. The authors develop very interesting imaging approaches to this end, and quantify the results in a convincing and elegant fashion. The system to measure 2-DG uptake and glucose uptake by electrochemical sensing in isolated fibers using the microfluidic pump is very ingenious.<br /> The main conclusion that microtubules are important for GLUT4 proper localization is important and adds mechanistic insight beyond that obtained from work in myoblasts and pre/adipocytes. The observation that microtubules are not engaged in GLUT4 traffic in the first round of insulin action but it is thereafter is also very revealing and should lead to more insights into the first and subsequent rounds of GLUT4 translocation.

Reviewer #2 (Public Review):

Schild et al. investigate the regulation of temporal control during neuroblast migration in the roundworm C. elegans. The authors find that expression of the Wnt pathway receptor Mig1 is regulated early through a specific noncoding conserved intronic element and later through two specific upstream conserved DNA elements. The expression levels of Mig1 in QR.pa cells are further regulated through Ced-3 and pig-1. The variability in the timing of later expression of Mig1 in QR.pa cells through bar-1 or a terminally truncated version of Bar1 was modulated but the mean expression did not change.

The single molecule RNA-FISH data is strong, and this method is sensitive enough to detect differences between different single cells and mutants. The mutants are very precise and straightforward to interpret. An additional strength is that many cells and replicas have been measured. The data analysis is simple.

The proposed model is simple with few intuitive parameters. This makes parameter identification straightforward. The qualitative predictions do make sense and are consistent with most experimental observations.

Overall the manuscript addresses the important question of timing regulation in transcription.

Reviewer #2 (Public Review):

The normally T cell restricted Src family tyrosine kinase Lck is ectopically expressed in most B cell Chronic Lymphocytic Leukemias. This, along with the fact that ectopic expression of other SFKs, such are Fyn and Fgr, are not seen, suggests that Lck may have some unique function, distinct from the endogenous Lyn SFK, that promotes malignant transformation. Using inducible expression in a human B cell lymphoma, the study explores this possibility. Studies reveal no qualitative functional differences in Lck and Lyn that are likely to explain its unique ectopic expression of Lck in CLL.

The strengths of this study include the use of Lentiviral transfer of genes encoding SFKs in conjunction with Doxycycline inducible expression. This allows comparative analysis of acute Lyn and Lck overexpression effects, free of cell resetting artifacts consequent to long term expression of the SFK. Strength is also seen in the authors fluorescent tagging of the SFK so analysis could be gated on ectopic expression level. Strength exists in the authors dissection of SFK effects on early events in the BCR signaling pathway, which reveal the ability of both overexpressed SFKs to drive receptor ITAM tyrosine phosphorylation and initiating BCR signaling. These studies reveal little difference in the function of the SFKs, though it appears that Lck may be less sensitive to phosphatase regulation.

It is unclear from the material and methods whether the overexpressed Lyn is LynA or Lyn B. It appears in the text (lines 130-133) that they overexpress LynB specifically. A recent paper from Tania Freedman (Sci Adv 2022 PMID:35452291) suggests that LynA is more activating whereas LynB is more balanced with an inhibitory bias. The point is that it is important to discuss this because they may not be making a relevant comparison.

If Lck promotes pathophysiology by transduction of a qualitatively unique signal, one would expect that transcriptome analysis should reveal this difference. The authors look for this signal using transcriptome analysis of bulk populations expressing similar levels of SFK. Although differences were seen in the transcriptome, finding were not consistent with a qualitatively unique function. However, bulk transcriptomic analysis may miss important differences. Single cell RNAseq, e.g., by 10x, may have been more incisive because gene expression could have been normalized to SFK expression in individual cells.

Finally, while some interesting differences are seen in the biology of Lyn and Lck, weakness exists in the failure to explore the causality of these differences in driving CLL phenotype. A final thought relevant to this comment. It is a truism that "absence of proof is not proof of absence".

Reviewer #2 (Public Review):

In this work, the authors aimed to understand the ion selectivity mechanism of a plant NRAMP-related aluminum transporter by structural and biochemical characterizations.

The authors successfully identified SiNRAT as a promising candidate for structural and biochemical analyses, showed that SiNRAT transport various divalent cations as well as binding to trivalent cations, determined the cryo-EM structure of SiNRAT, and performed structure-based mutational analysis to identify a potential binding site for metal ions. Unfortunately, the authors failed to show direct evidence of Al3- transport, due to technical problems. Furthermore, the structure of SiNRAT in complex with Al3+ was also not shown.

Despite such weakness, the structural comparison with other NRAMP members with different ion selectivity properties together with the extensive biochemical analyses would support the statement by the authors on a mechanism of ion selectivity for Al3+.

In the discussion section, the authors posed an important question. Considering the weak ion selectivity of SiNRAT over divalent cations, it is still unclear how NRAT proteins can function as an Al3+ transporter in a physiological condition where other divalent cations are also abundant. This would be an important question to be addressed in the related research field in the future.

The methods section is well written and the atomic coordinates and EM map file will be available to the community.

Reviewer #2 (Public Review):

The authors use data from 3 cross-sectional age-stratified serosurveys on Enterovirus D68 from England between 2006 and 2017 to examine the transmission dynamics of this pathogen in this setting. A key public health challenge on EV-D68 has been its implication in outbreaks of acute flaccid myelitis over the past decade, and past circulation patterns and population immunity to this pathogen are not yet well-understood. Towards this end, the authors develop and compare a suite of catalytic models as fitted to this dataset and incorporate different assumptions on how the force of infection varies over time and age. They find high overall EV-D68 seroprevalence as measured by neutralizing antibodies, and detect increased transmission during this time period as measured by the annual probability of infection and basic reproduction number. Interestingly, their data indicate very high seroprevalence in the youngest children (1 year-olds), and to accommodate this observation, the authors separate the force of infection in this age class from the other groups. They then reconstruct the historical patterns of EV-D68 circulation using their models and conclude that, while the serologic data suggest that transmissibility has increased between serosurvey rounds, additional factors not accounted for here (e.g., changes in pathogenicity) are likely necessary to explain the recent emergence of AFM outbreaks, particularly given the broader age-profile of reported AFM cases. The Discussion mentions important current unknowns on the biological interpretation of EV-D68 neutralizing antibody titers for protection against infection and disease. The analysis is rigorous and the conclusions are well-supported, but a few aspects of the work need to be clarified and extended, detailed below:

1) Due to the lack of a clear single cut-point for seropositivity on this assay, the authors sensibly present results for two cut-points in the main text (1:16 and 1:64). While some differences that stem from using different cut-points are fully expected (i.e., seroprevalence being higher using the less stringent cut-point), differences that are less expected should be further discussed. For instance, it was not clear in Figure 2 why the annual probability of infection decreased after 2010 using the 1:64 cut-point, while it continued to increase using the 1:16 cut-point. It would also be helpful to explain why overall seroprevalence and R0 continue to increase over this time period using the 1:64 cut-point. Lastly, it would be useful to see the x-axis in Figure 4 extended to the start of the time period that FOI is estimated, with accompanying credible intervals.

2) Additional context of EV-D68 in the study setting of England would be useful. While the Introduction does mention AFM cases "in the UK and elsewhere in Europe" (line 53), a summary of reported data on EV-D68/AFM in England prior to this study would provide important context. The Methods refers to "whether transmission had increased over time (before the first reported big outbreak of EV-D68 in the US in 2014)" (lines 133-134), rather than in this setting. It would be useful to summarize the viral genomic data from the region for additional context - particularly since the emergence of a viral clade is highlighted as a co-occurrence with the increased transmissibility detected in this analysis.

Reviewer #2 (Public Review):

This manuscript explores a novel technique to use dyes co-injected with various pharmaceutical reagents, like chemotherapic agents, to assess cellular effects in a cell culture model.

The major premise is that dye diffusion can be detected through fluorescent microscopy and be used as a measure of co-injected drug concentration. In chemotherapy commonly multiple drugs are given simultaneously, however, understanding how to tailor the concentrations of a multi-drug cocktail to each individual is largely trial and error. The authors surmise that perhaps using a cell culture model whereby cancer cells are cultured and then exposed to dye-tracked molecules an optimal multi-drug combination and concentrations can be determined. In other words, the intermixing of various connected drugs can then be fluorescently monitored to elucidate optimal concentrations of multi-drug combinations.

The concept overall is interesting but is relatively preliminary in its proof of concept. The authors note that varying free-diffusion of drugs out of the cell could complicate interpretation and that most of the analysis was done on a relatively short time basis and not longer evaluation periods that were more typical of chemotherapy.

Reviewer #2 (Public Review):

The authors unexpectedly found that the protein Grb2, an adaptor protein that mediates the recruitment of the Ras guanine-nucleotide exchange factor, SOS, to the EGF receptor, can be recruited to membranes by the immune cell tyrosine kinase Btk. The authors show, using total internal reflection fluorescence (TIRF) microscopy that the interaction with Grb2 is reversible, dependent on the proline-rich region of Btk, and independent of PIP3. These experiments are well performed and unambiguous.

The authors next asked whether Grb2 binding to Btk influences its kinase activity, by evaluating (i) Btk autophosphorylation and (ii) the phosphorylation of a peptide from the endogenous substrate PLC1. The readout relies on non-specific antibody-mediated detection of phosphotyrosine but nevertheless reveals a concentration-dependent increase in both Btk autophosphorylation and PLCy1 phosphorylation. The experiments, however, have only been performed in duplicate and, particularly in the case of PLCy1 phosphorylation, exhibit enormous variability which is not reflected in the example blot the authors have chosen to display in Figure 3C. Comparison of the same, duplicate experiment presented in Figure 3 Supplement 2 paints a very different picture.

The authors next sought to determine which domains of Grb2 are required for activation of Btk. Again, these experiments were only performed in duplicates, and the authors' claims that Grb2 can moderately stimulate the SH3-SH2-kinase module of Grb2 are not well supported by their data (Figure 4C-D).

The authors next asked whether Grb2 stimulates Btk by promoting its dimerization and trans-autophosphorylation. The authors measured the diffusion coefficient of Btk on PIP3-containing supported lipid bilayers in the presence and absence of Grb2. They noted that the diffusion coefficient of individual Btk particles decreases with increasing unlabeled Btk, which they interpret as Btk dimerization. Grb2 does not appear to influence the diffusion of Btk on the membrane (Figure 5A). Presumably, the diffusion coefficient reported here is the average of a number of single-molecule tracks, which should result in error bars. It is unclear why these have not been reported. Next, the authors assessed the ability of Grb2 to stimulate a mutant of Btk that is impaired in its ability to dimerize on PIP3-containing membranes. In contrast to wild-type Btk, autophosphorylation of dimerization-deficient Btk is not enhanced by Grb2. Whilst the data are consistent with this conclusion, again, the experiment has only been repeated once and the western blot presented in Figure 5 Supplement 2 is unreadable. It is also puzzling why Grb2 gets phosphorylated in this experiment, but not in the same experiment reported in Figure 3 Supplement 2.

Finally, the authors argue that Grb2 facilitates the recruitment of Btk to molecular condensates of adaptor and scaffold proteins immobilized on a supported lipid bilayer (SLB) (Figure 6). This is a highly complex series of experiments in which various components are added to supported lipid bilayers and the diffusion of labelled Btk is measured. When Btk is added to SLBs containing the LAT adaptor protein (phosphorylated in situ by Hck immobilized on the membrane via its His tag), it exhibits similar mobility to LAT alone, and its mobility is decreased by the addition of Grb2. The addition of the proline-rich region (PRR) of SOS further decreases this mobility. In this final condition, the authors incubate the reactions for 1 h until LAT undergoes a phase transition, forming gel-like, protein-rich domains on the membrane, shown in Figure 6B. The authors' conclusion that Btk is recruited into these phase-separated domains based on a slow-down in its diffusion is not well supported by the data, which rather indicates that Btk is excluded from these domains (Figure 6B - Btk punctae (green) are almost exclusively found in between the LAT condensates (red)). As such, the restricted mobility of Btk that the authors report may simply reflect the influence of barriers to diffusion on the membrane that result from LAT condensation into phase-separated domains. The authors also present data in Figure 6 Supplement 1 indicating that Grb2 recruitment to Btk is out-competed by SOS-PRR and that Btk does not support the co-recruitment of Grb2 and SOS-PRR to the membrane. These data would appear to suggest that the authors' interpretation of the decreased mobility of Btk on the membrane may not be correct.

Alternative explanations from Walfram Math world: here.

In brief, you can use an isometry map from a banach space to another subspace in a different Banach space such that it's dense.

Reviewer #2 (Public Review):

This is an exceptional study that provides conclusive evidence for the existence of a descending pathway from the brain that inhibits nociceptive behavioral outputs in larvae of Drosophila melanogaster. The authors identify molecular both molecular and neuronal/cellular components of this pathway. Converging lines of evidence and conclusive genetic experiments indicate that the neuropeptide, drosulfakinin (DSK), and its receptors (CCK1 and CCK2) function to inhibit nociception behaviors. Interestingly, the authors show that the relevant DSK neurons have cell bodies that are in the larval brain and that these neurons send projections into the thoracic ganglion and ventral nerve cord. Several lines of evidence support the hypothesis that fourth-order nociceptive neurons called Goro, are one relevant target for these outputs. RNAi knockdown of the CCK1 receptor in these cells sensitizes behavioral and physiological responses to noxious heat. Second, the axons of DSK neurons form physical contact with processes of Goro neurons as revealed by GRASP analysis. However, the authors' careful experiments indicate that the contacts between axons and Goro neurites might not be indicative of direct synapses and instead might operate through the bulk transmission of the peptidergic signals. The study raises many interesting questions for future study such as what behavioral contexts might depend on this pathway. Using the CAMPARI approach, the authors do not find that the DSK neurons are activated in response to nociceptive input but instead suggest that these cells may be tonically active in gating nociception. Future studies may find contexts in which the output of the DSK neurons is inhibited to facilitate nociception, or contexts in which the cells are more active to inhibit nociception.

Reviewer #2 (Public Review):

The voltage-gated potassium channel KCNQ1/KCNE1 (IKs) plays important physiological functions, for instance in the repolarization phase of the cardiac action potential. Loss-of-function of KCNQ1/KCNE1 is linked to disease. Hence, KCNQ1/KCNE1 is a highlighted pharmacological target and mechanistic insights into how channel modulators enhance the function of the channel is of great interest. The authors have through several previous studies provided mechanistic insights into how small-molecule activators like ML277 act on KCNQ1. However, less is known about the binding site and mechanism of action of other type of channel activators, which require KCNE1 for their effect. In this study, Chan and co-workers use molecular dynamics approaches, mutagenesis and electrophysiology to propose an overall similar binding site for the KCNQ1/KCNE1 activators mefenamic acid and DIDS, located at the extracellular interface of KCNQ1 and KCNE1. The authors propose an induced-fit model for the binding site, which critically engages residues in the N-terminus of KCNE1. Moreover, the authors discuss possible mechanisms of action of how drug binding to this site may enhance channel function.

The authors address an important question, of broad relevance to researchers in the field. The manuscript is generally well written and the text easy to follow. A strength of the work is the parallel use of experimental and simulation approaches, which enables both functional testing and mechanistic predictions and interpretations. For instance, the authors have experimentally assessed the putative relevance of a large set of residues based on simulation predictions. A limitation is that several methods need to be described in more detail to allow for evaluation of the presented data. Also, a more extensive presentation of representative data would be useful, along with discussions on the putative impact on drug effects of the diverse intrinsic properties of tested mutants.

Reviewer #2 (Public Review):

The authors provide comprehensive results showing that pharmacological inhibition of PI3K negatively affects heart tube formation via misoriented and slower cardiac movements. They used several cellular and molecular assays to demonstrate the potential mechanisms involved in PI3K-dependent cardiac fusion defects. Moreover, they use several imaging techniques and quantitative assessments to support their findings. Although the manuscript is well-written and most of their results support their conclusions, the manuscript and its findings heavily rely on high concentrations of PI3K small-molecule inhibitors, which will have off-target effects. The off-targets of PI3K pharmacological inhibition should be interpreted with caution and further evaluated. The authors suggest PI3K inhibition mediates heart tube formation throughout PI3K-mediated migration defects rather than PI3K-mediated proliferative defects. However, the authors did not further evaluate this later point; it should be considered carefully.

Reviewer #2 (Public Review):

In this study, Lamire et al. use a calcium imaging approach, behavioural tests, and pharmacological manipulations to identify the molecular mechanisms behind visual habituation. Overall, the manuscript is well-written but difficult to follow at times. They show a valuable new drug screen paradigm to assess the impact of pharmacological compounds on the behaviour of larval zebrafish, the results are convincing, but the description of the work is sometimes confusing and lacking details.

The volumetric calcium imaging of habituation to dark flashes is valuable, but the mix of responses to visual cues that are not relevant to the dark flash escape, such as the slow increase back to baseline luminosity, lowers the clarity of the results. The link between the calcium imaging results and free-swimming behaviour is not especially convincing, however, that is a common issue of head-restrained imaging with larval zebrafish.

The strong focus on GABA seems unwarranted based on the pharmacological results, as only Picrotoxinin gives clear results, but the other antagonists do not give a consistent results. On the other hand, the melatonin receptor agonists, and oestrogen receptor agonists give more consistent results, including more convincing dose effects.

The pharmacological manipulation of the habituation circuits mapped in the first part does not arrive at any satisfying conclusion, which is acknowledged by the authors. These results do reinforce the disconnect between the calcium imaging and the behavioural experiments and undercut somewhat the proposed circuit-level model.

Overall, the authors did identify interesting new molecular pathways that may be involved in habituation to dark flashes. Their screening approach, while not novel, will be a powerful way to interrogate other behavioural profiles. The authors identified circuit loci apparently involved in habituation to dark flashes, and the potentiation and no adaptation clusters have not been previously observed as far as I know.

The data will be useful to guide follow-up experiments by the community on the new pathway candidates that this screen has uncovered, including behaviours beyond dark flash habituation.

Reviewer #2 (Public Review):

The authors sequenced a clinical pathogen, Klebsiella FK688, and definitively establish the genetic basis of the carbapenem-resistance phenotype of this strain. They also show that the causal mutations confer reduced fitness under laboratory conditions, and that carbapenem sensitivity readily re-evolves in the lab due to the fitness costs associated with the resistance mutations in the clinical isolate. They also establish that subinhibitory concentrations of ceftazidime select for the otherwise deleterious blaDHA-1 gene. Based on this finding the authors speculate that prior beta-lactam selection faced by the ancestors of Klebsiella FK688 potentiated the evolution of the carbapenem-resistance phenotype of this strain. If this hypothesis is true, then prior history of beta-lactam exposure may generally potentiate the evolution of carbapenem resistance.

Strengths:

From a technical perspective, the findings in this paper are solid. In addition, the authors establish a simple genetic basis for carbapenem resistance in a clinical strain, which is a valuable and non-trivial finding (i.e. they show that the CRE phenotype in this strain is not an omnigenic trait distributed over hundreds of loci).

Weaknesses:

The main weakness of this paper is that the authors draw overly broad conclusions of a conceptual nature from narrow experimental findings. This could be addressed by drawing more modest and narrow implications from the findings.

1) The title of this paper is "Treatment history shapes the evolution of complex carbapenem-resistant phenotypes in Klebsiella spp." But they provide no data on the treatment history of the patient from whom this strain was isolated from. Therefore, the authors have no evidence to support their central claim. Indeed, it is completely possible that this strain never faced beta-lactam selection in the past, or that the patient's hypothetical history of betalactamase was irrelevant for the evolution of FK688. First, it is completely possible that this is a hospital-acquired infection, such that the history of this strain is due to selection in other contexts in the hospital that have little to do with the patient's treatment history. Second, it is completely possible that this strain (the chromosome anyway) has no prior history of beta-lactamase selection, and that it acquired the megaplasmid containing blaDHA-1 via conjugation from some other strain. In this second hypothetical scenario, it is possible that the fitness cost of the blaDHA-1 gene is not particularly high in a different source strain, but that it has some cost in the FK688 strain that it was isolated from. And of course, fitness costs in the human host could be very different than fitness costs in the laboratory, where strains are evolving under strong selection for fast growth. And given the benefit of resistance, it's clear that this strain clearly has a strong fitness advantage over faster-growing sensitive strains in the context of the source patient under antibiotic treatment.

My general point here is that the broad claims made about patient history or prior history shaping the evolution of this strain are largely indefensible because there is no data here to make solid inferences about *how* prior history shaped the evolution of this strain.

2) Historical contingency. The authors claim that their work shows how historical contingency shapes the evolution of resistance. One problem with this claim is that it is trivial- this is only a significant claim if the reader believes that prior history is not important in the evolution of antibiotic resistance, which is a straw-man null hypothesis, to mix a couple metaphors. To be more concrete, clearly strain background (prior history) matters-eliminating the plasmid with the resistance gene eliminates resistance. But that is not particularly surprising, given the past 50 years of evolutionary microbiology literature on plasmids and resistance. By contrast to this work, the major contribution of papers that examine the role of historical contingency in evolution (i.e. various Lenski papers) is that those works *quantitatively* measure the role of history in comparison to other factors (chance, adaptation). Since this work is a deep dive into a single clinical isolate, the data presented here do not and cannot shed light on the role of historical contingency in the emergence of this strain. The authors' claims about the prior history that led to the CRE phenotype are reasonable- but are fundamentally speculative. I have nothing against speculation, as long as it is clear what claims are speculative, and what are concrete implications. But the authors frame these speculative claims as concrete implications of their findings.

3) The authors claim that "[This work] suggests that the strategic combinations of antibiotics could direct the evolution of low-fitness, drug-resistant genotypes". I suppose this is true, but I also think this is a stretch of an implication given these findings. To be blunt, while I suppose it's better to have costly resistance variants that re-evolve sensitivity than to have low-cost high-resistance strains circulating, I think the patient's family would probably disagree that the evolution of a low-fitness drug-resistant genotype was good or strategic in the clinical context, even if better from a public health perspective. Low-fitness drug-resistant strains are just as lethal under clinical antibiotic concentrations!

The authors do show the plausibility of their hypothesis/model that prior beta-lactam selection is sufficient to potentiate the evolution of carbapenem-resistance (by the additional ompK loss-of-function mutation). I think those findings are very nice. But the authors undermine their results by extrapolating too far from their data. Hence, I think narrowing the scope of the implications would improve this paper.

In addition to narrowing the scope of the implications as written, I also would like to add that there may be other ways of framing this paper (other than historical contingency) that may make the significance of this work more apparent to a broader audience. This may be worth considering during the revision process.