Reviewer #3 (Public Review):

Induced pluripotent stem cells, or iPSCs, are cells that scientists can push to become new, more mature cell types like neurons. iPSCs have a high potential to transform how scientists study disease by combining precision medicine gene editing with processes known as high-content imaging and drug screening. However, there are many challenges that must be overcome to realize this overall goal. The authors of this paper solve one of these challenges: predicting cell types that might result from potentially inefficient and unpredictable differentiation protocols. These predictions can then help optimize protocols.

The authors train advanced computational algorithms to predict single-cell types directly from microscopy images. The authors also test their approach in a variety of scenarios that one may encounter in the lab, including when cells divide quickly and crowd each other in a plate. Importantly, the authors suggest that providing their algorithms with just the right amount of information beyond the cells' nuclei is the best approach to overcome issues with cell crowding.

The work provides many well-controlled experiments to support the authors' conclusions. However, there are two primary concerns: (1) The model may be relying too heavily on the background and thus technical artifacts (instead of the cells) for making CNN-based predictions, and (2) the conclusion that their nucleocentric approach (including a small area beyond the nucleus) is not well supported, and may just be better by random chance. If the authors were to address these two concerns (through additional experimentation), then the work may influence how the field performs cell profiling in the future.

Additionally, the impact of this work will be limited, given the authors do not provide a specific link to the public source code that they used to process and analyze their data.

Reviewer #3 (Public Review):

Summary:

The mechanical properties of DNA wrapped in nucleosomes affect the stability of nucleosomes and may play a role in the regulation of DNA accessibility in eukaryotes. In this manuscript, Ngo and coworkers study how the stability of a nucleosome is affected by the introduction of a CC mismatched base pair, which has been reported to increase the flexibility of DNA. Previously, the group has used a sophisticated combination of single-molecule FRET and force spectroscopy with an optical trap to show that the more flexible half of a 601 DNA segment provides for more stable wrapping as compared to the other half. Here, it is confirmed with a single-molecule cyclization essay that the introduction of a CC mismatch increases the flexibility of a DNA fragment. Consistent with the previous interpretation, it also increased the unwrapping force for the half of the 601 segment in which the CC mismatch was introduced, as measured with single-molecule FRET and force spectroscopy. Enhanced stability was found up to 56 bp into the nucleosome. The intricate role of mechanical stability of nucleosomes was further investigated by comparing force-induced unwrapping profiles of yeast and Xenopus histones. Intriguingly, asymmetric unwrapping was more pronounced for yeast histones.

Strengths:

(1) High-quality single-molecule data.

(2) Novel mechanism, potentially explaining the increased prominence of mutations near the dyads of nucleosomes.

(3) A clear mechanistic explanation of how mismatches affect nucleosome stability.

Weaknesses:

(1) Disconnect between mismatches in nucleosomes and measurements comparing Xenopus and yeast nucleosome stability.

(2) Convoluted data in cyclization experiments concerning the phasing of mismatches and biotin site.

Reviewer #3 (Public Review):

Summary:

In this study, Wang et al. have demonstrated that TMC7, a testis-enriched multipass transmembrane protein, is essential for male reproduction in mice. Tmc7 KO male mice are sterile due to reduced sperm count and abnormal sperm morphology. TMC7 co-localizes with GM130, a cis-Golgi marker, in round spermatids. The absence of TMC7 results in reduced levels of Golgi proteins, elevated abundance of ER stress markers, as well as changes of Ca2+ and pH levels in the KO testis. However, further confirmation is required because the analyses were performed with whole testis samples in spite of the differences in the germ cell composition in WT and KO testis. In addition, the causal relationships between the reported anomalies await thorough interrogation.

Strengths:<br /> The microscopic images are of great quality, all figures are properly arranged, and the entire manuscript is very easy to follow.

Weaknesses:<br /> Tmc7 KO male mice show multiple anomalies in sperm production and morphogenesis, such as reduced sperm count, abnormal sperm head, and deformed midpiece. Thus, it is confusing that the authors focused solely on impaired acrosome biogenesis. Further investigations are warranted to determine whether the abnormalities reported in this manuscript (e.g., changes in protein, Ca2+, and pH levels) are directly associated with the molecular function of TMC7 or are the byproducts of partially arrested spermiogenesis. Please find additional comments in "Recommendations for the authors".

Reviewer #3 (Public Review):

Summary:

A known feature of cilia in vertebrates and many, if not all, invertebrates is the striking heterogeneity of their lengths among different cell types. The underlying mechanisms, however, remain largely elusive. In the manuscript, the authors addressed this question from the angle of intraflagellar transport (IFT), a cilia-specific bidirectional transportation machinery essential to biogenesis, homeostasis, and functions of cilia, by using zebrafish as a model organism. They conducted a series of experiments and proposed an interesting mechanism. Furthermore, they achieved in situ live imaging of IFT in zebrafish larvae, which is a technical advance in the field.

Strengths:

The authors initially demonstrated that ectopically expressed Ift88-GFP through a certain heat-shock induction protocol fully sustained the normal development of mutant zebrafish that would otherwise be dead by 7 dpf due to the lack of this critical component of IFT-B complex. Accordingly, cilia formations were also fully restored in the tissues examined. By imaging the IFT using Ift88-GFP in the mutant fish as a marker, they unexpectedly found that both anterograde and retrograde velocities of IFT trains varied among cilia of different cell types and appeared to be positively correlated with the length of the cilia.

For insights into the possible cause(s) of the heterogeneity in IFT velocities, the authors assessed the effects of IFT kinesin Kif3b and Kif17, BBSome, and glycylation or glutamylation of axonemal tubulin on IFT and excluded their contributions. They also used a cilia-localized ATP reporter to exclude the possibility of different ciliary ATP concentrations. When they compared the size of Ift88-GFP puncta in crista cilia, which are long, and spinal cord cilia, which are relatively short, by imaging with a cutting-edge super-resolution microscope, they noticed a positive correlation between the puncta size, which presumably reflected the size of IFT trains, and the length of the cilia.

Finally, they investigated whether it is the size of IFT trains that dictates the ciliary length. They injected a low dose (0.5 ng/embryo) of ift88 MO and showed that, although such a dosage did not induce the body curvature of the zebrafish larvae, crista cilia were shorter and contained less Ift88-GFP puncta. The particle size was also reduced. These data collectively suggested mildly downregulated expression levels of Ift88-GFP. Surprisingly, they observed significant reductions in both retrograde and anterograde IFT velocities. Therefore, they proposed that longer IFT trains would facilitate faster IFT and result in longer cilia.

Weaknesses:

The current manuscript, however, contains serious flaws that markedly limit the credibility of major results and findings. Firstly, important experimental information is frequently missing, including (but not limited to) developmental stages of zebrafish larvae assayed (Figures 1, 3, and 5), how the embryos or larvae were treated to express Ift88-GFP (Figures 3-5), and descriptions on sample sizes and the number of independent experiments or larvae examined in statistical results (Figures 3-5, S3, S6). For instance, although Figure 1B appears to be the standard experimental scheme, the authors provided results from 30-hpf larvae (Figure 3) that, according to Figure 1B, are supposed to neither express Ift88-GFP nor be genotyped because both the first round of heat shock treatment and the genotyping were arranged at 48 hpf. Similarly, the results that ovl larvae containing Tg(hsp70l:ift88 GFP) (again, because the genotype is not disclosed in the manuscript, one can only deduce) display normal body curvature at 2 dpf after the injection of 0.5 ng of ift88 MO (Fig 5D) is quite confusing because the larvae should also have been negative for Ift88-GFP and thus displayed body curvature. Secondly, some inferences are more or less logically flawed. The authors tend to use negative results on specific assays to exclude all possibilities. For instance, the negative results in Figures 4A-B are not sufficient to "suggest that the variability in IFT speeds among different cilia cannot be attributed to the use of different motor proteins" because the authors have not checked dynein-2 and other IFT kinesins. In fact, in their previous publication (Zhao et al., 2012), the authors actually demonstrated that different IFT kinesins have different effects on ciliogenesis and ciliary length in different tissues. Furthermore, instead of also examining cilia affected by Kif3b or Kif17 mutation, they only examined crista cilia, which are not sensitive to the mutations. Similarly, their results in Figures 4C-G only excluded the importance of tubulin glycylation or glutamylation in IFT. Thirdly, the conclusive model is based on certain assumptions, e.g., constant IFT velocities in a given cell type. The authors, however, do not discuss other possibilities.

Reviewer #3 (Public Review):

Summary:

The authors studied the function of Hsp110 co-chaperones (e.g. yeast Sse1) in Hsp70-dependent protein disaggregation reactions. The study builds on former work by the authors (Wyszkowski et al., 2021, PNAS), analyzing the binding of Hsp70 and J-domain protein (JDP) cochaperones to protein aggregates using bio-layer interferometry (BLI). It was shown before by other groups that Hsp110 enhances Hsp70 disaggregation activity. The mechanism of Hsp110-stimulated disaggregation activity, however, remained poorly defined. Here, the authors show that yeast Hsp110 increases Hsp70 recruitment to the surface of protein aggregates. The effect is largely dependent on J-domain protein (JDP) identity and is particularly pronounced for class B JDPs (e.g. yeast Sis1), which are also more effective in disaggregation reactions. The authors also confirm former results, showing inhibition by increased Hsp110 levels, and provide novel evidence that the inhibitory effect is caused by competition between Hsp110 and JDPs for Hsp70 binding.

Strengths:

The work represents a very thoroughly executed study, which provides novel insights into the mechanism of Hsp70-mediated protein disaggregation. Key findings established for yeast chaperones are also documented for human counterparts. The observation that Hsp110 might displace JDPs from Hsp70 during the disaggregation reaction is very appealing. It will now become important to validate this initial finding and dissect how it propels the disaggregation reaction.

Weaknesses:

How exactly the interplay between JDPs and Hsp110 orchestrates protein disaggregation remains largely speculative and further analysis is required for a deeper mechanistic understanding. Enhanced recruitment of Hsp70 in the presence of Hsp110 was shown for amyloid fibrils before (Beton et al., EMBO J 2022) and should be acknowledged. The assay reporting on the refolding activity of Hsp70 seems problematic due to the high spontaneous refolding of the substrate Luciferase and should be modified.

Reviewer #3 (Public Review):

Overall:

ExoIII has been described and commercialized as a dsDNA-specific nuclease. Several lines of evidence, albeit incomplete, have indicated this may not be entirely true. Therefore, Wang et al comprehensively characterize the endonuclease and exonuclease enzymatic activities of ExoIII on ssDNA. A strength of the manuscript is the testing of popular kits that utilize ExoIII and coming up with and testing practical solutions (e.g. the addition of SSB proteins ExoIII variants such as K121A and varied assay conditions).

Comments:

(1) The footprint of ExoIII on DNA is expected to be quite a bit larger than 5-nt, see structure in manuscript reference #5. Therefore, the substrate design in Figure 1A seems inappropriate for studying the enzymatic activity and it seems likely that ExoIII would be interacting with the FAM and/or BHQ1 ends as well as the DNA. Could this cause quenching? Would this represent real ssDNA activity? Is this figure/data necessary for the manuscript?

(2) Based on the descriptions in the text, it seems there is activity with some of the other nucleases in 1C, 1F, and 1I other than ExoIII and Cas12a. Can this be plotted on a scale that allows the reader to see them relative to one other?

(3) The sequence alignment in Figure 2N and the corresponding text indicates a region of ExoIII lacking in APE1 that may be responsible for their differences in substrate specificity in regards to ssDNA. Does the mutational analysis support this hypothesis?

Reviewer #3 (Public Review):

This study aimed to explore the computational mechanisms of view invariance, driven by the observation that in some regions of monkey visual cortex, neurons show comparable responses to (1) a given face and (2) to the same face but horizontally flipped. Here they study this known phenomenon using AlexNet and other shallow neural networks, using an index for mirror symmetric viewpoint tuning based on representational similarity analyses. They find that this tuning is enhanced at fully connected- or global pooling layers (layers which combine spatial information), and that the invariance is prominent for horizontal- but not vertical- or rotational transformations. The study shows that mirror tuning can be learned when a given set of images are flipped horizontally and given the same label, but *not* if they are flipped and given different labels. They also show that networks learn this tuning by focusing on local features, not global configurations.

I found the study to be a mixed read. Some analyses were fascinating: for example, it was satisfying to see the use of well-controlled datasets to increase or decrease the rate of mirror-symmetry tuning. The insertion- and deletion¬ experiments were elegant tests to probe the mechanisms of mirror symmetry, asking if symmetry could arise from (1) global feature configurations (in a holistic sense) vs. (2) local features, with stronger evidence for the latter. These two sets of results were successful and interpretable. They stand in contrast with the first analysis, which relies on observations that do not seem justified. Specifically, Figure 2D shows mirror-symmetry tuning across 11 stages of image processing, from pixels space to fully connected layers. It shows that images from different object categories evoke considerably different tuning index values. The explanation for this result is that some categories, such as "tools," have "bilaterally symmetric structure," but this is not explicitly measured anywhere. "Boats" are described as having "front-back symmetry," more so than flowers. One imagines flowers being extremely symmetric, but perhaps that depends on the metric. What is the metric? At first I thought it was the mirror-symmetric viewpoint tuning index in the image (pixel) space, but this cannot be, as the index for faces and flowers is negative, cars have no symmetry, and boats are positive. To support these descriptions, one must have an independent variable (for object class symmetry) that can be related to the dependent variable (the mirror-symmetric viewpoint tuning index). If it exists, it is not a part of the Results section. This omission undermines other parts of the Results section: "some car models have an approximate front-back symmetry...however, a flower typically does not..." "Some," "typically:" how many in the dataset exactly, and how often? The description of CIFAR-10 as having bilaterally symmetric categories - are all these categories equally symmetric? If not, would such variability matter in terms of these results? These assessments of object category symmetry values are made before experiments are presented, so they are not interpretations of the results, and it would be circular to write it otherwise.

Overall, my bigger concern is that the framing is misleading or at best incomplete. The manuscript successfully showed that if one introduces left-right symmetry to a dataset, the network will develop population-level representations that are also bilaterally symmetric. But the study does not explain that the model's architecture and random weight distribution are sufficient for symmetry tuning to emerge, without training, just to a much more limited degree. Baek et al. showed in 2021 that viewpoint-invariant face-selective units and mirror-symmetric units emerge in untrained networks ("Face detection in untrained deep neural networks"; this current manuscript cites this paper but does not mention that mirror symmetry is a feature of the 2021 study). This current study also used untrained networks as controls (Fig. 3), and while they were useful in showing that learning boosts symmetry tuning, the results also clearly show that horizontal-reflection invariance is far from zero. So, the simple learning-driven explanation for the mirror-symmetric viewpoint tuning for faces is wrong: while (1) network training and (2) pooling are mechanisms that charge the development of mirror-symmetric tuning, the lottery ticket hypothesis is enough for its emergence. Faces and numbers are simple patterns, so the overparameterization of networks is enough to randomly create units that are tuned to these shapes and to wire many of them together. How learning shapes this process is an interesting direction, especially now that this current study has outlined its importance.

Finally, it would help to cite other previous demonstrations of equivariance and mirror symmetry in neural networks. Chris Olah, Nick Cammarata, Chelsea Voss, Ludwig Schubert, and Gabriel Goh of OpenAI wrote of this phenomenon in 2020 (Distill journal).

Some other observations that might help:

- I am enthusiastic about the experiments using different datasets to increase or decrease the rate of mirror-symmetry tuning (sets including CIFAR10, SVHN, symSVHN, asymSVHN); it is worth noting, however, that the lack of a ground truth metric for category symmetry is a problem here too. In the asymSVHN dataset, images are flipped and given different labels. If some categories are naturally symmetric after horizontal flips, such as images containing "0" or "8", then changing the label is likely to disturb training. This would explain why the training loss is larger for this condition (Figure S4D).

- It is puzzling why greyscale 3D rendered images are used. By using greyscale 3D render (at least as shown in the figures) the study proceeds as if the units are invariant under color transformations. Unfortunately, this is not true and using greyscale images impact the activations of different layers of Alexnet in a way that is not fully defined. Moreover, many units in shallow networks focus on color and exactly these units could be invariant to other transformation like the mirror symmetry, but grey scaling the images makes them inactive.

Reviewer #3 (Public Review):

It has recently been demonstrated that bacteria in biofilms show changes in membrane potential in response to changes in their environment, and that these can propagate signals through the biofilm to coordinate bacterial behavior. Akabuogu et al. contribute to this exciting research area with a study of blue light-induced membrane potential dynamics in E. coli biofilms. They demonstrate that Thioflavin-T (ThT) intensity (a proxy for membrane potential) displays multiphasic dynamics in response to blue light treatment. They additionally use genetic manipulations to implicate the potassium channel Kch in the latter part of these dynamics. Mechanosensitive ion channels may also be involved, although these channels seem to have blue light-independent effects on membrane potential as well. In addition, there are challenges to the quantitative interpretation of ThT microscopy data which require consideration. The authors then explore whether these dynamics are involved in signaling at the community level. The authors suggest that cell firing is both more coordinated when cells are clustered and happens in waves in larger, 3D biofilms; however, in both cases evidence for these claims is incomplete. The authors present two simulations to describe the ThT data. The first of these simulations, a Hodgkin-Huxley model, indicates that the data are consistent with the activity of two ion channels with different kinetics; the Kch channel mutant, which ablates a specific portion of the response curve, is consistent with this. The second model is a fire-diffuse-fire model to describe wavefront propagation of membrane potential changes in a 3D biofilm; because the wavefront data are not presented clearly, the results of this model are difficult to interpret. Finally, the authors discuss whether these membrane potential changes could be involved in generating a protective response to blue light exposure; increased death in a Kch ion channel mutant upon blue light exposure suggests that this may be the case, but a no-light control is needed to clarify this.

In a few instances, the paper is missing key control experiments that are important to the interpretation of the data. This makes it difficult to judge the meaning of some of the presented experiments.

(1) An additional control for the effects of autofluorescence is very important. The authors conduct an experiment where they treat cells with CCCP and see that Thioflavin-T (ThT) dynamics do not change over the course of the experiment. They suggest that this demonstrates that autofluorescence does not impact their measurements. However, cellular autofluorescence depends on the physiological state of the cell, which is impacted by CCCP treatment. A much simpler and more direct experiment would be to repeat the measurement in the absence of ThT or any other stain. This experiment should be performed both in the wild-type strain and in the ∆kch mutant.

(2) The effects of photobleaching should be considered. Of course, the intensity varies a lot over the course of the experiment in a way that photobleaching alone cannot explain. However, photobleaching can still contribute to the kinetics observed. Photobleaching can be assessed by changing the intensity, duration, or frequency of exposure to excitation light during the experiment. Considerations about photobleaching become particularly important when considering the effect of catalase on ThT intensity. The authors find that the decrease in ThT signal after the initial "spike" is attenuated by the addition of catalase; this is what would be predicted by catalase protecting ThT from photobleaching (indeed, catalase can be used to reduce photobleaching in time lapse imaging).

(3) It would be helpful to have a baseline of membrane potential fluctuations in the absence of the proposed stimulus (in this case, blue light). Including traces of membrane potential recorded without light present would help support the claim that these changes in membrane potential represent a blue light-specific stress response, as the authors suggest. Of course, ThT is blue, so if the excitation light for ThT is problematic for this experiment the alternative dye tetramethylrhodamine methyl ester perchlorate (TMRM) can be used instead.

(4) The effects of ThT in combination with blue light should be more carefully considered. In mitochondria, a combination of high concentrations of blue light and ThT leads to disruption of the PMF (Skates et al. 2021 BioRXiv), and similarly, ThT treatment enhances the photodynamic effects of blue light in E. coli (Bondia et al. 2021 Chemical Communications). If present in this experiment, this effect could confound the interpretation of the PMF dynamics reported in the paper.

(5) Figures 4D - E indicate that a ∆kch mutant has increased propidium iodide (PI) staining in the presence of blue light; this is interpreted to mean that Kch-mediated membrane potential dynamics help protect cells from blue light. However, Live/Dead staining results in these strains in the absence of blue light are not reported. This means that the possibility that the ∆kch mutant has a general decrease in survival (independent of any effects of blue light) cannot be ruled out.

(6) Additionally in Figures 4D - E, the interpretation of this experiment can be confounded by the fact that PI uptake can sometimes be seen in bacterial cells with high membrane potential (Kirchhoff & Cypionka 2017 J Microbial Methods); the interpretation is that high membrane potential can lead to increased PI permeability. Because the membrane potential is largely higher throughout blue light treatment in the ∆kch mutant (Fig. 3AB), this complicates the interpretation of this experiment.

Throughout the paper, many ThT intensity traces are compared, and described as "similar" or "dissimilar", without detailed discussion or a clear standard for comparison. For example, the two membrane potential curves in Fig. S1C are described as "similar" although they have very different shapes, whereas the curves in Fig. 1B and 1D are discussed in terms of their differences although they are evidently much more similar to one another. Without metrics or statistics to compare these curves, it is hard to interpret these claims. These comparative interpretations are additionally challenging because many of the figures in which average trace data are presented do not indicate standard deviation.

The differences between the TMRM and ThT curves that the authors show in Fig. S1C warrant further consideration. Some of the key features of the response in the ThT curve (on which much of the modeling work in the paper relies) are not very apparent in the TMRM data. It is not obvious to me which of these traces will be more representative of the actual underlying membrane potential dynamics.

A key claim in this paper (that dynamics of firing differ depending on whether cells are alone or in a colony) is underpinned by "time-to-first peak" analysis, but there are some challenges in interpreting these results. The authors report an average time-to-first peak of 7.34 min for the data in Figure 1B, but the average curve in Figure 1B peaks earlier than this. In Figure 1E, it appears that there are a handful of outliers in the "sparse cell" condition that likely explain this discrepancy. Either an outlier analysis should be done and the mean recomputed accordingly, or a more outlier-robust method like the median should be used instead. Then, a statistical comparison of these results will indicate whether there is a significant difference between them.

In two different 3D biofilm experiments, the authors report the propagation of wavefronts of membrane potential; I am unable to discern these wavefronts in the imaging data, and they are not clearly demonstrated by analysis.

The first data set is presented in Figures 2A, 2B, and Video S3. The images and video are very difficult to interpret because of how the images have been scaled: the center of the biofilm is highly saturated, and the zero value has also been set too high to consistently observe the single cells surrounding the biofilm. With the images scaled this way, it is very difficult to assess dynamics. The time stamps in Video S3 and on the panels in Figure 2A also do not correspond to one another although the same biofilm is shown (and the time course in 2B is also different from what is indicated in 2B). In either case, it appears that the center of the biofilm is consistently brighter than the edges, and the intensity of all cells in the biofilm increases in tandem; by eye, propagating wavefronts (either directed toward the edge or the center) are not evident to me. Increased brightness at the center of the biofilm could be explained by increased cell thickness there (as is typical in this type of biofilm). From the image legend, it is not clear whether the image presented is a single confocal slice or a projection. Even if this is a single confocal slice, in both Video S3 and Figure 2A there are regions of "haze" from out-of-focus light evident, suggesting that light from other focal planes is nonetheless present. This seems to me to be a simpler explanation for the fluorescence dynamics observed in this experiment: cells are all following the same trajectory that corresponds to that seen for single cells, and the center is brighter because of increased biofilm thickness.

The second data set is presented in Video S6B; I am similarly unable to see any wave propagation in this video. I observe only a consistent decrease in fluorescence intensity throughout the experiment that is spatially uniform (except for the bright, dynamic cells near the top; these presumably represent cells that are floating in the microfluidic and have newly arrived to the imaging region).

3D imaging data can be difficult to interpret by eye, so it would perhaps be more helpful to demonstrate these propagating wavefronts by analysis; however, such analysis is not presented in a clear way. The legend in Figure 2B mentions a "wavefront trace", but there is no position information included - this trace instead seems to represent the average intensity trace of all cells. To demonstrate the propagation of a wavefront, this analysis should be shown for different subpopulations of cells at different positions from the center of the biofilm. Data is shown in Figure 8 that reflects the velocity of the wavefront as a function of biofilm position; however, because the wavefronts themselves are not evident in the data, it is difficult to interpret this analysis. The methods section additionally does not contain sufficient information about what these velocities represent and how they are calculated. Because of this, it is difficult for me to evaluate the section of the paper pertaining to wave propagation and the predicted biofilm critical size.

There are some instances in the paper where claims are made that do not have data shown or are not evident in the cited data:

(1) In the first results section, "When CCCP was added, we observed a fast efflux of ions in all cells"- the data figure pertaining to this experiment is in Fig. S1E, which does not show any ion efflux. The methods section does not mention how ion efflux was measured during CCCP treatment.

(2) In the discussion of voltage-gated calcium channels, the authors refer to "spiking events", but these are not obvious in Figure S3E. Although the fluorescence intensity changes over time, it's hard to distinguish these fluctuations from measurement noise; a no-light control could help clarify this.

(3) The authors state that the membrane potential dynamics simulated in Figure 7B are similar to those observed in 3D biofilms in Fig. S4B; however, the second peak is not clearly evident in Fig. S4B and it looks very different for the mature biofilm data reported in Fig. 2. I have some additional confusion about this data specifically: in the intensity trace shown in Fig. S4B, the intensity in the second frame is much higher than the first; this is not evident in Video S6B, in which the highest intensity is in the first frame at time 0. Similarly, the graph indicates that the intensity at 60 minutes is higher than the intensity at 4 minutes, but this is not the case in Fig. S4A or Video S6B.

Reviewer #3 (Public Review):

The authors present in this study the characterization of two mutant lines of the filasterean Capsaspora owczarzaki, a unicellular holozoan with a key phylogenetic position to understand multicellular development in animals. The present study is built on a previous work from the same research group, on the mutant of the orthologue of the Yorki gene in C. owczarzaki. By knocking out the two upstream kinases of the same pathway, coHpo-/- and coWts-/-, in single cell and aggregates of C. owkzarzaki, they now have mutated the entire pathway and in different cellular contexts.

The authors obtain results in the same direction as the previous work, demonstrating that the Hippo pathway of the unicellular holozoan C. owczarzaki, is not involved in the control of cell proliferation but is related with cytoskeletal dynamics through the actin-myosin mechanism.

In this revised version of the study, the authors have addressed my concerns by providing additional experiments, references and discussing further the points of controversy.

I think the authors have done a great job improving the robustness of the paper proving further some of the claims raised in the previous version of the manuscript.

Reviewer #3 (Public Review):

Summary:

The manuscript examined the role of large versus small prediction errors (PEs) in creating a state-based memory distinction between acquisition and extinction. The premise of the paper is based on theoretical claims and empirical findings that gradual changes between acquisition and extinction would lead to the potential overwriting of the acquisition memory with extinction, resulting in a more durable reduction in conditioned responding (i.e. more durable extinction effect). The paper tests the hypotheses in a series of elegant experiments in which the shock intensity is decreased across extinction sessions before non-reinforced CS presentations are given. Additional manipulations include context change, shock devaluation, and controlling for lower shock intensity exposure. The critical comparison was standard non-reinforced extinction training. The critical tests were done in spontaneous recovery and reinstatement.

Strengths:<br /> The findings are of tremendous importance in understanding how memories can be updated and reveal a well-defined role of PE in this process. It is well-established that PE is critical for learning, so delineating how PE is critical for generating memory states and the role it serves in keeping memories dissociable (or not) is exciting and clever. As such the paper addresses a fundamental question in the field.

The studies test clear and defined predictions derived from simulations of the state-belief model of Cochran & Cisler (2019). The designs are excellent: well-controlled and address the question.

The authors have done an excellent job of explaining the value of the latent state models.

The authors have studied both sexes in the study presented, providing generality across the sexes in their findings. However, depicting the individual data points in the bar graphs and noting which data represent males and which represent females would be of great value.

Weaknesses:

(1) While it seems obvious that delivering a lower intensity shock will generate a smaller PE than say no shock, it would have been nice to see data from say a compound testing procedure that confirms this.

(2) The devaluation experiment is quite clever, but it also would be strengthened if there was evidence in the paper that this procedure does indeed lead to shock devaluation.

(3) It would have been very exciting to see even more parametric examinations of this idea, like maintaining shock intensity but gradually reducing shock duration, which would have increased the impact of the paper.

(4) Individual data points should be represented in the test figures (see above also).

Reviewer #3 (Public Review):

Summary:<br /> This manuscript describes the symptoms of patients harboring KCNQ2 mutation G256W, functional changes of the mutant channel in exogenous expression, and phenotypes of G256W/+ mice. The patients presented seizures, the mutation reduced currents of the channel, and the G256W/+ mice showed seizures, increased firing frequency in neurons, reduced KCNQ2 expression,<br /> and altered subcellular distribution.

Strengths:

This is a large amount of work and all results corroborated the pathogenicity of the mutation in KCNQ2, providing an interesting example of KCNQ2-associated neurological disorder's impact on functions at all levels including molecular, cellular, tissue, animal model, and patients.

Weaknesses:

The manuscript described observations of changes in association with the mutation at molecular cellular functions and animal phenotype, but the results in some aspects are not as strong as in others. Nevertheless, the manuscript made overarching conclusions even when the evidence was not sufficiently strong.

yeah. but the actual problem is pacifism and overpopulation.<br /> all other problems, including world wars, are only symptoms of pacifism and overpopulation.<br /> this is just another intelligence test, and again, most people fail, most people are idiots

Reviewer #3 (Public Review):

Stout et al investigate the link between prefrontal-hippocampal (PFC-HPC) theta-band coherence and accurate decisions in spatial decision making tasks. Previous studies show that PFC-HPC theta coherence positively correlates with task learning and correct decisions but the nature of this relation relies on correlations that cannot show whether coherence leads, coincides or is a consequence of decision making. To investigate more precisely this link, the authors devise a novel paradigm. In this paradigm the rat is blocked during a delay period preceding its choice and they control on a trial-by-trial basis the level of PFC-HPC theta coherence prior to the decision by allowing the rat to access the choice point only at a time when coherence reaches above or below a threshold. The design of the paradigm is very well controlled in many ways. First, using the PFC-HPC theta coherence during the delay period to gate when the rat accesses the choice zone clearly separates this coherence from the behavioural decision itself. Moreover, the behaviour of the animal is similar during high and low coherence periods. Finally, control trials are matched trial-by-trial to the time spent waiting by the rat when gated on theta coherence, which is crucial given that working memory performance depends on delay duration. All these features bolster the specificity of the author's main finding which is that PFC-HPC theta coherence prior to choice making is strongly predictive of accuracy in two tasks : one that requires working memory and another in which behaviour is cue-guided. Although this paradigm does not provide direct causal evidence, it convincingly supports the idea that PFC-HPC theta coherence prior to the behavioural decision is related to correct decision making and is not simply temporally coincidental or a consequence of the decision output.

The authors also investigate the mechanisms behind the increase in PFC-HPC coherence during the task and show that it likely involves the recruitment of a small population of PFC neurons, via interactions with the Ventral Midline Thalamus that could mediate prefrontal/hippocampal dialogue.

A key point of interest is the unexpected result showing a link between theta coherence even in the cue-driven version of the task. As the authors point out, muscimol inhibition of neither PFC nor HPC, nor the ventral midline thalamus impacts performance in this task. This raises the question of why coherence between two areas is predictive of choice accuracy when neither area appears to be causally involved. The authors discuss various options and explanations for this discrepancy which clearly adds to the current debate. Moreover their novel paradigm provides new tools to interrogate when inter-area synchrony is associated with information transfer and when this information is then used to drive behavioural decisions.

"we have been winning battles... but losing the war

big environmentalist shifting toward anti-capitalism

Reviewer #3 (Public Review):

Summary and strengths:

In the manuscript, Abd El Hay et al investigate the role of thermally sensitive ion channels TRPM2 and TRPV1 in warm preference and their dynamic response features to thermal stimulation. They develop a novel thermal preference task, where both the floor and air temperature are controlled, and conclude that mice likely integrate floor with air temperature to form a thermal preference. They go on to use knockout mice and show that TRPM2-/- mice play a role in the avoidance of warmer temperatures. Using a new approach for culturing DRG neurons they show the involvement of both channels in warm responsiveness and dynamics. This is an interesting study with novel methods that generate important new information on the different roles of TRPV1 and TRPM2 on thermal behavior.

Open questions and weaknesses:

(1) Differences in the response features of cells expressing TRPM2 and TRPV1 are central and interesting findings but need further validation (Figures 3 and 4). To show differences in the dynamics and the amplitude of responses across different lines and stimulus amplitudes more clearly, the authors should show the grand average population calcium response from all responsive neurons with error bars for all 3 groups for the different amplitudes of stimuli (as has been presented for the thermal stimuli traces). The authors should also provide a population analysis of the amplitude of the responses in all groups to all stimulus amplitudes. Prior work suggests that thermal detection is supported by an enhancement or suppression of the ongoing activity of sensory fibers innervating the skin. The authors should present any data on cells with ongoing activity.

(2) The authors should better place their findings in context with the literature and highlight the novelty of their findings. The introduction builds a story of a 'disconnect' or 'contradictory' findings about the role of TRPV1 and TRPM2 in warm detection. While there are some disparate findings in the literature, Tan and McNaughton (2016) show a role for TRPM2 in the avoidance of warmth in a similar task, Paricio et al. (2020) show a significant reduction in warm perception in TRPM2 and TRPV1 knock out lines and Yarmolinksy et al. (2016) show a reduction in warm perception with TRPV1 inactivation. All these papers are therefore in agreement with the authors finding of a role for these channels in warm behavior. The authors should change their introduction and discussion to more correctly discuss the findings of these studies and to better pinpoint the novelty of their own work.

(3) The responses of 60 randomly selected cells are shown in Figure 2B. But, looking at the TRPM2-/- data, warm responses appear more obvious than in WTs and the weaker responders of the WT group appear weaker than the equivalent group in the TRPV1-/- and TRPM2-/- data. This does not necessarily invalidate the results, but it may suggest a problem in the data selection. Because the correct classification of warm-sensitive neurons is central to this part of the study more validation of the classifier should be presented. For example, the authors could state if they trained the classifier using equal amounts of cells, show some randomly selected cells that are warm-insensitive for all genotypes, and show the population average responses of warm-insensitive neurons.

(4) The interpretation of the main behavioral results and justification of the last figure is presented as the result of changes in sensing but differences in this behavior could be due to many factors and this needs clarification and discussion. (i) The authors mention that 'crucially temperature perception is not static' and suggest that there are fluctuating changes in perception over time and conclude that their modelling approach helps show changes in temperature detection. They imply that temperature perceptual threshold changes over time, but the mouse could just as easily have had exactly the same threshold throughout the task but their motivation (or some other cognitive variable) might vary causing them to change chamber. The authors should correct this. (ii) Likewise, from their fascinating and high-profile prior work the authors suggest a model of internal temperature sensing whereby TRPM2 expression in the hypothalamus acts as an internal sensory of body temperature. Given this, and the slow time course of the behavior in chambers with different ambient temperatures, couldn't the reason for the behavioral differences be due to central changes in hypothalamic processing rather than detection by skin temperature? If TRPM2-/- were selectively ablated from the skin or the hypothalamus (these experiments are not necessary for this paper) it might be possible to conclude whether sensation or body temperature is more likely the root cause of these effects but, without further experiments it is tough to conclude either way. (iii) Because the ambient temperature is controlled in this behavior, another hypothesis is that warm avoidance could be due to negative valence associated with breathing warm air, i.e. a result of sensation within the body in internal pathways, rather than sensing from the external skin. Overall, the authors should tone down conclusions about sensation and present a more detailed discussion of these points.

(5) It is an excellent idea to present a more in-depth analysis of the behavioral data collected during the preference task, beyond 'the mouse is on one side or the other'. However, the drift-diffusion approach is complex to interpret from the text in the results and the figures. The results text is not completely clear on which behavioral parameters are analyzed and terms like drift, noise, estimate, and evidence are not clearly defined. Currently, this section of the paper slightly confuses and takes the paper away from the central findings about dynamics and behavioral differences. It seems like they could come to similar conclusions with simpler analysis and simpler figures.

(6) In Figure 2D the % of warm-sensitive neurons are shown for each genotype. Each data point is a field of view, however, reading the figure legend there appear to be more FOVs than data points (eg 10 data points for the TRPV1-/- but 17 FOVs). The authors should check this.

(7) Can the authors comment on why animals with over-expression of TRPV1 spend more time in the warmest chamber to start with at 38C and not at 34C?

Reviewer #3 (Public Review):

Reviewing Editor's Summary:

The revised manuscript has clarified concerns raised by the reviewers concerning the analysis method in constructing the correlation matrix. These key results are now readily comprehensible. They have also added a final section to the Discussion, sketching some important questions for future research (e.g., number/resolution of channels and extension of the logic used here to look at number channels in other tasks).

Reviewer 1 was satisfied with these changes and has updated their review. Reviewer 2 did not think the revision tackled the theoretical issues raised in their initial review; as such, this reviewer has opted to leave their initial public review unchanged.

I also believe that the revision does not adequately address a major theoretical issue, namely whether the current data provide evidence of sensorimotor number channels, the central claim of the paper. The authors argue that since perception is noise free (stimuli were given symbolically), then the task variance comes from processes associated with sensorimotor transformation. Let's consider the task: A number is presented, the participant then attempts to produce that number of taps. To preclude counting, they are required to say the syllable "ba" as fast as possible while tapping. The sensorimotor channel idea would suppose that the symbolic stimulus activates a set of channels, each of which specifies the number of taps that should be produced. For example, a "6" channel likes to produce 6 outputs (with variability), a "10" channel 10 outputs (with variability), etc., with the actual production of the (weighted) integration of these outputs.

An alternative is that, since explicit counting is prevented by the secondary task, the participant makes an internal estimation of the number of produced taps. These judgments could be based on the output of amodal number channels. For example, the same process would be at play if the task were changed such that the participants watched a dot flash and had to estimate the number of flashes (while concurrently saying "ba"). The authors indicate in their response letter that they are conducting experiments along these lines and that the results are similar. They suggest that this provides support for the existence of both sensory and sensorimotor number channels. Extending this, if the experiment were tones instead of flashes, the argument would be that there are auditory, visual, and sensorimotor number channels. It seems more parsimonious to interpret such a pattern as reflective of amodal number channels.

I recognize there are other intriguing reasons to think there may be intimate links between our sense of number and movement, but I remain unconvinced that the current results provide evidence for sensorimotor number channels.

Reviewer #3 (Public Review):

Summary:

This manuscript reports two neuroimaging experiments assessing commonalities and differences in activation loci across mechanical problem-solving, technical reasoning, fluid cognition, and "mentalizing" tasks. Each task includes a control task. Conjunction analyses are performed to identify regions in common across tasks. As Area PF (a part of the supramarginal gyrus of the inferior parietal lobe) is involved across 3 of the 4 tasks, the investigators claim that it is the hub of technical cognition.

Strengths:

The aim of finding commonalities and differences across related problem-solving tasks is a useful and interesting one.

The experimental tasks themselves appear relatively well-thought-out, aside from the concern that they are differentially difficult.

The imaging pipeline appears appropriate.

Weaknesses:

(1) Methodological<br /> As indicated in the supplementary tables and figures, the experimental tasks employed differ markedly in 1) difficulty and 2) experimental trial time. Response latencies are not reported (but are of additional concern given the variance in difficulty). There is concern that at least some of the differences in activation patterns across tasks are the result of these fundamental differences in how hard various brain regions have to work to solve the tasks and/or how much of the trial epoch is actually consumed by "on-task" behavior. These difficulty issues should be controlled for by 1) separating correct and incorrect trials, and 2) for correct trials, entering response latency as a regressor in the Generalized Linear Models, 3) entering trial duration in the GLMs.

A related concern is that the control tasks also differ markedly in the degree to which they were easier and faster than their corresponding experimental task. Thus, some of the control tasks seem to control much better for difficulty and time on task than others. For example, the control task for the psychotechnical task simply requires the indication of which array contains a simple square shape (i.e., it is much easier than the psychotechnical task), whereas the control task for mechanical problem-solving requires mentally fitting a shape into a design, much like solving a jigsaw puzzle (i.e., it is only slightly easier than the experimental task).

(2) Theoretical<br /> The investigators seem to overlook prior research that does not support their perspective and their writing seems to lack scientific objectivity in places. At times they over-reach in the claims that can be made based on the present data. Some claims need to be revised/softened.

Reviewer #2 (Public Review):

Summary:

Hoogstraaten et al investigated the involvement of rabphilin-3A RPH3A in DCV fusion in neurons during calcium-triggered exocytosis at the synapse and during neurite elongation. They suggest that RPH3A acts as an inhibitory factor for LDV fusion and this is mediated partially via its interaction with SNAP25 and not Rab3A/Rab27. It is a very elegant study although several questions remain to be clarified.

Strengths:

The authors use state-of-the-art techniques like tracking NPY-PHluorin exocytosis and FRAP experiments to quantify these processes providing novel insight into LDCs exocytosis and the involvement of RPH3A.

Weaknesses:

At the current state of the manuscript, further supportive experiments are necessary to fully support the authors' conclusions.

Reviewer #3 (Public Review):

Summary:

In this work, the authors use a Hidden Markov Model (HMM) to describe dynamic connectivity and amplitude patterns in fMRI data, and propose to integrate these features with the Fisher Kernel to improve the prediction of individual traits. The approach is tested using a large sample of healthy young adults from the Human Connectome Project. The HMM-Fisher Kernel approach was shown to achieve higher prediction accuracy with lower variance on many individual traits compared to alternate kernels and measures of static connectivity. As an additional finding, the authors demonstrate that parameters of the HMM state matrix may be more informative in predicting behavioral/cognitive variables in this data compared to state-transition probabilities.

Strengths:

- Overall, this work helps to address the timely challenge of how to leverage high-dimensional dynamic features to describe brain activity in individuals.<br /> - The idea to use a Fisher Kernel seems novel and suitable in this context.<br /> - Detailed comparisons are carried out across the set of individual traits, as well as across models with alternate kernels and features.<br /> - The paper is well-written and clear, and the analysis is thorough.

Potential weaknesses:

- One conclusion of the paper is that the Fisher Kernel "predicts more accurately than other methods" (Section 2.1 heading). I was not certain this conclusion is fully justified by the data presented, as it appears that certain individual traits may be better predicted by other approaches (e.g., as shown in Figure 3) and I found it hard to tell if certain pairwise comparisons were performed -- was the linear Fisher Kernel significantly better than the linear Naive normalized kernel, for example?

- While 10-fold cross-validation is used for behavioral prediction, it appears that data from the entire set of subjects is concatenated to produce the initial group-level HMM estimates (which are then customized to individuals). I wonder if this procedure could introduce some shared information between CV training and test sets. This may be a minor issue when comparing the HMM-based models to one another, but it may be more important when comparing with other models such as those based on time-averaged connectivity, which are calculated separately for train/test partitions (if I understood correctly).

Reviewer #3 (Public Review):

Summary:

This paper seeks to understand the role of alveolar myofibroblasts in abnormal lung development after saccular stage injury.

Strengths:

Multiple models of neonatal injury are used, including hyperoxia and transgenic models that target alveolar myofibroblasts.

Weaknesses:

There are several weaknesses that leave the conclusions significantly undersupported by the data as presented:

(1) There is no validation of the decreased number of myofibroblasts suggested by flow cytometry/scRNAseq at the level of the tissue. Given that multiple groups have reported increased myofibroblasts (aSMA+ fibroblasts) in humans with BPD and in mouse models, demonstrating a departure from prior findings with tissue validation in the mouse models is essential. There are many reasons for decreased numbers of a subpopulation by flow cytometry, most notably that injured cells may be less likely to survive the cell sorting process.

(2) The hallmark genes used to define the subpopulations are not given in single-cell data. As the definition of fibroblast subtypes remains an area of unsettled discussion in the field, it is possible that the decreased number by classification and not a true difference. Tissue validation and more transparency in the methods used for single-cell sequencing would be critical here.

(3) There is an oversimplification of neonatal hyperoxia as a "BPD model" used here without a reference to detailed prior work demonstrating that the degree and duration of hyperoxia dramatically change the phenotype. For example, Morty et al have shown that hyperoxia of 85% or more x 14 days is required to demonstrate the septal thickening observed in severe human BPD. Other than one metric of lung morphometry (MLI), which is missing units on the y-axis and flexivent data, the authors have not fully characterized this model. Prior work comparing 75% O2 exposure for 5, 8, or 14 days shows that in the 8-day exposed group (similar to the model used here), much of the injury was reversible. What evidence do the authors have that hyperoxia alone is an accurate model of the permanent structural injury seen in human BPD?

(4) Thibeault et al published a single-cell analysis of neoantal hyperoxia in 2021, with seemingly contrasting findings. How does this dataset compare in context?

Reviewer #3 (Public Review):

Summary:

This is a well-prepared manuscript that presented interesting research results. The only defect is that the authors should further revise the English language.

Strengths:

The omics method produced unbiased results.

Weaknesses:

LPS neutralization is not a new method for treating leptospiral infection.

Reviewer #3 (Public Review):

Summary:<br /> This study provides a comparison of TCR gene segment usage between foetal and adult thymus.

Strengths:<br /> Interesting computational analyses was performed to find interesting differences in TCR gene usage within unpaired TCRa and TCRb chains between foetal and adult thymus.

Weaknesses:<br /> This study was significantly lacking insight and interpretation into what the data analysed actually means for the biology. The dataset discussed in the paper is from only two experiments. One comparing foetal and adult thymi from 4 mice per group and another which involved hydrocortisone treatment. The paper uses TCR sequencing methodology that sequences each TCR alpha and beta chains in an unpaired way, meaning that the true identity of the TCR heterodimer is lost. This also has the added problem of overestimating clonality, and underestimating diversity.<br /> Limited detail in the methods sections also limits the ability for readers to properly interpret the dataset. What sex of mice were used? Are there any sex differences? What were the animal ethics approvals for the study?

Reviewer #3 (Public Review):

This manuscript reports a detailed genetic and epigenomic analysis of diagnostic and relapsed AML. The study is mostly correlative and some of the initial findings, such as the stability of mutations in epigenetic regulators at diagnosis and relapse and in signaling pathway modulators such as FLT3 and NRAS being lost - not novel.

The authors show that in a large fraction (approximately half) of the relapsed AMLs they study, there are no alterations in the AML driver mutations. The authors conclude that this indicates that these patients show non-genetic mechanisms of relapse, for which the authors embark on a series of epigenomic experiments to try and pin down the correlative or causative epigenetic mechanisms. In 9 (out of 25) patient samples with stable driver mutations ( i.e. no change in clonality or novel AML driver mutation accumulation) the study shows that there is high epigenetic variability as measured by chromatin accessibility changes and that these changes resemble less differentiated state in the relapsed compared to the diagnostic sample. The manuscript makes some key observations: 1) non-genetic mechanisms are likely to account for relapse in a substantial proportion of patients. 2) some of the clones that emerge following relapse are likely present in prior diagnosis samples indicating that chemotherapy selects for them, 3) Of note, the authors also look at the LSC and non-LSC compartments and show that the LSC compartment is more rigid in terms of epigenetic evolution towards relapse than the non-LSC cells. 4) Using a small number of patients (but justifiable since the assays used are rigorous and demanding) - the authors present the most interesting finding of the study - that epigenetic evolution of relapse in several different AML patients seems to be convergent.<br /> This is based on the epigenetic similarities in clones (as defined by mitochondrial Atac-seq) between different epigenetic relapsed clones, even though they were distinct at diagnosis. Thus, this study has several important observations. Some of these observations are incremental - it has been shown that epigenetic mechanisms drive relapse in AML but several are not. I think this study - although descriptive and not showing causal relationships - is an important study for advancing our understanding of AML relapse.

Reviewer #3 (Public Review):

Summary:<br /> In the manuscript by Valenzisi et al., the authors report on the role of WRNIP1 to prevent R-loop and TRC-associated DNA damage. The authors claim WRNIP1 localizes to TRCs in response to replication stress and prevents R-loop accumulation, TRC formation, replication fork stalling, and subsequent DNA damage. While the findings are of potential significance to the field, the strength of evidence in support of the conclusions is lacking.

In the revised submission by Velenzisi et al., the manuscript is still missing the controls that were requested in the original review. One cannot conclude the D37A mutant is unable to rescue DNA damage unless it is shown in the same experiment that the WT is able to rescue it. This is also true for the fork speed, stalled forks, and restarting forks experiments. Below is a list of Figures missing key controls.

Figure 1B -missing the shWRNIP1WT control<br /> Figure 1C - missing the MRC5SV control<br /> Figure 1D - missing the shWRNIP1WT control<br /> Figure 3C - missing the shWRNIP1WT control<br /> Figure 5A - missing the shWRNIP1WT control<br /> Figure 5B - missing the shWRNIP1WT control<br /> Figure 5C - missing the shWRNIP1WT control<br /> Figure 5D - missing the shWRNIP1WT control<br /> Figure 6C - missing the shWRNIP1WT control

Also, the authors did not explain the result showing shWRNIP1 decreases DNA damage compared to MRC5SV in Figure 1D (compare lanes 4 and 8). Again, this suggests WRNIP1 actually increases DNA damage in response to Aph and DRB. This concern was raised in the original peer review, and it remains unaddressed in the revised manuscript.

The use of RNaseHIII increases the specificity of the S9.6 antibody and improves confidence in the DNA-RNA hybrid data throughout the manuscript.

Reviewer #3 (Public Review):

Summary:

The vesicular monoamine transporter is a key component in neuronal signaling and is implicated in diseases such as Parkinson's. Understanding of monoamine processing and our ability to target that process therapeutically has been to date provided by structural modeling and extensive biochemical studies. However, structural data is required to establish these findings more firmly.

Strengths:

Dalton et al resolved a structure of VMAT2 in the presence of an important inhibitor, tetrabenazine, with the protein in detergent micelles, using cryo-EM and with the aid of protein domains fused to its N- and C-terminal ends, including one fluorescent protein that facilitated protein screening and purification. The resolution of the maps allows clear assignment of the amino acids in the core of the protein. The structure is in good agreement with a wealth of experimental and structural prediction data, and provides important insights into the binding site for tetrabenazine and selectivity relative to analogous compounds. The authors provide additional biochemical analyses that further support their findings. The comparison with AlphaFold models is enlightening.

Reviewer #3 (Public Review):

Summary:

This manuscript describes a study of the olfactory tubercle in the context of reward representation in the brain. The authors do so by studying the responses of OT neurons to odors with various reward contingencies and compare systematically to the ventral pallidum. Through careful tracing, they present convincing anatomical evidence that the projection from the olfactory tubercle is restricted to the lateral portion of the ventral pallidum.

Using a clever behavioral paradigm, the authors then investigate how D1 receptor- vs. D2 receptor-expressing neurons of the OT respond to odors as mice learn different contingencies. The authors find that, while the D1-expressing OT neurons are modulated marginally more by the rewarded odor than the D2-expressing OT neurons as mice learn the contingencies, this modulation is significantly less than is observed for the ventral pallidum. In addition, neither of the OT neuron classes shows conspicuous amount of modulation by the reward itself. In contrast, the OT neurons contained information that could distinguish odor identities. These observations have led the authors to conclude that the primary feature represented in the OT may not be reward.

Strengths:

The highly localized projection pattern from olfactory tubercle to ventral pallidum is a valuable finding and suggests that studying this connection may give unique insights into the transformation of odor by reward association.

Comparison of olfactory tubervle vs. ventral pallidum is a good strategy to further clarify the olfactory tubercle's position in value representation in the brain.

Weaknesses:

The study comes to a different conclusion about the olfactory tubercle regarding reward representations from several other prior works. Whether this stems from a difference in the experimental configurations such as behavioral paradigms used or indeed points to a conceptually different role for the olfactory tubercle remains to be seen.

Reviewer #3 (Public Review):

Summary:<br /> This study indicates that connections across human cortical pyramidal cells have identical latencies despite a larger mean dendritic and axonal length between somas in the human cortex. A precise demonstration combining detailed electrophysiology and modeling indicates that this property is due to faster propagation of signals in proximal human dendrites. This faster propagation is itself due to a slightly thicker dendrite, a larger capacitive load, and stronger hyperpolarizing currents. Hence, the biophysical properties of human pyramidal cells are adapted such that they do not compromise information transfer speed.

Strengths:<br /> The manuscript is clear and very detailed. The authors have experimentally verified a large number of aspects that could affect propagation speed and have pinpointed the most important one. This paper provides an excellent comparison of biophysical properties between rat and human pyramidal cells. Thanks to this approach a comprehensive description of the mechanisms underlying the acceleration of propagation in human dendrite is provided.

Weaknesses:<br /> Several aspects having an impact on propagation speed are highlighted (dendritic diameter, ionic channels, capacitive load) and there is no clear ranking of their impact on signal propagation speed. It seems that the capacitive load plays a major role, much more than dendritic diameter for which only a 10% increase is observed across species. Both aspects actually indicate that there is an increase in passive signal propagation speed with bigger cells at least close to the soma. This suggests that bigger cells are mechanically more rapid. An intuitive reason why capacitive load increases speed would also help the reader follow the demonstration.

Reviewer #3 (Public Review):

Summary:<br /> This work offers a novel perspective on the question of how hippocampal networks can adaptively generate different spatial maps and replays/preplays of the corresponding place cells, without any such maps pre-existing in the network architecture or its inputs. Unlike previous modeling attempts, the authors do not pre-tune their model neurons to any particular place fields. Instead, they build a random, moderately-clustered network of excitatory (and some inhibitory) cells, similar to CA3 architecture. By simulating spatial exploration through border-cell-like synaptic inputs, the model generates place cells for different "environments" without the need to reconfigure its synaptic connectivity or introduce plasticity. By simulating sleep-like random synaptic inputs, the model generates sequential activations of cells, mimicking preplays. These "preplays" require small-world connectivity, so that weakly connected cell clusters are activated in sequence. Using a set of electrophysiological recordings from CA1, the authors confirm that the modeled place cells and replays share many features with real ones. In summary, the model demonstrates that spontaneous activity within a small-world structured network can generate place cells and replays without the need for pre-configured maps.

Strengths:<br /> This work addresses an important question in hippocampal dynamics. Namely, how can hippocampal networks quickly generate new place cells when a novel environment is introduced? And how can these place cells preplay their sequences even before the environment is experienced? Previous models required pre-existing spatial representations to be artificially introduced, limiting their adaptability to new environments. Other models depended on synaptic plasticity rules which made remapping slower than what is seen in recordings. This modeling work proposes that quickly-adaptive intrinsic spiking sequences (preplays) and spatially tuned spiking (place cells) can be generated in a network through randomly clustered recurrent connectivity and border-cell inputs, avoiding the need for pre-set spatial maps or plasticity rules. The proposal that small-world architecture is key for place cells and preplays to adapt to new spatial environments is novel and of potential interest to the computational and experimental community.

The authors do a good job of thoroughly examining some of the features of their model, with a strong focus on excitatory cell connectivity. Perhaps the most valuable conclusion is that replays require the successive activation of different cell clusters. Small-world architecture is the optimal regime for such a controlled succession of activated clusters.

The use of pre-existing electrophysiological data adds particular value to the model. The authors convincingly show that the simulated place cells and preplay events share many important features with those recorded in CA1 (though CA3 ones are similar).

Weaknesses:<br /> To generate place cell-like activity during a simulated traversal of a linear environment, the authors drive the network with a combination of linearly increasing/decreasing synaptic inputs, mimicking border cell-like inputs. These inputs presumably stem from the entorhinal cortex (though this is not discussed). The authors do not explore how the model would behave when these inputs are replaced by or combined with grid cell inputs which would be more physiologically realistic.

Even though the authors claim that no spatially-tuned information is needed for the model to generate place cells, there is a small location-cue bias added to the cells, depending on the cluster(s) they belong to. Even though this input is relatively weak, it could potentially be driving the sequential activation of clusters and therefore the preplays and place cells. In that case, the claim for non-spatially tuned inputs seems weak. This detail is hidden in the Methods section and not discussed further. How does the model behave without this added bias input?

Unlike excitation, inhibition is modeled in a very uniform way (uniform connection probability with all E cells, no I-I connections, no border-cell inputs). This goes against a long literature on the precise coordination of multiple inhibitory subnetworks, with different interneuron subtypes playing different roles (e.g. output-suppressing perisomatic inhibition vs input-gating dendritic inhibition). Even though no model is meant to capture every detail of a real neuronal circuit, expanding on the role of inhibition in this clustered architecture would greatly strengthen this work.

For the modeling insights to be physiologically plausible, it is important to show that CA3 connectivity (which the model mimics) shares the proposed small-world architecture. The authors discuss the existence of this architecture in various brain regions but not in CA3, which is traditionally thought of and modeled as a random or fully connected recurrent excitatory network. A thorough discussion of CA3 connectivity would strengthen this work.

Reviewer #3 (Public Review):

In this manuscript, Ma et al seek to identify stem cell niche factors. They perform an RNAi screen in glial cells and screen for candidates that support and maintain neuroblasts (NBs) in the developing fly brain. Through this, they identify two subunits of ferritin, which is a conserved protein that can store iron in cells in a non-toxic form and release it in a controlled manner when and where required. They present data to support the conclusion that ferritin produced in glia is released and taken up by NBs where it is utilised by enzymes in the Krebs cycle as well as in the electron transport chain. In its absence from glia, NBs are unable to generate sufficient energy for division and therefore prematurely differentiate via nuclear prospero resulting in small brains. The work will be of interest to those interested in neural stem cells and their non-cell autonomous control by niches.

The past decade has seen a growing appreciation of how glial cells support and maintain NBs during development. The authors' discovery of glial-derived ferritin providing essential iron atoms for energy production is interesting and important. They have employed a variety of genetic tools and assays to uncover how ferritin in glia might support NBs. This is particularly challenging because there are no direct ways of assaying for iron or energy consumption in a cell-specific manner.

There are however instances where conclusions are drawn to support the story being developed without considering the equally plausible alternative explanations that should ideally be addressed.

For example, the data supporting the transfer of ferritin from glia to NBs was weak given the misexpression system used; the Shi[ts] experiment was also not convincing (perhaps they have more representative images?).

The iron manipulation experiments are in the whole animal and it is likely that this affects general feeding behaviour, which is known to affect NB exit from quiescence and proliferative capacity. The loss of ferritin in the gut and iron chelators enhancing the NB phenotype are used as evidence that glia provide iron to NB to support their number and proliferation. Since the loss of NB is a phenotype that could result from many possible underlying causes (including low nutrition), this specific conclusion is one of many possibilities.

Similarly, knockdown of the FeS protein assembly components phenocopy glial ferritin knock down. Since iron is so important for the TCA and the ETC, this is not surprising, but the similarities in the two phenotypes seem insufficient to say that it's glial ferritin that's causing the lack of iron in the NB and therefore resulting in loss of NBs.

Pros RNAi will certainly result in an increase in NB numbers because the loss of pros results in an inability of NB progeny to differentiate. This (despite the slight increase in nuclear pros) is not sufficient to infer that glial ferritin knockdown results in premature differentiation of NBs via nuclear pros.

I recognise these are challenging to prove irrefutably, however, the frequency of such expansive interpretations of data is of concern.

Reviewer #3 (Public Review):

Summary:<br /> Loewinger et al., extend a previously described framework (Cui et al., 2021) to provide new methods for statistical analysis of fiber photometry data. The methodology combines functional regression with linear mixed models, allowing inference on complex study designs that are common in photometry studies. To demonstrate its utility, they reanalyze datasets from two recent fiber photometry studies into mesolimbic dopamine. Then, through simulation, they demonstrate the superiority of their approach compared to other common methods.

Strengths:<br /> The statistical framework described provides a powerful way to analyze photometry data and potentially other similar signals. The provided package makes this methodology easy to implement and the extensively worked examples of reanalysis provide a useful guide to others on how to correctly specify models.

Modeling the entire trial (function regression) removes the need to choose appropriate summary statistics, removing the opportunity to introduce bias, for example in searching for optimal windows in which to calculate the AUC. This is demonstrated in the re-analysis of Jeong et al., 2022, in which the AUC measures presented masked important details about how the photometry signal was changing.

Meanwhile, using linear mixed methods allows for the estimation of random effects, which are an important consideration given the repeated-measures design of most photometry studies.

Weaknesses:<br /> While the availability of the software package (fastFMM), the provided code, and worked examples used in the paper are undoubtedly helpful to those wanting to use these methods, some concepts could be explained more thoroughly for a general neuroscience audience.

While the methodology is sound and the discussion of its benefits is good, the interpretation and discussion of the re-analyzed results are poor:

In section 2.3, the authors use FLMM to identify an instance of Simpson's Paradox in the analysis of Jeong et al. (2022). While this phenomenon is evident in the original authors' metrics (replotted in Figure 5A), FLMM provides a convenient method to identify these effects while illustrating the deficiencies of the original authors' approach of concatenating a different number of sessions for each animal and ignoring potential within-session effects. The discussion of this result is muddled. Having identified the paradox, there is some appropriate speculation as to what is causing these opposing effects, particularly the decrease in sessions. In the discussion and appendices, the authors identify (1) changes in satiation/habitation/motivation, (2) the predictability of the rewards (presumably by the click of a solenoid valve) and (3) photobleaching as potential explanations of the decrease within days. Having identified these effects, but without strong evidence to rule all three out, the discussion of whether RPE or ANCCR matches these results is probably moot. In particular, the hypotheses developed by Jeong et al., were for a random (unpredictable) rewards experiment, whereas the evidence points to the rewards being sometimes predictable. The learning of that predictability (e.g. over sessions) and variation in predictability (e.g. by attention level to sounds of each mouse) significantly complicate the analysis. The FLMM analysis reveals the complexity of analyzing what is apparently a straightforward task design. If this paper is not trying to arbitrate between RPE and ANCCR, as stated in the text, the post hoc reasoning of the authors of Jeong et al 2022 provided in the discussion is not germane. Arbitrating between the models likely requires new experimental designs (removing the sound of the solenoid, satiety controls) or more complex models (e.g. with session effects, measures of predictability) that address the identified issues.

Of the three potential causes of within-session decreases, the photobleaching arguments advanced in the discussion and expanded greatly in the appendices are not convincing. The data being modeled is a processed signal (ΔF/F) with smoothing and baseline correction and this does not seem to have been considered in the argument. Furthermore, the photometry readout is also a convolution of the actual concentration changes over time, influenced by the on-off kinetics of the sensor, which makes the interpretation of timing effects of photobleaching less obvious than presented here and more complex than the dyes considered in the cited reference used as a foundation for this line of reasoning.

Within this discussion of photobleaching, the characterization of the background reward experiments used in part to consider photobleaching (appendix 7.3.2) is incorrect. In this experiment (Jeong et al., 2022), background rewards were only delivered in the inter-trial-interval (i.e. not between the CS+ and predicted reward as stated in the text). Both in the authors' description and in the data, there is a 6s before cue onset where rewards are not delivered and while not described in the text, the data suggests there is a period after a predicted reward when background rewards are not delivered. This complicates the comparison of this data to the random reward experiment.

The discussion of the lack of evidence for backpropagation, taken as evidence for ANCCR over RPE, is also weak. A more useful exercise than comparing FLMM to the methods and data of Jeong et al., 2022, would be to compare against the approach of Amo et al., 2022, which identifies backpropagation (data publicly available: DOI: 10.5061/dryad.hhmgqnkjw). The replication of a positive result would be more convincing of the sensitivity of the methodology than the replication of a negative result, which could be a result of many factors in the experimental design. Given that the Amo et al. analysis relies on identifying systematic changes in the timing of a signal over time, this would be particularly useful in understanding if the smoothing steps in FLMM obscure such changes.

Reviewer #3 (Public Review):

Summary:<br /> The authors present a Python tool, Heron, that provides a framework for defining and running experiments in a lab setting (e.g. in behavioural neuroscience). It consists of a graphical editor for defining the pipeline (interconnected nodes with parameters that can pass data between them), an API for defining the nodes of these pipelines, and a framework based on ZeroMQ, responsible for the overall control and data exchange between nodes. Since nodes run independently and only communicate via network messages, an experiment can make use of nodes running on several machines and in separate environments, including on different operating systems.

Strengths:<br /> As the authors correctly identify, lab experiments often require a hodgepodge of separate hardware and software tools working together. A single, unified interface for defining these connections and running/supervising the experiment, together with flexibility in defining the individual subtasks (nodes) is therefore a very welcome approach. The GUI editor seems fairly intuitive, and Python as an accessible programming environment is a very sensible choice. By basing the communication on the widely used ZeroMQ framework, they have a solid base for the required non-trivial coordination and communication. Potential users reading the paper will have a good idea of how to use the software and whether it would be helpful for their own work. The presented experiments convincingly demonstrate the usefulness of the tool for realistic scientific applications.

Weaknesses:<br /> In my opinion, the authors somewhat oversell the reproducibility and "self-documentation" aspect of their solution. While it is certainly true that the graph representation gives a useful high-level overview of an experiment, it can also suffer from the same shortcomings as a "pure code" description of a model - if a user gives their nodes and parameters generic/unhelpful names, reading the graph will not help much. Making the link between the nodes and the actual code is also not straightforward, since the code for the nodes is spread out over several directories (or potentially even machines), and not directly accessible from within the GUI. The authors state that "[Heron's approach] confers obvious benefits to the exchange and reproducibility of experiments", but the paper does not discuss how one would actually exchange an experiment and its parameters, given that the graph (and its json representation) contains user-specific absolute filenames, machine IP addresses, etc, and the parameter values that were used are stored in general data frames, potentially separate from the results. Neither does it address how a user could keep track of which versions of files were used (including Heron itself).

Another limitation that in my opinion is not sufficiently addressed is the communication between the nodes, and the effect of passing all communications via the host machine and SSH. What does this mean for the resulting throughput and latency - in particular in comparison to software such as Bonsai or Autopilot? The paper also states that "Heron is designed to have no message buffering, thus automatically dropping any messages that come into a Node's inputs while the Node's worker function is still running."- it seems to be up to the user to debug and handle this manually?

As a final comment, I have to admit that I was a bit confused by the use of the term "Knowledge Graph" in the title and elsewhere. In my opinion, the Heron software describes "pipelines" or "data workflows", not knowledge graphs - I'd understand a knowledge graph to be about entities and their relationships. As the authors state, it is usually meant to make it possible to "test propositions against the knowledge and also create novel propositions" - how would this apply here?

Reviewer #3 (Public Review):

Summary:

This paper reexamined large excitatory neurons in the stratum radiatum with optogenetics. The findings are valuable because prior studies of the circuitry were confounded by the use of stimulating electrodes placed in different layers where multiple inputs were stimulated at one time. The strength of the evidence for the conclusions is incomplete because of several concerns with the manuscript.

Strengths:

The strength of the study is the very nice presentation of data. Also, there is a nice combination of patching, LFPs, paired recordings, and microelectrode arrays.

Weaknesses:

The limitations are in the conclusions which don't seem fully justified.

Reviewer #3 (Public Review):

Summary:

Xiong et al. investigated the debated mechanism of PKA activation using hippocampal CA1 neurons under pharmacological and synaptic stimulations. Examining the two PKA major isoforms in these neurons, they found that a portion of PKA-C dissociates from PKA-R and translocates into dendritic spines following norepinephrine bath application. Additionally, their use of a non-dissociable form of PKC demonstrates its essential role in structural long-term potentiation (LTP) induced by two-photon glutamate uncaging, as well as in maintaining normal synaptic transmission, as verified by electrophysiology. This study presents a valuable finding on the activation-dependent re-distribution of PKA catalytic subunits in CA1 neurons, a process vital for synaptic functionality. The robust evidence provided by the authors makes this work particularly relevant for biologists seeking to understand PKA activation and its downstream effects essential for synaptic plasticity.

Strengths:

The study is methodologically robust, particularly in the application of two-photon imaging and electrophysiology. The experiments are well-designed with effective controls and a comprehensive analysis. The credibility of the data is further enhanced by the research team's previous works in related experiments. The conclusions of this paper are mostly well supported by data. The research fills a significant gap in our understanding of PKA activation mechanisms in synaptic functioning, presenting valuable insights backed by empirical evidence.

Weaknesses:

The physiological relevance of the findings regarding PKA dissociation is somewhat weakened by the use of norepinephrine (10 µM) in bath applications, which might not accurately reflect physiological conditions. Furthermore, the study does not address the impact of glutamate uncaging, a well-characterized physiologically relevant stimulation, on the redistribution of PKA catalytic subunits, leaving some questions unanswered.

Reviewer #3 (Public Review):

Summary:

Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also show that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. Though interesting, the findings are not all integrated into a single narrative, and some of the results are only replications of earlier findings using modern methods. Overall, these findings provide incremental advance.

Strengths:

(1) Predator saliva is a stimulus of high ethological relevance

(2) The authors performed a careful quantification of fos induction across the anterior-posterior axis in figure 6

Weaknesses:

(1) It is unclear if fresh and old saliva indeed alter the perceived imminence of predation, as claimed by the authors. Prior work indicates that lower imminence induces anxiety-related actions, such as re-organization of meal patterns and avoidance of open spaces, while slightly higher imminence produces freezing. Here, the authors show that fresh and old predator saliva only provoke different amounts of freezing, rather than changing the topography of defensive behaviors, as explained above. Another prediction of predatory imminence theory would be that lower imminence induced by old saliva should produce stronger cortical activation, while fresh saliva would activate amygdala, if these stimuli indeed correspond to significantly different levels of predation imminence.

(2) It is known that predator odors activate and require AOB, VNO and VMH, thus replications of these findings are not novel, decreasing the impact of this work.

(3) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing neural activity of relevant cell bodies and axonal projections, significantly decreasing the mechanistic insights generated by this work

(4) The correlation shown in Figure 5c may be spurious. It appears that the correlation is primarily driven by a single point (the green square point near the bottom left corner). All correlations should be calculated using Spearman correlation, which is non-parametric and less likely to show a large correlation due to a small number of outliers. Regardless of the correlation method used, there are too few points in Figure 5c to establish a reliable correlation. Please add more points to 5c.

(5) Please cite recent relevant papers showing VMH activity induced by predators, such as https://pubmed.ncbi.nlm.nih.gov/33115925/ and https://pubmed.ncbi.nlm.nih.gov/36788059/

(6) Add complete statistical information in the figure legends of all figures, which should include n, name of test used and exact p values.

(7) Some of the findings are disconnected from the story. For example, the authors show V2R-A4-expressing cells are activated by predator odors. Are these cells more likely to be connected to the rest of the predatory defense circuit than other VNO cells?

(8) Please paste all figure legends directly below their corresponding figure to make the manuscript easier to read

(9) Were there other behavioral differences induced by fresh compared to old saliva? Do they provoke differences in stretch-attend risk evaluation postures, number of approaches, average distance to odor stimulus, velocity of movements towards and away the odor stimulus, etc?

Reviewer #3 (Public Review):

This study from the Flores group aims at understanding neuronal circuit changes during adolescence which is an ill-defined, transitional period involving dramatic changes in behavior and anatomy. They focus on DA innervation of the prefrontal cortex, and their interaction with the guidance cue Netrin-1. They propose DA axons in the PFC increase in the postnatal period, and their density is reduced in a Netrin 1 knockdown, suggesting that Netrin abets the development of this mesocortical pathway. In such mice impulsivity gauged by a go-no go task is reduced. They then provide some evidence that Unc5c is developmentally regulated in DA axons. Finally they use an interesting hamster model, to study the effect of light hours on mesocortical innervation, and make some interesting observations about the timing of innervation and Unc5c expression, and the fact that females housed in winter day length conditions display an accelerated innervation of the prefrontal cortex.

Comments on the revision. Several points were addressed; some remain to be addressed.

4. It's not clear to me that TH doesnt stain noradrenergic axons in the PFC. See Islam and Blaess, 2021, and references therein.

6. The Netrin knockdown data provided is from a previous study/samples.

8. While the authors make the argument that the behavior is linked to DA, they still haven't formally tested it, in my opinion.

13. Fig 3, UNc 5c levels are not yet quantified. Furthermore, I agree with the previous reviewer that Unc5C knockdown would corroborate key aspects of the model.

New - Developmental trajectory of prefrontal TH-positive axons from early adolescence to adulthood is similar in male and female rats, (Willing Juraska et al., 2017). This needs discussion.

Reviewer #3 (Public Review):

Summary:<br /> This paper by Portela Catani et al examines the antigenic relationships (measured using monotypic ferret and mouse sera) across a panel of N2 genes from the past 14 years, along with the underlying sequence differences and phylogenetic relationships. This is a highly significant topic given the recent increased appreciation of the importance of NA as a vaccine target, and the relative lack of information about NA antigenic evolution compared with what is known about HA. Thus, these data will be of interest to those studying the antigenic evolution of influenza viruses. The methods used are generally quite sound, though there are a few addressable concerns that limit the confidence with which conclusions can be drawn from the data/analyses.

Strengths:<br /> -The significance of the work, and the (general) soundness of the methods.<br /> -Explicit comparison of results obtained with mouse and ferret sera

Weaknesses:<br /> - Approach for assessing influence of individual polymorphisms on antigenicity does not account for potential effects of epistasis (this point is acknowledged by the authors).<br /> - Machine learning analyses neither experimentally validated nor shown to be better than simple, phylogenetic-based inference.

Reviewer #3 (Public Review):

Summary:

This study studied the neural mechanisms underlying the shift of ocular dominance induced by "dichoptic-backward-movie" adaptation. The study is self-consistent.

Strengths:

The experimental design is solid and progressive (relationship among three studies), and all of the raised research questions were well answered.<br /> The logic behind the neural mechanisms is solid.<br /> The findings regarding the cTMS (especially the position/site can be useful for future medical implications).<br /> The updated Exp4 eliminates some concerns and thus makes the results even more solid.

Reviewer #3 (Public Review):

Summary:

This manuscript reveals an important mechanism of KCNQ1/IKs channel gating such that the open state of the pore is unstable and undergoes intermittent closed and open conformations. PUFA enhances the maximum open probability of IKs by binding to a crevice adjacent to the pore and stabilizing the open conformation. This mechanism is supported by convincing single-channel recordings that show empty and open channel traces and the ratio of such traces is affected by PUFA. In addition, mutations of the pore residues alter PUFA effects, convincingly supporting that PUFA alters the interactions among these pore residues.

Strengths:<br /> The data are of high quality and the description is clear.

Weaknesses:<br /> Some comments about the presentation.

(1) The structural illustrations in this manuscript in general need to be more clarified.

(2) The manuscript heavily relies on the comparison between the S4-down and S4-up structures (Figures 3, 4, and 7) to illustrate the difference between the extracellular side of the pore and to lead to the hypothesis of open-state stability being affected by PUFA. This may mislead the readers to think that the closed conformation of the channel in the up-state is the same as that in the down-state.

Reviewer #3 (Public Review):

Summary:<br /> The authors demonstrate the activation of Polycystin-1 (PC1), a G-protein coupled receptor, using small peptides derived from its original agonist, the stalk TA protein. In the experimental part of the study, the authors performed cellular assays to check the peptide-induced reactivation of a mutant form of PC1 which does not contain the stalk agonist. The experimental data is supported by computational studies using state-of-the-art Gaussian accelerated Molecular Dynamics (GaMD) and bioinformatics analysis based on sequence covariance. The computer simulations revealed the mechanistic details of the binding of the said peptides with the mutant PC1 protein and discovered different bound, unbound, and intermediate conformations depending on the peptide size and sequence. The use of reliable and well-established molecular simulation algorithms and the physiological relevance of this protein autosomal dominant polycystic kidney disease (ADPKD) make this work particularly valuable.

Strengths:<br /> This work is exploratory and its goal is to establish that small peptides can be used to probe the PC1 signaling process. The authors have provided sufficient evidence to justify this claim. Their GaMD simulations have produced free-energy landscapes that differentiate the interaction of PC1 with three different synthetic peptides and demonstrate the associated conformational dynamics of the receptor protein. Their trajectory analysis and sequence covariance analysis could identify residue-specific interactions that facilitate this process.

Weaknesses:<br /> The following minor weaknesses should be taken into account by the reader when interpreting the results:

(1) No control has been used for the computational (GaMD) study as the authors only report the free energy surface for 3 highly agonistic peptides but for none of the other peptides that did not induce an agonistic effect. Therefore, in the current version, the reliability of the computational results is not foolproof.

(2) All discussions about the residue level interactions focused only on geometric aspects (distance, angle, etc) but not the thermodynamic aspect (e.g. residue-wise interaction energy). Considering they perform a biased simulation, the lack of interaction energy analysis only provides a qualitative picture of the mechanism.

(3) It is not mentioned clearly whether the reader should interpret the free energy landscapes quantitatively or qualitatively. Considering no error analysis or convergence plots are reported for the GaMD free energy surfaces, it may be assumed the results are qualitative. The readers should consider this caveat and not try to quantitatively reproduce these free energy landscapes with other comparable techniques.

Reviewer #3 (Public Review):

Summary:

Jojoa-Cruz et al provide a new structure of At-OSCA3.1. The structure of OSCA 3.1 is similar to previous OSCA cryo-em structures of both OSCA3.1 and other homologues validating the new structure. Using the novel structure of OSCA3.1 as a guide they created several point mutations to investigate two different mechanosensitive modalities: poking and stretching. To investigate the ability of OSCA channels to gate in response to poking they created point mutations in OSCA1.2 to reduce sensitivity to poking based on the differences between the OSCA1.2 and 3.1 structures. Their results suggest that two separate regions are responsible for gating in response to poking and stretching.

Strengths:

Through a detailed structure based analysis, the authors identified structural differences between OSCA3.1 and OSCA1.2. The use of technically sound data supports the hypothesis that poking and stretching are sensed by two unique regions in the protein. These subtle structural changes between homologues identify regions in the amphipathic helix and near the pore that are essential for gating of OSCA1.2 in response to poking and stretching. Mutations in the AH of OSCA1.2 decrease the sensitivity to poking stimulus however these mutations have similar stretch activated currents to the WT. The point mutations described in the manuscript will set the foundation for investigations into how these two channels sense tension using different regions of structurally similar proteins.

Weaknesses:

Mutations in the amphipathic helix at W75 and L80 show reduced gating in response to poking stimuli. The gating observed occurs at poking depths similar to cellular rupture, the similarity in depths suggests that these mutations could be a complete loss of functions.

Reviewer #3 (Public Review):

In their manuscript, Schneider et al. aim to develop voyAGEr, a web-based tool that enables the exploration of gene expression changes over age in a tissue- and sex-specific manner. The authors achieved this goal by calculating the significance of gene expression alterations within a sliding window, using their unique algorithm, Shifting Age Range Pipeline for Linear Modelling (ShARP-LM), as well as tissue-level summaries that calculated the significance of the proportion of differentially expressed genes by the windows and calculated enrichments of pathways for showing biological relevance. Furthermore, the authors examined the enrichment of cell types, pathways, and diseases by defining the co-expressed gene modules in four selected tissues. Although their algorithm ShARP-LM has limited statistical power due to its calculation within a 16-year window, the voyAGEr was developed as a discovery tool, giving researchers easy access to the vast amount of transcriptome data from the GTEx project. Overall, the research design is unique and well-performed in simulating age-dependent changes in gene expression. The interesting results provide useful resources for the field of human genetics of aging.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, entitled "Telomere length sensitive regulation of Interleukin Receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment", Dr. Mukherjee and colleagues pointed out clarifying the extra-telomeric role of TRF2 in regulating IL1R1 expression with consequent impact on TAMs tumor-infiltration.

Strengths:<br /> Upon careful manuscript evaluation, I feel that the presented story is undoubtedly well conceived. At the technical level, experiments have been properly performed and the obtained results support the authors' conclusions.

Weaknesses:<br /> Unfortunately, the covered topic is not particularly novel. In detail, the TRF2 capability of binding extratelomeric foci in cells with short telomeres has been well demonstrated in a previous work published by the same research group. The capability of TRF2 to regulate gene expression is well-known, the capability of TRF2 to interact with p300 has been already demonstrated and, finally, the capability of TRF2 to regulate TAMs infiltration (that is the effective novelty of the manuscript) appears as an obvious consequence of IL1R1 modulation (this is probably due to the current manuscript organization).

Reviewer #3 (Public Review):

Summary:

The work submitted by Dr. Jeong-Oh Shin and co-workers aims to investigate the therapeutic efficacy of rhPTH(1-34) and R25CPTH(1-34) on bone regeneration and osseointegration of titanium implants using a postmenopausal osteoporosis animal model.<br /> In my opinion the findings presented are not strongly supported by the provided data since the methods utilized do not allow to significantly support the primary claims.

Strengths:

Strengths include certain good technologies utilized to perform histological sections (i.e. the EXAKT system).

Weaknesses:

Certain weaknesses significantly lower the enthusiasm for this work. Most important: the limited number of samples/group. In fact, as presented, the work has an n=4 for each treatment group. This limited number of samples/group significantly impairs the statistical power of the study. In addition, the implants were surgically inserted following a "conventional implant surgery", implying that no precise/guided insertion was utilized. This weakness is, in my opinion, particularly significant since the amount of bone osteointegration may greatly depend on the bucco-lingual positioning of each implant at the time of the surgical insertion (which should, therefore, be precisely standardized across all animals and for all surgical procedures).<br /> On a minor note: not sure why the authors present a methodology to evaluate the dynamic bone formation (line 272) but do not present results (i.e. by means of histomorphometrical analyses) utilizing this methodology.

Reviewer #3 (Public Review):

Chen et al. investigated how intermittent fasting causes metabolic benefits in obese mice and find that intestinal ILC3 and IL-22-IL-22R signaling contribute to the beiging of white adipose tissue (WAT) and consequent metabolic benefits including improved glucose and lipid metabolism in diet-induced obese mice. They demonstrate that intermittent fasting causes increased IL22+ILC3 in small intestines of mice. Adoptive transfer of purified intestinal ILC3 or administration of exogenous IL-22 can lead to increases in UCP1 gene expression and energy expenditure as well as improved glucose metabolism. Importantly, the above metabolic benefits caused by intermittent fasting are abolished in IL-22R-/- mice. Using an in vitro experiment, the authors show that ILC3-derived IL-22 may directly act on adipocytes to promote SVF beige differentiation. Finally, by performing sc-RNA-seq analysis of intestinal immune cells from mice with different treatments, the authors indicate a possible way of intestinal ILC3 being activated by intermittent fasting. Overall, this study provides a new mechanistic explanation for the metabolic benefits of intermittent fasting and reveals the role of intestinal ILC3 in the enhancement of the whole-body energy expenditure and glucose metabolism likely via IL-22-induced beige adipogenesis.

Although this study presents some interesting findings, particularly IL-22 derived from intestinal ILC3 could induce beiging of WAT by directly acting on adipocytes, the experimental data are not sufficient to support the key claims in the manuscript.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Lim and colleagues use an innovative CDRA chip platform to derive and mechanistically elucidate the molecular wiring of doxorubicin-resistant (DOXR) MDA-MB-231 cells. Given their enlarged morphology and polyploidy, they termed these cells as Large-DOXR (L-DORX). Through comparative functional omics, they deduce the NUPR1/HDAC11 axis to be essential in imparting doxorubicin resistance and, consequently, genetic or pharmacologic inhibition of the NUPR1 to restore sensitivity to the drug.

Strengths:

The study focuses on a major clinical problem of the eventual onset of resistance to chemotherapeutics in patients with triple-negative breast cancer (TNBC). They use an innovative chip-based platform to establish as well as molecularly characterize TNBC cells showing resistance to doxorubicin and uncover NUPR1 as a novel targetable driver of the resistant phenotype.

Weaknesses:

Critical weaknesses are the use of a single cell line model (i.e., MDA-MB-231) for all the phenotypic and functional experiments and absolutely no mechanistic insights into how NUPR1 functionally imparts resistance to doxorubicin. It is imperative that the authors demonstrate the broader relevance of NUPR1 in driving dox resistance using independent disease models.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Yujiro Umezaki and colleagues aims to describe how taste stimuli influence temperature preference in Drosophila. Under starvation flies display a strong preference for cooler temperatures than under fed conditions that can be reversed by refeeding, demonstrating the strong impact of metabolism on temperature preference. In their present study, Umezaki and colleagues observed that such changes in temperature preference are not solely triggered by the metabolic state of the animal but that gustatory circuits and peptidergic signalling play a pivotal role in gustation-evoked alteration in temperature preference.

The study of Umezaki is definitively interesting and the findings in this manuscript will be of interest to a broad readership.

Strengths:<br /> The authors demonstrate interesting new data on how taste input can influence temperature preference during starvation. They propose how gustatory pathways may work together with thermosensitive neurons, peptidergic neurons and finally try to bridge the gap between these neurons and clock genes. The study is very interesting and the data for each experiment alone are very convincing.

Weaknesses:<br /> In my opinion, the authors have opened many new questions but did not fully answer the initial question - how do taste-sensing neurons influence temperature preferences? What are the mechanisms underlying this observation? Instead of jumping from gustatory neurons to thermosensitive neurons to peptidergic neurons to clock genes, the authors should have stayed within the one question they were asking at the beginning. How does sugar sensing influence the physiology of thermos-sensation in order to change temperature preference? Before addressing all the following questions of the manuscript the authors should first directly decipher the neuronal interplay between these two types of neurons.

Reviewer #3 (Public Review):

Summary:<br /> The authors examined the effects of glutamate release from PMv LepR neurons in the regulation of puberty and reproduction in female mice.

Strengths:<br /> Multiple genetic mouse models were utilized to either manipulate PMv LepR neuron activities, or to delete glutamate vesicle transporters from LepR neurons. The authors have been quite rigorous in validating these models and exploring potential contaminations. Most of the data presented are solid and convincing and support the conclusion.

Weaknesses:<br /> Some results are hard to interpret.

Reviewer #3 (Public Review):

This work is carried out by the research group led by Shuiqiao Yuan, who has a long interest and significant contribution in the field of male germ cell development. The authors study a protein for which limited information existed prior to this work, a component of the E3 ubiquitin ligase complex, FBXO24. The authors generated the first FBXO24 KO mouse model reported in the literature using CRISPR, which they complement with HA-tagged FBXO24 transgenic model in the KO background. The authors begin their study with a very careful examination of the expression pattern of the FBXO24 gene at the level of mRNA and the HA-tagged transgene, and they provide conclusive evidence that the protein is expressed exclusively in the mouse testis and specifically in post-meiotic spermatids of stages VI to IX, which include early stages of spermatid elongation and nuclear condensation. The authors report a fully sterile phenotype for male mice, while female mice are normal. Interestingly, the testis size and the populations of spermatogenic cells in the KO mutant mice show small (but significant) reduction compared to the WT testis. Importantly, the mature sperm from KO animals show a series of defects that were very thoroughly documented in this work by scanning and transmission electron microscopy; this data constitutes a very strong point in this paper. FBXO24 KO sperm have severe defects in the mitochondrial sheath with missing mitochondria near the annulus, and missing outer dense fibers. Collectively these defects cause abnormal bending of the flagellum and severely reduced sperm motility. Moreover, defects in nuclear condensation are observed with faint nuclear staining of elongating and elongated spermatids, and reduction of protein levels of protamine 2 combined with increased levels of histones and transition protein 1. All the above are in line with the observed male sterility phenotype.

The authors also performed RNASeq in the KO animal, and found profound changes in the abundance of thousands of mRNAs; changes in mRNA splicing patterns were noted as well. This data reveals deeply affected gene expression patterns in the FBXO24 KO testis, which further supports the essential role that this factor serves in spermiogenesis. Unfortunately, a molecular explanation of what causes these changes is missing; it is still possible that they are an indirect consequence of the absence of FBXO24 and not directly caused by it.

The finding that Miwi protein levels are increased in the FBXO24 KO testis is an important point in this work, and it is in agreement with the observed increased size of the chromatoid body, where most of Miwi protein is accumulated in round spermatids. This finding is well supported with experiments performed in 293T cells showing that Miwi ubiquitination is FBXO24 dependent in this ectopic system. Moreover, the authors detect reduced ubiquitination of endogenous Miwi protein immunoprecipitated from FBXO24 KO testis. Consistent with an increase in Miwi protein levels, Miwi-sized piRNAs show increased abundance in total RNA from FBXO24 KO testis. It has been documented that Piwi proteins stabilize their piRNA cargo, so the increase in piRNA levels in 29-32 nt sizes is most likely not a result of altered biogenesis, but increased half-life of the piRNAs as a result of Miwi upregulation. piRNAs have been involved in the regulation of mRNAs in the post-meiotic spermatid, but it is unclear how increased Miwi protein and its piRNA cargo at the levels observed in this study contribute to the complete infertility phenotype of the FBXO24 KO male mice.

Therefore, a well-reasoned narrative on if and how the absence of FBXO24 as an E3 ubiquitin ligase is responsible for the observed mRNA and protein differential expression is largely absent. If FBXO24-mediated ubiquitination is required for normal protein degradation during spermiogenesis, protein level increase should be the direct consequence of genuine FBXO24 targets in the KO testis. Apart from Miwi, the possible involvement of ubiquitination was not shown for any other proteins that the authors found interact with FBXO24 such as splicing factors SRSF2, SRSF3, SRSF9, or any of the other proteins whose levels were found to be changed (reduced, thus the change in the KO is less likely due to absence of ubiquitination) such as ODF2, AKAP3, TSSK4, PHF7, TSSK6 and RNF8. Interestingly, the authors do observe increased amounts of histones and transition proteins, but reduced amounts of protamines, which directly shows that histone to protamine transition is indeed affected in the FBXO24 KO testis, consistent with the observed less condensed nuclei of spermatozoa. Could histones and transition proteins be targets of the proposed ubiquitin ligase activity of FBXO24, and in its absence, histone replacement is abrogated? Providing experimental evidence to address this possibility would greatly expand our understanding on why FBXO24 is essential during spermiogenesis.

Reviewer #3 (Public Review):

Summary:

The authors used a novel technique to treat male infertility. In a proof-of-concept study, the authors were able to rescue the phenotype of a knockout mouse model with immotile sperm using this technique. This could also be a promising treatment option for infertile men.

Strengths:

In their proof-of-concept study, the authors were able to show that the novel technique rescues the infertility phenotype in vivo.

Weaknesses:

Some minor weaknesses, especially in the discussion section, could be addressed to further improve the quality of the manuscript.

It is very convincing that the phenotype of Armc2 KO mice could (at least in part) be rescued by injection of Armc2 RNA. However, a central question remains about which testicular cell types have been targeted by the constructs. From the pictures presented in Figures 7 and 8, this issue is hard to assess. Given the more punctate staining of the DNA construct a targeting of Sertoli cells is more likely, whereas the more broader staining of seminiferous tubules using RNA constructs is talking toward germ cells.

Further, the staining for up to 119 days (Figure 5) would point toward an integration of the DNA construct into the genome of early germ cells such as spermatogonia and/or possibly to Sertoli cells. Given the expression after RNA transfection for up to 21 days (Figure 4) and the detection of motile sperm after 21 days (Figure 11), this would point to either round spermatids or spermatocytes.

These aspects need to be discussed more carefully (discussion section: lines 549-574).

It would also be very interesting to know in which testicular cell type Armc2 is endogenously expressed (lines 575-591).

Reviewer #3 (Public Review):

Summary:<br /> This work uses tools and concepts from co-phylogenetic analyses to reconstruct the evolutionary and diversification history of coronaviruses in mammals. It concludes that cross-species transmissions from bats to humans are a relatively common event (compared to bats to other species). Across all mammals, the diversification history of coronaviruses suggests that there is potential for further evolutionary diversification.

Strengths:<br /> The article uses an interesting approach based on jointly looking at the extant network of coronaviruses-mammals interactions, and the phylogenetic history of both these organisms. The authors do an impressive job of explaining the challenges of reconstructing evolutionary dynamics for RNA viruses, and this helps readers appraise the relevance of their approach.

Weaknesses:<br /> I remain unconvinced by the argument that sampling does not introduce substantial biases in the analyses. As the authors highlight, incomplete knowledge of the extant interactions would lead to a biased reconstruction of the diversification history. In a recent paper (Poisot et al. 2023, Patterns), we look at sampling biases in the virome of mammals and suggest that is a fairly prominent issue, that is furthermore structured by taxonomy, space, and phylogenetic position. Case in point, even for betacoronaviruses, there have been many newly confirmed hosts in recent years. For organisms that have received less intense scrutiny, I think a thorough discussion of potential gaps in data would be required (see for example Cohen et al. 2022, Nat. Comms).

I was also surprised to see little discussion of the differences between alpha and beta coronaviruses - there is evidence that they may differ in their cross-species transmission (see Caraballo et al. 2022 Micr. Spectr.), which could call into question the relevance of treating all coronaviruses as a single, homogeneous group.

Some of the discussions in this paper also echo previous work by e.g. Geoghegan et al. (see 2017, PLOS Pathogens), which I was surprised to not see discussed, as it is a much earlier investigation of the relative frequencies of co-divergence and host switches for different viral families, with a deep discussion of how this may structure future evolutionary dynamics.

Reviewer #3 (Public Review):

Summary:

This work describes a new pathway by which malaria parasites, P. falciparum, may regulate their growth and virulence (i.e. their expression of virulence-linked cytoadhesins). This is a topic of considerable interest in the field - does this important parasite sense factor(s) in its host bloodstream and regulate itself accordingly? Several fragments of evidence have come out on this topic in the past decade, showing, for example, reduced parasite growth under calorie restriction (in mice); parasite dormancy in response to amino acid starvation (in culture and in mice), and also reduced virulence in dry-season, low-parasitaemia infections in humans. The molecular mechanisms that may underlie this interesting biology remain only poorly understood.

Here, the authors show that dry-season P. falciparum parasites have reduced expression of Pol3-transcribed tRNAs and ncRNAs that positively regulate virulence gene expression. They link the level of Pol3 activity to PfMaf1, a remnant of the largely-absent nutrient-sensing TOR pathway in this parasite. They propose that in the dry season, human hosts may be calorie-restricted, leading to Maf1 moving to the nucleus and suppressing Pol3, thus downregulated growth and virulence of parasites. The evidence is intriguing and the idea is conceptually elegant.

Strengths:

The use of dry/wet-season field samples from The Gambia is a strength, showing potential real-world relevance. The generation of an inducible knockdown of Maf1 in lab-cultured parasites is also a strength, allowing this pathway to be studied somewhat in isolation.

Weaknesses:

(1) The signals upstream of Maf1 remain rather a black box. 4 are tested - heat shock and low-glucose, which seem to suppress ALL transcription; low-Isoleucine and high magnesium, which suppress Pol3. Therefore the authors use Mg supplementation throughout as a 'starvation type' stimulus. They do not discuss why they didn't use amino acid limitation, which could be more easily rationalised physiologically. It may be for experimental simplicity (no need for dropout media) but this should be discussed, and ideally, sample experiments with low-IsoLeu should be done too, to see if the responses (e.g. cytoadhesion) are all the same.

(2) The proteomics, conducted to seek partners of Maf1, is probably the weakest part. From Figure S3: the proteins highlighted in the text are clearly highly selected (as ones that might be relevant, e.g. phosphatases), but many others are more enriched. It would be good to see the whole list, and which GO terms actually came top in enrichment.

(3) Figure 3 shows the Maf1-low line has very poor growth after only 5 days but it is stated that no dead parasites are seen even after 8 cycles and the merozoites number is down only ~18 to 15... is this too small to account for such poor growth (~5-fold reduced in a single cycle, day 3-5)? It would additionally be interesting to see a cell-cycle length assessment and invasion assay, to see if Maf1-low parasites have further defects in growth.

Reviewer #3 (Public Review):

Liang and colleagues set out to test whether the human brain uses distance and grid-like codes in social knowledge using a design where participants had to navigate in a two-dimensional social space based on competence and warmth during an fMRI scan. They showed that participants were able to navigate the social space and found distance-based codes as well as grid-like codes in various brain regions, and the grid-like code correlated with behavior (reaction times).

On the whole, the experiment is designed appropriately for testing for distant-based and grid-like codes, and is relatively well powered for this type of study, with a large amount of behavioral training per participant. They revealed that a number of brain regions correlated positively or negatively with distance in the social space, and found grid-like codes in the frontal polar cortex and posterior medial entorhinal cortex, the latter in line with prior findings on grid-like activity in entorhinal cortex. The current paper seems quite similar conceptually and in design to previous work, most notably Park et al., 2021, Nature Neuroscience.

(1) The authors claim that this study provides evidence that humans use a spatial / grid code for abstract knowledge like social knowledge.

This data does specifically not add anything new to this argument. As with almost all studies that test for a grid code in a similar "conceptual" space (not only the current study), the problem is that, when the space is not a uniform, square/circular space, and 2-dimensional then there is no reason the code will be perfectly grid like, i.e., show six-fold symmetry. In real world scenarios of social space (as well as navigation, semantic concepts), it must be higher dimensional - or at least more than two dimensional. It is unclear if this generalizes to larger spaces where not all part of the space is relevant. Modelling work from Tim Behrens' lab (e.g., Whittington et al., 2020) and Bradley Love's lab (e.g., Mok & Love, 2019) have shown/argued this to be the case. In experimental work, like in mazes from the Mosers' labs (e.g., Derdikman et al., 2009), or trapezoid environments from the O'Keefe lab (Krupic et al., 2015), there are distortions in mEC cells, and would not pass as grid cells in terms of the six-fold symmetry criterion.

The authors briefly discuss the limitations of this at the very end but do not really say how this speaks to the goal of their study and the claim that social space or knowledge is organized as a grid code and if it is in fact used in the brain in their study and beyond. This issue deserves to be discussed in more depth, possibly referring to prior work that addressed this, and raise the issue for future work to address the problem - or if the authors think it is a problem at all.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Salazar-Lázaro et al. presented interesting data that C-terminal half of the Syx1 SNARE domain is responsible for clamping of spontaneous release, stabilizing RRP, and also Ca2+-evoked release. The authors routinely utilized the chimeric approach to replace the SNARE domain of Syx1 with its paralogue Syx2 and analyzed the neuronal activity through electrophysiology. The data are straightforward and fruitful. The conclusions are reasonable.

Strengths:<br /> The electrophysiology data that illustrate the important functions of Syx1 in clamping of spontaneous release, stabilizing RRP, and also Ca2+-evoked release were clear and convincing.

Weaknesses:<br /> One weakness is that the authors did not go deep into the underlying molecular mechanisms experimentally, either because of a variety of complicated possibilities or limited space of the manuscript.

Reviewer #3 (Public Review):

Summary: The authors aim to solve how landscape context impacts the community BEF relationship. They found habitat loss and fragmentation per se have inconsistent effects on biodiversity and ecosystem function. And habitat loss rather than fragmentation per se can weaken the positive BEF relationship by decreasing the degree of habitat specialization of the community.

Strengths: The authors provide a good background, and they have a good grasp of habitat fragmentation and BEF literature.<br /> A major strength of this study is separating the impacts of habitat loss and fragmentation per se using the convincing design selection of landscapes with different combinations of habitat amount and fragmentation per se.<br /> Another strength is considering the role of specialists and generalists in shaping the BEF relationship.

Weaknesses:<br /> No<br /> In the revised manuscript, the authors have provided more detailed information about the ecological significance of these fragmentation indices. and also integrated two environmental factors related to water and temperature (soil water content and land surface temperature) into the data analysis to control their potential impact.

Reviewer #3 (Public Review):

Summary:

Using a combination of approaches, including automated feature selection and hierarchical clustering, the author identified a set of genes persistently associated with extrachromosomal DNA (ecDNA) presence across cancer types. The authors further validated the gene set identified using gene ontology enrichment analysis and identified that upregulated genes in extrachromosomal DNA-containing tumors are enriched in biological processes like DNA damage and cell proliferation, whereas downregulated genes are enriched in immune response processes.

Comments for the previous version:

Major comments:

(1) The authors presented a solid comparative analysis of ecDNA-containing and ecDNA-free tumors. An established automated feature selection approach, Boruta, was used to select differentially expressed genes (DEG) in ecDNA(+) and ecDNA(-) TCGA tumor samples, and the iterative selection process and two-tier multiple hypothesis testing ensured the selection of reliable DEGs. The author showed that the DEG selected using Boruta has stronger predictive power than genes with top log-fold changes.

(2) The author performed a thorough interpretation of the findings with GO enrichment analysis of biological processes enriched in the identified DEG set and presented interesting findings, including the enrichment in DNA damage process among the genes upregulated in ecDNA(+) tumors.

(3) Overall, the authors achieved their aims with solid data mining and analysis approaches applied to public data tumor data sets.

(4) While it may not be the scope of this study, it will be interesting to at least have some justification for choosing Boruta over other feature selection methods, such as Recursive Feature Elimination (RFE) and backward stepwise selection.

(5) The authors showed that DESEQ-selected DEGs with top log-fold changes have less strong predictive power and speculated that this may be due to the fact that genes with top log-fold changes (LFC) are confined only to a small subset of samples. It will be interesting to select DEGs with top log-fold changes after first partitioning the tumor samples. For example, randomly partition the tumor samples, identify the DEGs with top LFC, combine the DEGs identified from each partition, then evaluate the predictive power of these DEGs against the Boruta-selected DEGs.

(6) While the authors showed that the presence of mutations was not able to classify ecDNA(+) and (-) tumor samples, it will be interesting to see if variant allele frequencies of the genes containing these mutations have predictive power.

Comments for the revised version:

The authors addressed the comments and recommendations with solid analysis and explanations in the revision. The added analysis using GLM is especially appreciated and provides convincing evidence for the predicting power of the Boruta-selected genes. The only comment is at this point is that it is recommended that the author provide some justification for choosing Boruta over other feature selection methods. It is not necessary to provide benchmarking results - justification based on the review of previous literature is sufficient, as it is not well explained in the paper why Boruta was chosen in the first place. Is it state-of-the-art? Has it demonstrated better performance in other settings? A few sentences answering these questions should suffice.

Reviewer #3 (Public Review):

In this manuscript, the authors define the developmental trajectory resulting in a diverse mTEC compartment. Using a variety of approaches, including a novel CCL21-fate mapping model, data is presented to argue that embryonic CCL21-expressing thymocyte attracting mTECs naturally convert to into self-antigen displaying mTEC subsets, including Aire+ mTECs and thymic tuft cells. Perhaps somewhat surprisingly, a large fraction of cTECs were also marked for having expressed CCL21, suggesting that there exists some conversion of mTEC (progenitors) into cTEC, a developmentally interesting observation that could be followed up later. Overall, the experimental setup, writing, and conclusions, are all outstanding. The one question I have, which may be more of a curiosity of this reviewer than a requirement for the manuscript, is whether thymocytes themselves are required for the conversion/maturation of attracting TECs to mTECs? For example, in CD3e-/- (or Rag-/-) mice, are mTECs arrested at the thymocyte attracting stage, or is the conversion process 'pre-programed'? In the same vein, do cTECs (or the immature cTECs) maintain CCL21 expression in the absence of mature thymocytes? These are not critical studies but are fairly straightforward (effort- and time-wise) that would aid in placing this process in the overall scope of thymus development.

Reviewer #3 (Public Review):

I very much like this approach and the idea of incorporating hypervariable markers. The method is intriguing, and the ability to e.g. estimate recombination rates, the size of DMRs, etc. is a really nice plus. I am not able to comment on the details of the statistical inference, but from what I can evaluate it seems reasonable and in principle the inclusion of highly mutable sties is a nice advance. This is an exciting new avenue for thinking about inference from genomic data. I remain a bit concerned about how well this will work in systems where much less is understood about methylation,

The authors include some good caveats about applying this approach to other systems, but I think it would be helpful to empiricists outside of thaliana or perhaps mammalian systems to be given some indication of what to watch out for. In maize, for example, there is a non-bimodal distribution of CG methlyation (35% of sites are greater than 10% and less than 90%) but this may well be due to mapping issues. The authors solve many of the issues I had concerns with by using gene body methylation, but this is only briefly mentioned on line 659. I'm assuming the authors' hope is that this method will be widely used, and I think it worth providing some guidance to workers who might do so but who are not as familiar with these kind of data.

Reviewer #3 (Public Review):

Summary: The paper "Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction" reports the combination of prior knowledge signaling networks, multiparametric cell-based data on the activation status of 14 crucial proteins emblematic of the cell state downstream of FLT3 obtained under a variety of perturbation conditions and Boolean logic modeling, to gain mechanistic insight into drug resistance in acute myeloid leukemia patients carrying the internal tandem duplication in the FLT3 receptor tyrosine kinase and predict drug combinations that may reverse pharmacoresistant phenotypes. Interestingly, the utility of the approach was validated in vitro and using real-world data.

Strengths:

The model predictions have been validated in vitro and using external data.

This is a complex study, but readability is enhanced by the inclusion of a section that summarizes the study design, plus relevant figures. The availability of data as supplementary material and the availability of code in GitHub are also high points.

Weaknesses:

There are some apparent discrepancies between predicted and observed data that have been seemingly overlooked.

Reviewer #3 (Public Review):

Summary:<br /> In this study, the authors collected a large set of data on root traits and root-associated microbes in the root endosphere and rhizosphere in order to integrate these important organisms in the root economics spectrum. By sampling a relatively large set of species from the subtropics along an elevation gradient, they tested whether microbial functions covary with root traits and root trait axes and if so, aimed to discuss what this could tell us about the (belowground) functioning of trees and forests.

Strengths:<br /> The strengths of this study lie mostly in the impressive dataset set the authors compiled: they sampled belowground properties of a relatively large number of tree species from an understudied region: i.e., the subtropics, where species-level root data are notoriously scarce. Secondly, their extensive sampling of associated microbes to integrate them in the root economics space is an important quality, because of the strong associations between roots and fungi and bacteria: soil microbes are directly related to root form (e.g., mycorrhizal fungi and root diameter and SRL), and function (e.g., taking up soil nutrients from various sources). Thirdly, the PCA figures (Figures 2 and 3) look very nice and intuitive and the paper is very well written.

Weaknesses:<br /> That said, this study also has several methodological weaknesses that make the results, and therefore the impact of this study difficult to evaluate and interpret.

(1) Design: The design of this study needs further explanation and justification in the Introduction and Methods sections in order to understand the ecological meaning of the results. Root traits and microbial community composition differ with their environment, and therefore (likely) also with elevation. Elevation is included in the redundancy analysis as a main effect, but without further environmental information, its impact is not ecologically meaningful. What is the rationale for including an elevation gradient in the design and as a main effect in the analyses? Do environmental conditions vary across altitudes and how, and if so, how would this impact the data?

What is the rationale behind sampling endosphere and rhizosphere microbial communities - why do both? And why also include pathogens - what are their expected roles in the RES? What do we know about this already? The introduction needs a more extensive literature review of these additional variables that are included in the analyses.

(2) Units of replication and analysis in the model: What are the units of replication and analyses, e.g., how many trees were sampled per species, how many species or trees per elevation, and how many plots per elevation? Were all 11 plots at different elevations and if so, which ones? The level of analysis for the redundancy analyses is not entirely clear: L. 404 mentions that the analyses were done 'across the rhizosphere and root tissue samples', but is that then at the individual-tree level? If so, it seems that these analyses should then also account for dependencies between trees from the same species and phylogeny (as (nested) covariates or random factors). With the information provided, I cannot tell whether there was sufficient replication for statistical interpretations.

(3) PCA: The results of the parallel analyses are not described: which components were retained? Because the authors aim to integrate microbial functions in a root economics space, I recommend first demonstrating the existence of a root economics space across the 52 subtropical species before running a PCA that includes the microbial traits. The PCA shown in this study does not exactly match the RES and this could be because traits of these species covary differently, but may also simply result from including additional traits to the PCA.

Also, the PCA's shown are carried out at the individual-tree level. I would recommend, however, including the species-level PCA's in the main text, because the individual-level PCA may not only reflect species-inherent ecological strategies (that e.g., the RES by Bergmann et al. 2020 describe) but also plasticity (Figures 2 and 3 both show an elevation effect that may be partly due to plasticity). While the results here are rather similar, intraspecific differences in root traits may follow different ecological principles and therefore not always be appropriate to compare with an interspecific RES (see for example Weemstra & Valverde-Barrantes, 2022, Annals of Botany).

I could not deduce whether tree species in the "fungal PCA" (Figure 2) were assigned as AM or EcM based on Table 1, or based on their observed fungal community composition. In the former case, the fungal functional guild gradient (from EcM to saprotrophs and AM) is partially an artificial one, because EcM tree species are not AM species (according to Table 1) and therefore, by definition, constitute a tradeoff or autocorrelation. And, as the authors also discuss, AM tree species may host EcM fungal species. Before I can evaluate the ecological meaning of PC1, and whether or not it really represents a mineral/organic nutrient gradient, information is needed on which data are used here.

I do not agree with the term 'gradient of bacterial guilds' (i.e., PC1 in Figure 3). All but 1 bacterial 'function' positively loaded on PC1 and 'fermentation' was only weakly negatively correlated with PC1. I do not think this constitutes a 'bacterial gradient'.

(4) Soil samples: Were they collected from the surrounding soil of each tree (L. 341), or from the root zone (L. 110). The former seems to refer to bulk soil samples, but the latter could be interpreted as rhizosphere soils. It is therefore not entirely clear whether these are the same soil samples, and if so, where they were sampled exactly.

Aims:<br /> The authors aimed to integrate endospheric and rhizospheric microbial and fungal community composition in the root economics space. Owing to statistical concerns (i.e., lacking parallel analysis results and the makeup of the PCs (AM versus EcM classification), I am not sure the authors succeeded in this. Besides that, the interpretation of the axes seems rather oversimplified and needs some consideration.

Root N is discussed as an important driver of fungal functional composition. Indeed, it was one of the significant variables in the redundancy models predicting microbial community composition, but its contribution to community composition was small (2 - 3 %), and the mechanistic interpretation was rather speculative. Specifically, the role of root N in root (and tree) functioning remains highly uncertain: the link with respiration and exudation is increasingly demonstrated but its actual meaning for nutrient uptake is not well understood (Freschet et al. 2021. New Phytologist). If and how root economics (represented by root N) and the fungal-driven nutrient economy (EcM versus AM, saprotrophs) can indeed be integrated into a unified framework (L. 223 - 224) seems a relevant question that is worth pursuing based on this paper, but in my opinion, this study does not clearly answer it, because the statistical analyses might need further work (or explanation) and underlying mechanisms are not well explained and supported by evidence.

In addition, the root morphology axis was indeed independent of the "fungal gradient", but this is in itself not an interesting finding. What is interesting, but not discussed is that, generally, AM species are expected to have thicker roots than EcM tree species (Gu et al. 2014 Tree Physiology; Kong et al. 2014 New Phytologist). I am therefore curious to see why this is not the case here? Did the few EcM species sampled just happen to have very thick roots? Or is there a phylogenetic effect that influences both mycorrhizal type and root thickness that is not accounted for here (Baylis, 1975; Guo et al., 2008 New Phytologist; Kubisch et al., 2015 Frontiers in Plant Science; Valverde-Barrantes et al., 2015 Functional Ecology; 2016 Plant and Soil)?

I also do not agree with the conclusion that this integrated framework 'explained' tree distributions along the elevation gradient. First of all, it is difficult to interpret because the elevation gradient is not well explained (e.g., in terms of environmental variation). Secondly, the framework might coincide with the framework, but the framework does not explain it: an environmental gradient probably underlies the elevation gradient that may be selected for species with certain root traits or mycorrhizal types, but this is not tested nor clearly demonstrated by the data. It thus remains rather speculative, and it should be more thoroughly explained based on the data observed. Similarly, I do not understand from this study how root traits like root N can influence the abundance of EcM and pathogenic fungi (L. 242 - 243). Which data show this causality? It seems a strong statement, but not well supported (or explained).

Impact:<br /> The data collected for this study are timely, valuable, and relevant. Soilborne microbes (fungi and bacteria; symbionts and pathogens) play important roles in root trait expressions (e.g., root diameter) and below-ground functioning (e.g., resource acquisition). They should therefore not be excluded from studies into the belowground functioning of forests, but they mostly are. This dataset therefore has the potential to improve our understanding of this subject. Making these data publicly available in large-scale datasets that have recently been initiated (e.g., FRED) will also allow further study in comparative (with other biomes) or global (across biomes) studies.

Technically, the methodology seems sound, although I lack the expertise to judge the Molecular Methods (L. 349 - 397). However, owing to some statistical uncertainties mentioned above (that the authors might well clarify or improve) and the oversimplified discussion, I am hesitant to determine the impact of the contents of this work. Statistical improvements and/or clearer explanation/justification of statistical choices made can make this manuscript highly interesting and impact, however.

Context:<br /> As motivated above, I am not sure to what extent the EcM - AM/saprotroph presents a true ecological tradeoff. However, if it does, this work would fit very well in the context of the mycorrhizal-associated nutrient economy (Phillips et al. 2013 New Phytology). This theory postulates that EcM trees generally produce low-quality litter (associated with 'slow traits') that can be more readily accessed by EcM but not AM fungi, thereby slowing down nutrient cycling rates at their competitive advantage, and vice versa for AM tree species. This study did not aim to test the MANE, so it was beyond its scope to study litter quality, and the number of EcM and AM species was unbalanced (8 EcM versus 44 AM species): nonetheless, the denser roots of EcM species and higher root N of AM species indicates that the MANE may also apply to this subtropical forest and may be an interesting impetus for future work on this topic. It might also offer one way to bridge the root economics space and the MANE.

What I also found interesting is the sparse observations of EcM fungal taxa in the root endosphere of species typically identified as AM hosts (L. 212 - 214). While their functionality remains to be tested (fungal structures in the endosphere were not studied here), this observation might call for renewed attention to classifying species as AM, EcM, or both.

Reviewer #3 (Public Review):

This paper uses behavior and functional brain imaging to understand how neural and cognitive representations of visual and auditory stimuli change as participants learn associations among them. Prior work suggests that areas in the anterior temporal (ATL) and perirhinal cortex play an important role in learning/representing cross-modal associations, but the hypothesis has not been directly tested by evaluating behavior and functional imaging before and after learning cross-modal associations. The results show that such learning changes both the perceived similarities amongst stimuli and the neural responses generated within ATL and perirhinal regions, providing novel support for the view that cross-modal learning leads to a representational change in these regions.

This work has several strengths. It tackles an important question for current theories of object representation in the mind and brain in a novel and quite direct fashion, by studying how these representations change with cross-modal learning. As the authors note, little work has directly assessed representational change in ATL following such learning, despite the widespread view that ATL is critical for such representation. Indeed, such direct assessment poses several methodological challenges, which the authors have met with an ingenious experimental design. The experiment allows the authors to maintain tight control over both the familiarity and the perceived similarities amongst the shapes and sounds that comprise their stimuli so that the observed changes across sessions must reflect learned cross-modal associations among these. I especially appreciated the creation of physical objects that participants can explore and the approach to learning in which shapes and sounds are initially experienced independently and later in an associated fashion. In using multi-echo MRI to resolve signals in ventral ATL, the authors have minimized a key challenge facing much work in this area (namely the poor SNR yielded by standard acquisition sequences in ventral ATL). The use of both univariate and multivariate techniques was well-motivated and helpful in testing the central questions. The manuscript is, for the most part, clearly written, and nicely connects the current work to important questions in two literatures, specifically (1) the hypothesized role of the perirhinal cortex in representing/learning complex conjunctions of features and (2) the tension between purely embodied approaches to semantic representation vs the view that ATL regions encode important amodal/crossmodal structure.

There are some places in the manuscript that would benefit from further explanation and methodological detail. I also had some questions about the results themselves and what they signify about the roles of ATL and the perirhinal cortex in object representation.

A) I found the terms "features" and "objects" to be confusing as used throughout the manuscript, and sometimes inconsistent. I think by "features" the authors mean the shape and sound stimuli in their experiment. I think by "object" the authors usually mean the conjunction of a shape with a sound---for instance, when a shape and sound are simultaneously experienced in the scanner, or when the participant presses a button on the shape and hears the sound. The confusion comes partly because shapes are often described as being composed of features, not features in and of themselves. (The same is sometimes true of sounds). So when reading "features" I kept thinking the paper referred to the elements that went together to comprise a shape. It also comes from ambiguous use of the word object, which might refer to (a) the 3D-printed item that people play with, which is an object, or (b) a visually-presented shape (for instance, the localizer involved comparing an "object" to a "phase-scrambled" stimulus---here I assume "object" refers to an intact visual stimulus and not the joint presentation of visual and auditory items). I think the design, stimuli, and results would be easier for a naive reader to follow if the authors used the terms "unimodal representation" to refer to cases where only visual or auditory input is presented, and "cross-modal" or "conjoint" representation when both are present.

B) There are a few places where I wasn't sure what exactly was done, and where the methods lacked sufficient detail for another scientist to replicate what was done. Specifically:

(1) The behavioral study assessing perceptual similarity between visual and auditory stimuli was unclear. The procedure, stimuli, number of trials, etc, should be explained in sufficient detail in methods to allow replication. The results of the study should also minimally be reported in the supplementary information. Without an understanding of how these studies were carried out, it was very difficult to understand the observed pattern of behavioral change. For instance, I initially thought separate behavioral blocks were carried out for visual versus auditory stimuli, each presented in isolation; however, the effects contrast congruent and incongruent stimuli, which suggests these decisions must have been made for the conjoint presentation of both modalities. I'm still not sure how this worked. Additionally, the manuscript makes a brief mention that similarity judgments were made in the context of "all stimuli," but I didn't understand what that meant. Similarity ratings are hugely sensitive to the contrast set with which items appear, so clarity on these points is pretty important. A strength of the design is the contention that shape and sound stimuli were psychophysically matched, so it is important to show the reader how this was done and what the results were.

(2) The experiences through which participants learned/experienced the shapes and sounds were unclear. The methods mention that they had one minute to explore/palpate each shape and that these experiences were interleaved with other tasks, but it is not clear what the other tasks were, how many such exploration experiences occurred, or how long the total learning time was. The manuscript also mentions that participants learn the shape-sound associations with 100% accuracy but it isn't clear how that was assessed. These details are important partly b/c it seems like very minimal experience to change neural representations in the cortex.

(3) I didn't understand the similarity metric used in the multivariate imaging analyses. The manuscript mentions Z-scored Pearson's r, but I didn't know if this meant (a) many Pearson coefficients were computed and these were then Z-scored, so that 0 indicates a value equal to the mean Pearson correlation and 1 is equal to the standard deviation of the correlations, or (b) whether a Fisher Z transform was applied to each r (so that 0 means r was also around 0). From the interpretation of some results, I think the latter is the approach taken, but in general, it would be helpful to see, in Methods or Supplementary information, exactly how similarity scores were computed, and why that approach was adopted. This is particularly important since it is hard to understand the direction of some key effects.

C) From Figure 3D, the temporal pole mask appears to exclude the anterior fusiform cortex (or the ventral surface of the ATL generally). If so, this is a shame, since that appears to be the locus most important to cross-modal integration in the "hub and spokes" model of semantic representation in the brain. The observation in the paper that the perirhinal cortex seems initially biased toward visual structure while more superior ATL is biased toward auditory structure appears generally consistent with the "graded hub" view expressed, for instance, in our group's 2017 review paper (Lambon Ralph et al., Nature Reviews Neuroscience). The balance of visual- versus auditory-sensitivity in that work appears balanced in the anterior fusiform, just a little lateral to the anterior perirhinal cortex. It would be helpful to know if the same pattern is observed for this area specifically in the current dataset.

D) While most effects seem robust from the information presented, I'm not so sure about the analysis of the perirhinal cortex shown in Figure 5. This compares (I think) the neural similarity evoked by a unimodal stimulus ("feature") to that evoked by the same stimulus when paired with its congruent stimulus in the other modality ("object"). These similarities show an interaction with modality prior to cross-modal association, but no interaction afterward, leading the authors to suggest that the perirhinal cortex has become less biased toward visual structure following learning. But the plots in Figures 4a and b are shown against different scales on the y-axes, obscuring the fact that all of the similarities are smaller in the after-learning comparison. Since the perirhinal interaction was already the smallest effect in the pre-learning analysis, it isn't really surprising that it drops below significance when all the effects diminish in the second comparison. A more rigorous test would assess the reliability of the interaction of comparison (pre- or post-learning) with modality. The possibility that perirhinal representations become less "visual" following cross-modal learning is potentially important so a post hoc contrast of that kind would be helpful.

E) Is there a reason the authors did not look at representation and change in the hippocampus? As a rapid-learning, widely-connected feature-binding mechanism, and given the fairly minimal amount of learning experience, it seems like the hippocampus would be a key area of potential import for the cross-modal association. It also looks as though the hippocampus is implicated in the localizer scan (Figure 3c).

F) The direction of the neural effects was difficult to track and understand. I think the key observation is that TP and PRh both show changes related to cross-modal congruency - but still it would be helpful if the authors could articulate, perhaps via a schematic illustration, how they think representations in each key area are changing with the cross-modal association. Why does the temporal pole come to activate *less* for congruent than incongruent stimuli (Figure 3)? And why do TP responses grow less similar to one another for congruent relative to incongruent stimuli after learning (Figure 4)? Why are incongruent stimulus similarities *anticorrelated* in their perirhinal responses following cross-modal learning (Figure 6)?

This work represents a key step in our advancing understanding of object representations in the brain. The experimental design provides a useful template for studying neural change related to the cross-modal association that may prove useful to others in the field. Given the broad variety of open questions and potential alternative analyses, an open dataset from this study would also likely be a considerable contribution to the field.

Reviewer #3 (Public Review):

D-type retroviruses, which include M-PMV assemble in the cytosol, however, do not efficiently start their maturation before membrane binding. There is very little known about the structural changes leading to maturation of D-type retroviruses and this manuscript presents compelling structural changes of the M-PMV matrix domain in mutations abrogating the myristol exposure or mutation which reasonably argue that myristol group is exposed (The relationship between these mutants and myristol exposure is argued based on structure of the matrix and liposome binding, however is not directly shown in structure). Assuming that the authors are correct about their mutations affect on myristol exposure, they have measured very interesting M-PMV matrix domain conformational changes which exposes the MAPP site to the protease.

Oligomerization of the matrix is probed by formation of disulfide bridges in a matrix mutant on liposomes with engineered cystine where authors suspect monomers of the matrix would be touching each other. The oligomerization data is very weak, does not directly support trimer formation and since 2D diffusion on liposomes would increase matrix-matrix interactions, can be non-specific, a point supported by presence of a stronger dimer band than trimer and tetramer. The main issue with the manuscript is that the authors do not show any evidence that the proposed mechanism actually works in the context of full M-PMV assembled particles.

Reviewer #3 (Public Review):

The study by Li et al investigates the role of type I interferon in suppressing ADAM17-mediated release of EGFR, the pathway previously implicated by this group in photosensitive skin reactions. Understanding the relevance of lupus murine models to the human disease is very important and the studies address this important gap in the knowledge. The most significant findings are: 1) the same high IFN and low Langerhans cell (LC) signatures seen in a lupus patient's skin, exist in the non-lesional skin of lupus mouse models; 2) IFN-Is and IFN-I signaling suppress ADAM17 activity in LCs in vitro and in vivo; 3) Blocking IFN-I signaling ameliorates photosensitive reactions, in an EGFR-dependent manner. These three conclusions are largely supported by the presented evidence but could be distilled as well as strengthened by additional data.

One of the strengths of the study is that the authors defined the relevance of lupus skin mouse models to human disease in the context of the Interferon-LC axis. The extensive computational approaches represent useful tools to compare cellular and molecular signatures across samples as well as species. This is highly relevant to the studies of lupus, a highly complex disease, for which the relevance of murine models has remained undefined. Major strengths related to the Aims of the study are that the authors established a role of interferon in suppressing Adam17 activity in the skin and showed that blocking interferon can reduce sunlight-induced skin inflammation in the lupus murine models. Interestingly, the authors observed that blocking IFN signaling in the absence of a high IFN-signature worsened sunlight-induced skin injury. The specificity of Adam17 in LCs for TNFR1 shedding provides an elegant approach to probing Adam17 activity in these cells.

While the three conclusions stated above are largely supported by the presented evidence, the data supporting a direct role of ADAM17 in IFN-triggered photosensitive reactions could be strengthened. Some of the concerns are outlined below:<br /> (1) Computational analyses in Figures 1 and 2 emphasize the co-occurrence of a high IFN-I signature and a low LC and/or DC signatures. It is not clear if the downregulation of the DC gene set indicates diminished presence of LCs in the non-lesional skin of the lupus mouse models or "reflects decreased LC function" as the authors suggest.

(2) Given the hypothesis that IFN-I may be the cause of a decreased DC signature in the mouse skin, it would be relevant to ask if this signature is also decreased in the IMQ model, which is a known model of IFN-induction as confirmed by the authors. Likewise, asking how anti-IFNAR treatment affects the DC signature / LC numbers would be important, in the absence and presence of UV. The authors indicate in Fig. 5I that IMQ reduces LC numbers.

(3) Decreased inflammation in LCad17 mice in the IMQ+UV model is unexpected. Previous studies by this group showed increased UV-induced inflammation in the absence of LC-ADAM17 (Shipman et al 2018). Therefore, it is not surprising that anti-IFNAR did not have an impact in these mice as ADAM17 deficiency appears to have normalized the response. These results are not addressed in the context of the previously published findings.

(4) Including the data that demonstrate the specificity of LCs for Adam17 expression in the epidermis and shedding of TNFR1 as a readout of LC-ADAM17-specific activity in the main figures would be important.

(5) UV light is an important inducer of IFN. Authors have previously shown that UV also induces Adam17 expression. Therefore, the question remains whether a high baseline IFN signature in lupus skin suppresses UV-induced Adam17 expression?

(6) A direct mechanistic link between high IFN-I and loss of Adam17 activity driving photosensitive reactions could be strengthened. Would blocking Adam17 with a blocking antibody suppress photosensitive reactions in lupus mouse models? Would treating LCAd17 mice with IFN fail to enhance or diminish UV-induced inflammation?

Reviewer #3 (Public Review):

This study used novel ingestible pills to measure pH and other gastric signals, and related these measures to self-report ratings of emotions induced by video clips. The main finding was that when participants viewed videos of disgust, there was an association between gastric pH and feelings of disgust and fear, and (in the opposite direction) happiness. These findings may be the first to relate objective measures of gastric physiology to emotional experience. The methods open up many new questions that can be addressed by future studies and are thus likely to have an impact on the field.

My main concern is with the reliability of the results. The study associates many measures (pH, temperature, pressure, EGG) in stomach, small bowel, and large bowel with multiple emotion ratings. This amounts to many statistical tests. Only one of these measures (pH in the stomach) shows a significant effect. Furthermore, the key findings, as displayed in Figure 4 do not look particularly convincing. Perhaps this is a display issue, but the relations between stomach pH and Vas ratings of disgust, fear, and happiness were not apparent from the scatter plot and may be influenced by outliers (e.g., happiness).

Reviewer #3 (Public Review):

In this manuscript, the authors seek to shed light on the role of awake hippocampal replay during memory tasks that are claimed to be short-term memory. For this, they make use of a real-time detection and disruption system of awake hippocampal ripples, which are used as a proxy for awake neuronal replay. The manuscript describes extensively the tasks as well as the disruption system and controls used during the experiments. The authors present numerous and solid analyses of the behavioral data acquired during the tasks. Nonetheless, the current version of the manuscript is lacking a more complete discussion in which the results are contrasted to previous similar findings, as well as mentioning the role of the awake ripple in the stabilization of hippocampal maps. Some extra analyses are also suggested below. The manuscript would also be enriched if the authors suggested alternative mechanisms for memory rehearsal. Finally, some claims of "we are first" seem inappropriate when compared to the previous literature.

Major comments:

How does one define short-term memory (STM) in rodents? The examples and papers cited in the first paragraphs refer mostly to human working memory tasks, from which it is known that a non-rehearsed STM lasts typically 20-30 seconds. Could the authors mention how this concept is translated to rodents? Could you clarify until what point memory is considered STM and what is the criteria to consider it has turned into long-term memory or when is it simply working memory or habit/skill? Further, why should these tasks be classified as testing STM while Jadhav et al. tasks are working memory or as they now mention in this article rule learning? In humans, the retention of memory after a certain time is achieved by retrieving a long-term memory. How do we know if the considerable training the rats received has not allowed the use of a long-term memory strategy which allows the rats to perform well even in the absence of rehearsal (replay)? These are conceptual explanations that would help understand the key concept of STM in greater detail.

Further, claims of "first" should be adjusted, since I do not see a large difference between the w (m) maze of Jadhav and these tasks. The main difference between the two projects would rather be that Jadhav tests when animals are still newer to the task while here overtrained animals are used. In Jadhav, it's unlikely that just rule learning is affected since the inbound component is not affected by disruption, which also tests rule learning. Therefore, it is still likely that the effect seen in Jadhav et al is a deficit in working memory/short-term memory. And here it is more likely, that no effect was seen since with overtrained animals other strategies (cortical, striatal, etc) were used. The authors should compare in more detail how overtrained animals were in these different projects as well as in the articles they cite for replay analysis.

The main conclusion of the authors is that hippocampal replay is not the rehearsal mechanism expected in STM given that its disruption doesn't lead to behavioral changes. Could the authors hypothesize in their discussion what other neural mechanisms different from hippocampal replay may be involved in this rehearsal? The discussion also lacks closure with respect to how the findings fit in the study of STM in human memory. This would make the article more interesting to a larger audience and highlight its translational aspect.

The results describe deeply the behavioral performance of the rats and the validation of the ripple detection/disruption system. However, one important aspect missing is how the hippocampal activity and its encoding of space may be affected by the awake ripple disruption. The authors don't cite the work by Roux et al., Nature Neuroscience. 2017 where optogenetic stimulation of hippocampal neurons provided evidence that neuronal activity associated with awake hippocampal ripples during goal-directed behavior is required for both stabilizing and refining hippocampal place fields, while memory performance was not affected during ripple-locked stimulations compared to a ripple-delayed stimulation control (See supplementary Figure 7 of the mentioned article). I would like the authors to comment on their own findings and contrast them with those of Roux et al.

Line 64: Could the authors clarify what they mean by "indirect" causal evidence when discussing the contribution of papers by Jadhav, Igata, and Fernandez? Is it the fact that rodents' learning speed changed instead of showing a complete absence of learning? Or is it the fact that the disruption/prolongation is done on the hippocampal ripple and not strictly in the replay sequence? I would also highlight this latter difference, given that the above-mentioned authors describe their methodological approaches in terms of ripples and not in terms of replay content. For example, the use of "replay" instead of "ripple" in Line 61 results in methodological inaccurate terms such as replay disruption and replay prolongation.

Despite its apparent lack of statistical significance, the reported mean ripple detection rate during the trial and non-trial periods tend to be always higher in the disruption condition of all tasks by observing the median of the boxplots in Figure 1J, Figure 2H, and Figure 3J. It is worth investigating this further using the same linear regression method as Girardeau et al. Journal of Neuroscience, 2014 which may reduce the variability and allow comparing slopes of a cumulative number of ripples over time. This may reveal a compensatory homeostatic-like increase in the rate of ripples during the disrupted sessions, which may suggest a need for the ripple/replay occurrence in spite of it not having an effect on the rats' performance during the task.

In line 425, the authors report a median relative delay of 52.9 of their disruption system. Such a value would indicate that only around 47% of the ripple is being blocked. Is there any data from the authors or others that could reassure the reader that the 52.9% of the ripple that "leaks" is not enough for the replay phenomenon to occur? Considering the findings of Fernandez-Ruiz et al. 2019 on large-duration ripples, could the authors report the relative delay for both short and long ripples (>100 ms) separately? Line 494: The authors define long ripples as (>120 ms) but this doesn't coincide with the 100ms threshold from Fernandez Ruiz et al. 2019.

The online ripple detector used filtered the traces in the 135-255 Hz range. This is a narrower frequency range compared to online detectors used by Jadhav et al. 2012 (100-400 Hz) and Fernandez-Ruiz et al. 2019 (80-300 Hz). What motivated the use of this narrow range? Would the omittance of ripples below 135 Hz have implications in the results? Could the authors add to the supplement a figure similar to Figure 4B (FDR vs TPR) using a wider frequency range similar to the authors above in the offline detection of ripples?

It is unclear what criterion was used to train the rats in the NMTS task. Line 216 specifies a learning criterion of 80% fully correct trials in one session for three days in a row, while the methods in line 852 mention an average performance below 50% for at least three days in a row.

In the methods section, it is not mentioned if there was a specific region in the cortex where the tetrode was placed (Line 908). The detections in this tetrode were used to mark events as "false positives". The authors should be careful in line 933 when they make the statement "ripples are not present in the cortex". There have been recent publications that challenge this affirmation. See Khodagholy, Science. 2017, Nitzan, Nature Comm. 2020.

Reviewer #3 (Public Review):

This is a potentially quite interesting paper that defines E2 and E3 genes in Drosophila that can impact the accumulation of the Q72-GFP protein in the fly eye. The authors then focus on the eff gene, showing which human homolog can rescue fly knockdown. They extend to skeletal muscle, from the htt protein, to show that eff by TMT mass spec decreases with age normally in the fly muscle and that there is a significant overlap of proteins that are disrupted with eff knockdown in young animals in muscle vs aged animals normally in muscle.

Overall these data suggest eff decrease with age may contribute to the increase in ubiquitinated proteins in muscle with age, and that upregulation of eff activity might be of interest to extending lifespan. Because eff function can be performed by a human homologue, the findings may also apply to human situations of aging.

These data are overall interesting and are of relevance for those interested in neurodegenerative disease and aging, although a number of points from the figures seem confusing and need more explanation or clarity.

Reviewer #3 (Public Review):

Summary: The paper aims at providing a neurocomputational account on how social perception translates in prosocial behaviors. Participants first completed a novel social perception task during fMRI scanning, in which were asked to judge the merit or need of people depicted in different situations. Second , a separate altruistic choice task was used to examine how the perception of merit and need influences the weights people place on themselves, others and fairness when deciding to provide help. Finally, a link between perception and action was drawn in those participants who completed both tasks.

Strengths: The paper is overall very well written and presented, leaving the reader at ease when describing complex methods and results. The approach used by the author is very compelling, as it combines computational modeling of behavior and neuroimaging data analyses. Despite not being able to comment on the computational model, I find the approach used (to disentangle sensitivity and biases, for merit and need) very well described and derived from previous theoretical work. Results are also clearly described and interpreted.

Weaknesses: in the social perception task, merit and need are evaluated by means of very different cues that rely on different cognitive processes (more abstract thinking for merit than need). Despite this limitation of the task, the authors were able to argue convincingly in the revised version about the solidity of their findings. Sample size is quite small for study 2, nevertheless the results provide convincing evidence.

Reviewer #3 (Public Review):

Summary:

Amacrine cells are a heterogeneous collection of retinal interneurons. Most are inhibitory, and like inhibitory neurons in other neural circuits, strongly shape retinal function. With a few exceptions, the role of amacrine cells in retinal signaling is poorly understood. This paper introduces an approach to study a set of wide-field amacrine cells that extend processes over large regions of the retina.

Strengths:

A substantial strength of the paper is the combination of genetic manipulations, electrophysiology, optogenetics and electron microscopy used to study these cells. As a result of that broad set of techniques, the results cover many properties of how the cells work and provide a nice overview. The paper is also (with a few exceptions below) clearly presented and the experiments look to be carefully executed with clean results.

Weaknesses:

My largest concern with the paper is that overall the results provided an initial view of an interesting set of issues about the function of these cells, but the interesting initial results are not pursued in more depth.

Spatial spread of signals in neurites<br /> An immediate question about axonless WACs is the extent of spread of signals along their processes, and hence whether they act as a collection of independent or semi-independent elements. This bears directly on interpretation of the responses to oriented stimuli for example. Did you do any experiments that might provide additional information about this issue? For example, if you stimulate one of the WACs peripherally do you see a strong modulation of the somatic voltage? Or in the imaging experiments, if you mask a region of the processes so that it is not receiving a stimulus, do you see responses "leak" into that occluded region from surrounding stimulated regions?

Orientation tuning and connectivity<br /> The most developed functional results in the paper relate to the sensitivity of the WAC processes to oriented stimuli. Interpretation of these results depends on a few factors. First is the spread of signals in the WAC processes - as noted above. Second is connectivity. The paper shows that the B/K WAC activity increases inhibitory input to Off-delta and On-alpha ganglion cells. These cells, as noted in the paper, are not orientation tuned. But the orientation tuned ganglion cells stratify in a similar location within the IPL, and hence are situated in an appropriate place to receive input from the B/K WACs. Did you focus exclusively on connections to the Off-delta and On-alpha cells (along with the Off-alpha) or did you look at any other ganglion cell types? This should at least get discussed in more detail.

In several places it is unclear whether the paper intends to be a methods paper or a basic research paper. One example is the last sentence of the abstract. If it is intended to be a basic research paper (which is my overall impression) then I suggest shifting the emphasis in some of those key locations towards results and away from methods.

Reviewer #3 (Public Review):

Summary:<br /> In the current work, Howell et al studied the connectivity between the cortex and thalamus using DTI tractorgraphy per parcel to all voxels in the thalamus. Following they performed various dimensional reduction techniques to uncover how differences in connectivity to the thalamus vary across cortical parcels. Following they explore the spatial correlation of these variations with cortical myelin and functional organization, thalamic nuclei, gene expression derived core-matrix cell differentiation, and extend the model towards macaques. Overall, the authors find a differentiation between sensory and association areas in terms of the association with the thalamus, which reflects differences in cortical microstructure and function, and links to core-matrix differences and can be replicated in macaques.

Strengths:<br /> A clear strength of the current work is the combination of different models and approaches to study the link between the cortex and the thalamus. This approach nicely bridges different approaches to describe the role of the thalamus in cortical organisation using a diffusion-based approach. Especially the extension of the model to the macaque is quite nice.

Weaknesses:<br /> Potential weaknesses of the study are that it seems to largely integrate aspects of the thalamus that have been already described before. The differentiation between sensory and association systems across thalamic subregions is something that has been described before (see: Oldham and Ball, 2023; Zheng et al., 2023; Yang et al., 2020 Mueller, 2020; Behrens, 2003).

Appraisal:<br /> However, the aim of the study: 'to investigate the spatial extent of anatomical connectivity patterns within the thalamus in both humans and non-human primates and determine if such patterns differ between sensorimotor and association cortical areas' has been met.

Discussion:<br /> Overall, I think the study is a nice addition to the growing literature studying the anatomical connectivity between the thalamus and cortex and its functional implications.

Reviewer #3 (Public Review):

Summary:<br /> The authors proposed a normative principle for how the brain's internal estimate of an observed sensory variable should be updated during each individual observation. In particular, they propose that the update size should be inversely proportional to the variance of the variable. They then proposed a microcircuit model of how such an update can be implemented, in particularly incorporating two types of interneurons and their subtractive and divisive inhibition onto pyramidal neurons. One type should represent the estimated mean while another represents the estimated variance. The authors used simulations to show that the model works as expected.

Strengths:<br /> The paper addresses two important issues: how uncertainty is represented and used in the brain, and the role of inhibitory neurons in neural computation. The proposed circuit and learning rules are simple enough to be plausible. They also work well for the designated purposes. The paper is also well-written and easy to follow.

Weaknesses:<br /> I have concerns with two aspects of this work.

(1) The optimality analysis leading to Eq (1) appears simplistic. The learning setting the authors describe (estimating the mean of a stationary Gaussian variable from a stream of observations) is a very basic problem in online learning/streaming algorithm literature. In this setting, the real "optimal" estimate is simply the arithmetic average of all samples seen so far. This can be implemented in an online manner with \hat{\mu}_{t} = \hat{\mu}_{t-1} +(s_t-\hat{\mu}_{t-1})/t. This is optimal in the sense that the estimator is always the maximum likelihood estimator given the samples seen up to time t. On the other hand, doing gradient descent only converges towards the MLE estimator after a large number of updates. Another critique is that while Eq (1) assumes an estimator of the mean (\hat{mu}), it assumes that the variance is already known. However, in the actual model, the variance also needs to be estimated, and a more sophisticated analysis thus needs to take into account the uncertainty of the variance estimate and so on. Finally, the idea that the update should be inverse to the variance is connected to the well-established idea in neuroscience that more evidence should be integrated over when uncertainty is high. For example, in models of two-alternative forced choices it is known to be optimal to have a longer reaction time when the evidence is noisier.

(2) While the incorporation of different inhibitory cell types into the model is appreciated, it appears to me that the computation performed by the circuit is not novel. Essentially the model implements a running average of the mean and a running average of the variance, and gates updates to the mean with the inverse variance estimate. I am not sure about how much new insight the proposed model adds to our understanding of cortical microcircuits.

Reviewer #3 (Public Review):

Summary:

Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine the activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during the approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength of their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of learning and memory. The conclusions of this manuscript are mostly well supported by the data, but some additional analysis and/or experiments may be required to fully support the author's conclusions.

Weaknesses:

(1) During teleportation between familiar to novel environments the authors report a decrease in the freezing ratio when combining the mice in the two experimental groups (Figure 3aiii). A major conclusion from the manuscript is the difference in VTA and LC activity following environment change, given VTA and LC activity were recorded in separate groups of mice, did the authors observe a similar significant reduction in freezing ratio when analyzing the behavior in LC and VTA groups separately?

(2) The authors satisfactorily apply control analyses to account for the unequal axon numbers recorded in the LC and VTA groups (e.g. Figure 1). However, given the heterogeneity of responses observed in Figures 3c, 4b and the relatively low number of VTA axons recorded (compared to LC), there are some possible limitations to the author's conclusions. A conclusion that LC-CA1 axons, as a general principle, heighten their activity during novel environment presentation, would require this activity profile to be observed in some of the axons recorded in most all LC-CA1 mice. Additionally, if the general conclusion is that VTA-CA1 axons ramp activity during the approach to reward, it would be expected that this activity profile was recorded in the axons of most all VTA-CA1 mice. Can the authors include an analysis to demonstrate that each LC-CA1 mouse contained axons that were activated during novel environments and that each VTA-CA1 mouse contained axons that ramped during the approach to reward?

(3) A primary claim is that LC axons projecting to CA1 become activated during novel VR environment presentation. However, the experimental design did not control for the presentation of a familiar environment. As I understand, the presentation order of environments was always familiar, then novel. For this reason, it is unknown whether LC axons are responding to novel environments or environmental change. Did the authors re-present the familiar environment after the novel environment while recording LC-CA1 activity?

Reviewer #3 (Public Review):

Summary:<br /> In the manuscript titled "Large pan-cancer cell screen coupled to (phospho-)proteomics underscores high-dose vitamin C (VitC) as a potent anti-cancer agent," the authors use a combination of proteomics and cell viability assays to understand the effect of Vitamin C on different solid tumor models in 51 different cancer cell lines. They found that many cancer cell lines are sensitive to high-dose Vitamin C, with IC50 values in the micromolar to millimolar range. Given that Vitamin C, when administered intravenously, can reach 20mM, this suggests that Vitamin C could provide some benefits to patients. The authors also generate and analyze bulk proteomic data for all 51 cell lines. They perform statistical analysis of these data to identify proteins that are up or downregulated in sensitive vs resistant cell lines in the same tumor and commonly across tumors. They then focus on PDAC cell lines and measure bulk and phosphoproteomics of PDAC cell lines 2, 4 and 24 hours after Vitamin C treatment.

Strengths: The strengths of the study are the rather large datasets accumulated on bulk proteomics of 51 different cancer cell lines. The IC50 values of these cell lines in response to Vitamin C is also useful.

Weaknesses:<br /> Though identifying targets to sensitize cancer cells to Vitamin C treatment is interesting, I felt the manuscript delved too much into listing off genes they found, with speculation on why the particular protein would be enriched in sensitive or resistant cell lines without testing any key claims experimentally.

Major Issues

(1) The overall premise of the study is that proteins that are enriched in Vitamin C-sensitive cell lines point to mechanisms of sensitivity and those enriched in Vitamin C resistant lines underlie mechanisms of resistance. Yet this is never directly tested. To show that the authors would need to knockdown/knockout a gene enriched in resistant lines and show this sensitizes cells to Vitamin C treatment or overexpress a protein associated with resistance and show that this leads to resistance in an otherwise sensitive cell line.

(2) One of the key strengths of this study is the large datasets generated, namely the proteomics data for 51 different cell lines. Yet the data is not included as a supplement or uploaded to a public repository.

Reviewer #3 (Public Review):

Summary:<br /> In this study, Brenes and colleagues carried out proteomic analysis of several human induced pluripotent (hiPSC) and human embryonic stem cell (hESC) lines. The authors found quantitative differences in the expression of several groups of cytoplasmic and mitochondrial proteins. Overall, hiPSC expressed higher levels of proteins such as glutamine transporters, mitochondrial metabolism proteins, and proteins related to lipid synthesis. Based on the protein expression differences, the authors propose that hiPSC lines differ from hESC in their growth and metabolism.

Strengths:<br /> The number of generated hiPSC and hESC lines continues to grow, but potential differences between hiPSC and hESC lines remain to be quantified and explained. This study is a promising step forward in understanding of the differences between different hiPSC and hESC lines.

Weaknesses:<br /> It is unclear whether changes in protein levels relate to any phenotypic features of cell lines used. For example, the authors highlight that increased protein expression in hiPSC lines is consistent with the requirement to sustain high growth rates, but there is no data to demonstrate whether hiPSC lines used indeed have higher growth rates.

The authors claim that the cell cycle of the lines is unchanged. However, no details of the method for assessing the cell cycle were included so it is difficult to appreciate if this assessment was appropriately carried out and controlled for.

Details and characterisation of iPSC and ESC lines used in this study were overall lacking. The lines used are merely listed in methods, but no references are included for published lines, how lines were obtained, what passage they were used at, their karyotype status, etc. For details of basic characterisation, the authors should refer to the ISSC Standards for the use of human stem cells in research. In particular, the authors should consider whether any of the changes they see may be attributed to copy number variants in different lines.

The expression data for markers of undifferentiated state in Figure 1a would ideally be shown by immunocytochemistry or flow cytometry as it is impossible to tell whether cultures are heterogeneous for marker expression.

TEM analysis should ideally be quantified.

All figure legends should explicitly state what graphs are representing (e.g. average/mean; how many replicates (biological or technical), which lines)? Some data is included in Methods (e.g. glutamine uptake), but not for all of the data (e.g. TEM).

Validation experiments were performed typically on one or two cell lines, but the lines used were not consistent (e.g. wibj_2 versus H1 for respirometry and wibj_2, oaqd_3 versus SA121 and SA181 for glutamine uptake). Can the authors explain how the lines were chosen?

The authors should acknowledge the need for further functional validation of the results related to immunosuppressive proteins.

Differences in H1 histone abundance were highlighted. Can the authors speculate as to the meaning of these differences?

Reviewer #3 (Public Review):

This reviewer appreciates the responses to previous notes. The authors attempted to address concerns mostly in writing, avoiding performing some of the experiments suggested in my previous review. Although some of the points were clarified, and the revised manuscript presents valuable insights into the implications of YBR238C and RMD9 on cellular function and yeast aging, my major concern still needs to be addressed. The gene expression signature significantly changes under different metabolic conditions. The media condition under which samples are collected for RNAseq analyses should match the media condition under which the lifespans of those KO strains are tested. This is the major confounding effect, and the conclusions are not informative based on the analysis done in this study.

To avoid experiments, the authors responded that yeast culture results in low optical density and does not reach the stationary phase under rapamycin treatment conditions; however, the simple solution is to grow the yeast cells until they reach the stationary phase and then rapamycin treatment can be done for certain hours - collect the cells for transcriptomics analysis then it can be compared to the CLS gene set.

Another example is chromosome copy number alteration, which can be easily analyzed using transcriptome data, and it is an important aspect to understand whether observed expression changes are also affected by this alteration in YBR238C KO cells. However, the authors ignore this important point as well.

After all, this is an interesting study "limited by subfield" and will be of general interest in the yeast aging field, again considering the lack of homology of the genes of interest in higher eukaryotes.

Reviewer #3 (Public Review):

Summary:<br /> Kobayashi et al identify MER21C as a common promoter of GPR1-AS/Liz in Euarchontoglires, which establishes a somatic DMR that controls ZFDB2 imprinting. In mice, MER21C appears to have diverged significantly from its primate counterparts and is no longer annotated as such.

Strengths:<br /> The authors used high-quality cross-species RNA-seq data to characterise GPR1-AS-like transcripts, which included generating new data in five different species. The association between MER21C/B elements and the promoter of GPR1-AS in most species is clear and convincing. The expression pattern of MER21C/B elements overall further supports their role in enabling correct temporal expression of GPR1-AS during embryonic development.

Weaknesses:<br /> A deeper comparison of syntenic regions to the GPR1-AS promoter could be performed to provide a clearer picture of how the MER21C/B element evolved. The use of alternative TE annotation software may also be helpful. These analyses would be particularly useful to drive home the conclusion that the mouse (Liz) promoter is derived from the same insertion.

Reviewer #3 (Public Review):

Summary:<br /> The authors use several quantitative approaches to characterize the feeding ecologies of bohaiornithid enantiornithines, including allometric data, mechanical advantage and finite element analyses of the jaw, and morphometric analyses of the claws. The authors combine their results with data for other enantiornithines collected from the literature to shed new insight on the ecological evolution of Enantiornithes as a clade.

Although the authors have taken steps to improve their paper, I generally find improvements unsatisfying, especially regarding my comments.

My remaining concerns:

Teeth: My concern here is not whether having teeth limits available niche space compared to having a keratinous beak. Rather, my concern regards how exploitation of the same niche space might be differently reflected in parameter space between birds with teeth and birds with beaks. Can we reliably expect two species that both eat seeds to occupy the same parameter space if, for example, distribution of stress/strain is across a series of teeth vs. across a more uniform beak? In this manuscript, the authors are clearly making this assumption, but that assumption is not made explicit, let alone justified. The authors should discuss this.

Cranial kinesis: As with teeth, my concern here regards our ability to compare data between birds with and without a flexible beak to mitigate forces when foraging. I appreciate that the functional complexity of the kinetic neognath skull precludes our ability to account for it in analyses such as these, but when comparisons are made using these analyses *specifically among neognaths*, we can reliably assume that we are comparing like to like - that is, we can assume that both have kinetic skulls, and so kinesis is reflected similarly in the data for each bird. Similarly, even if a comparison between two neognaths focuses exclusively on the mandible - in which cranial kinesis is not directly reflected - we can assume that those mandibles serve as comparisons between functionally similar systems. However, we cannot necessarily make those same assumptions when comparing the kinetic skull of a neognath to the akinetic skull of an enantiornithine. Indeed, even when focusing just on the mandible, can we reliably assume that data collected from an akinetic enantiornithine reflect the same comparative context as data collected from kinetic neognaths? I appreciate that the authors added a call for better functional understanding of bird cranial kinesis - a call I enthusiastically endorse - but the authors should still discuss how that current lack of understanding impacts interpretations of the comparisons they draw.

Finally, I still find the discussion to be overly long and lacking clear focus and organization. I again urge the authors to minimally consider adding subheadings to better allow the reader to follow the flow of ideas, and I second Reviewer #1's suggestion to add a "Conclusions" section.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Coberski et al describes a combined experimental and computational study aimed to shed light on the catalytic mechanism in a methyltransferase that transfers a methyl group from S-adenosylmethionine (SAM) to a substrate adenosine to form N6-methyladenosine (m6A).

Strengths:<br /> The authors determine crystal structures in complex with so-called bi-substrate analogs that can bridge across the SAM and adenosine binding sites and mimic a transition state or intermediate of the methyl-transfer reaction. The crystal structures suggest dynamical motions of the substrate(s) that are examined further using classical MD simulations. The authors then use QM/MM calculations to study the methyl-transfer process. Together with biochemical assays of ligand/substrate binding and enzyme turnover, the authors use this information to suggest what the key steps are in the catalytic cycle. The manuscript is in most places easy to read.

Weaknesses:<br /> After revising the manuscript, there are few weaknesses beyond those listed in the paper.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors use inhibitors and mimetics of juvenile hormone (JH) to demonstrate that JH has a key role in late embryonic development in Thermobia, specifically in gut and eye development but also resorption of the extraembryonic fluid and hatching. They then exogenously apply JH early in development (when it is not normally present) to examine the biological effects of JH at these stages. This causes a plethora of defects including developmental arrest, deposition of chitin, limb development, and enhanced muscle differentiation. The authors interpret these early effects on development as JH being important for the shift from morphogenetic growth to differentiation - a role that they speculate may have facilitated the evolution of metamorphosis (hemi- and holo-metaboly). This paper will be of interest to insect evo-devo researchers, particularly those with interests in the evolution of metamorphosis.

Strengths:

The experiments are generally conducted very well with appropriate controls and the authors have included a very detailed analysis of the phenotypes.<br /> The manuscript significantly advances our understanding of Thermobia development and the role of JH in Thermobia development.<br /> The authors interpret this data to present some hypotheses regarding the role of JH in the evolution of metamorphosis, some aspects of which can be addressed by future studies.

Weaknesses:

The results are based on using inhibitors and mimetics of JH and there was no attempt to discern immediate effects of JH from downstream effects. The authors show, for instance, that the transcription of myoglianin is responsive to JH levels, it would have been interesting to see if any of the phenotypic effects are due to myoglianin upregulation/suppression (using RNAi for example). These kinds of experiments will be necessary to fully work out if and how the JH regulatory network has been co-opted into metamorphosis.

The results generally support the authors' conclusions. However, the discussion contains a lot of speculation and some far-reaching conclusions are made about the role of JH and how it became co-opted into controlling metamorphosis. There are some interesting hypotheses presented and the author's speculations are consistent with the data presented. However, it is difficult to make evolutionary inferences from a single data point as although Thermobia is a basally branching insect, the lineage giving rise to Thermobia diverged from the lineages giving rise to the holo- and hemimetabolous insects approx.. 400 mya and it is possible that the effects of JH seen in Thermobia reflect lineage-specific effects rather than the 'ancestral state'. The authors ignore the possibility that there has been substantial rewiring of the networks that are JH responsive across these 400 my. I would encourage the authors to temper some of the discussion of these hypotheses and include some of the limitations of their inferences regarding the role of JH in the evolution of metamorphosis in their discussion.

Reviewer #3 (Public Review):

Summary:<br /> After the previous identification that the Streptococcus agalactiae MprF enzyme can synthesize also lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), besides the already known lysyl-phosphatidylglycerol (Lys-PG), the authors aim for the current manuscript was to investigate the molecular determinants of MprF lipid substrate specificity in a variety of bacterial species.

Strengths:<br /> - In general, the manuscript is well constructed and easy to follow, especially taking into account the multidisciplinary aspect of it (computational machine learning combined with lipid biology).<br /> -The added value of the Restricted Boltzmann machines (RBM) approach, in comparison to standard computational pairwise sequence statistics, becomes evident. This is exemplified by a successful, although not perfect, classification and categorization of MprF activity.<br /> - The MS analysis (monoisotopic mass, plus fragmentation pattern), convincingly shows the identification of a novel lipid species Lys-Glc2-DAG.

Weaknesses:<br /> -In many of the analyzed strains, the presence of the lipid species Lys-PG, Lys-Glc-DAG, and Lys-Glc2-DAG is correlated to the presence of the MprF enzyme(s), but one should keep in mind that a multitude of other membrane proteins are present that in theory could be involved in the synthesis as well. Therefore, there is no direct evidence that the MprF enzymes are linked to the synthesis of these lipid species. Although, it is unlikely that other enzymes are involved, this weakens the connection between the observed lipids and the type of MprF.<br /> -Related to this, in a few cases MprF activity is tested, but the manuscript does not contain any information on protein expression levels. Heterologous expression of membrane proteins is in general challenging and due to various reasons, proteins end up not being expressed at all. As an example, the absence of activity for the E. faecalis MprF1 and E. faecium MprF2 could very well be explained by the entire absence of the protein.

Overall, the authors largely achieved their goals, as the applied RBM approach led to specific sequence determinants in MprF enzymes that could categorize the specificity of these enzymes. The experimental data could largely confirm this categorization, although a stronger connection between synthesized lipids and enzyme activity would have further strengthened the observations.

The work now focuses only on MprF enzymes, but could in theory be expanded to other categories of lipid-synthesizing enzymes. In other words, the RBM approach could have an impact on the lipid synthesis field, if it would be a tool that is easily applicable. Moreover, the lipids synthesized by MprF (Lys-PG, but also other cationic lipids) play an important role in bacterial resistance against certain antibiotics.

Reviewer #3 (Public Review):

The revised study provides better evidence to suggest that loss of Aprt activity in Drosophila provides a model for the loss of HGPRT activity in humans, which is causative for LND. Analysis of Drosophila Aprt mutations and RNAi-mediated knockdown reveals similar phenotypes to LND, particularly neurological defects, reduced nighttime sleep, and potentially seizures. LND is currently resistant to treatments and screening of a limited number of compounds in Drosophila has not identified a compound that can reduce all of the associated phenotypes. It is appropriate, therefore, that claims to have developed a clinically exploitable model for human LND have been toned down. Future drug screening may well prove profitable, but currently the evidence that Drosophila Aprt will be a suitable model for LND remains speculative.

The second approach adopted is to express a 'humanised mutated' form of HGPRT in Drosophila, which holds more promise for the development of a pharmacological screen. In particular, the locomotor defect is recapitulated but the seizure-like activity, whilst reported as being recapitulated, is debatable. A recovery time of 2.3 seconds is very much less than timings for typical seizure mutants. Nevertheless, the SING behaviour could be sufficient to screen against. However, this is not explored. With respect the short seizure duration, the authors cite similar findings for porin loss of function, but the cited study similarly did not employ anti-seizure drug exposure to validate that this phenotype is seizure related.

In summary, this is a largely descriptive study reporting the behavioural effects of an Aprt loss-of-function mutation. RNAi KD and rescue expression studies suggest that a mix of neuronal (particularly dopaminergic and possibly adenosinergic signalling pathways) and glia are involved in the behavioural phenotypes affecting locomotion, sleep and seizure. There remains insufficient evidence to have full confidence that the Arpt fly model will prove valuable for understanding / treating LND.

Reviewer #3 (Public Review):

Summary:<br /> This paper aims to investigate how the human brain represents different forms of value and uncertainty that participate in active inference within a free-energy framework, in a two-stage decision task involving contextual information sampling, and choices between safe and risky rewards, which promotes a shift from exploration to exploitation. They examine neural correlates by recording EEG and comparing activity in the first vs second half of trials and between trials in which subjects did and did not sample contextual information, and perform a regression with free-energy-related regressors against data "mapped to source space." Their results show effects in various regions, which they take to indicate that the brain does perform this task through the theorised active inference scheme.

Strengths:<br /> This is an interesting two-stage paradigm that incorporates several interesting processes of learning, exploration/exploitation, and information sampling. Although scalp/brain regions showing sensitivity to the active-inference-related quantities do not necessarily suggest what role they play, it can be illuminating and useful to search for such effects as candidates for further investigation. The aims are ambitious, and methodologically it is impressive to include extensive free-energy theory, behavioural modelling, and EEG source-level analysis in one paper.

Weaknesses:<br /> Though I could surmise the above general aims, I could not follow the important details of what quantities were being distinguished and sought in the EEG and why. Some of this is down to theoretical complexity - the dizzying array of constructs and terms with complex interrelationships, which may simply be part and parcel of free-energy-based theories of active inference - but much of it is down to missing or ambiguous details.

In general, an insufficient effort has been made to make the paper accessible to readers not steeped in the free energy principle and active inference. There are critical inconsistencies in key terminology; for example, the introduction states that aim 1 is to distinguish the EEG correlates of three different types of uncertainty: ambiguity, risk, and unexpected uncertainty. But the abstract instead highlights distinctions in EEG correlates between "uncertainty... and... risk" and between "expected free energy .. and ... uncertainty." There are also inconsistencies in mathematical labelling (e.g. in one place 'p(s|o)' and 'q(s)' swap their meanings from one sentence to the very next).

Some basic but important task information is missing, and makes a huge difference to how decision quantities can be decoded from EEG. For example:<br /> - How do the subjects press the left/right buttons - with different hands or different fingers on the same hand?<br /> - Was the presentation of the Stay/cue and safe/risky options on the left/right sides counterbalanced? If not, decisions can be formed well in advance especially once a policy is in place.<br /> - What were the actual reward distributions ("magnitude X with probability p, magnitude y with probability 1-p") in the risky option? 

The EEG analysis is not sufficiently detailed and motivated. For example,<br /> - why the high lower-filter cutoff of 1 Hz, and shouldn't it be acknowledged that this removes from the EEG any sustained, iteratively updated representation that evolves with learning across trials?<br /> - Since the EEG analysis was done using an array of free-energy-related variables in a regression, was multicollinearity checked between these variables?<br /> - In the initial comparison of the first/second half, why just 5 clusters of electrodes, and why these particular clusters? How many different variables are systematically different in the first vs second half, and how do you rule out less interesting time-on-task effects such as engagement or alertness? In what time windows are these amplitudes being measured? In the comparison of asked and not-asked trials, what trial stage and time window is being measured? Again, how many different variables, of the many estimated per trial in the active inference model, are different in the asked and not-asked trials, and how can you know which of these differences is the one reflected in the EEG effects? The authors choose to interpret that on not-asked trials the subjects are more uncertain because the cue doesn't give them the context, but you could equally argue that they don't ask because they are more certain of the possible hidden states.<br /> - The EEG regressors are not fully explained. For example, an "active learning" regressor is listed as one of the 4 at the beginning of section 3.3, but it is the first mention of this term in the paper and the term does not arise once in the methods.<br /> - In general, it is not clear how one can know that the EEG results reflect that the brain is purposefully encoding these very parameters while implementing this very mechanism, and not other, possibly simpler, factors that correlate with them since there is no engagement with such potential confounds or alternative models. For example, a model-free reinforcement learning model is fit to behaviour for comparison. Why not the EEG?

Reviewer #3 (Public Review):

Summary:<br /> This work uses multiscale molecular dynamics simulations to demonstrate molecular mechanism(s) for phosphatidylinositol regulation of voltage gated sodium channel (Nav1.4) gating. Recent experimental work by Gada et al. JGP 2023 showed altered Nav1.4 gating when Nav1.4 current was recorded with simultaneous application of PI(4,5)P2 dephosphorylate. Here the authors revealed probable molecular mechanism that can explain PI(4,5)P2 modulation of Nav1.4 gating. They found PIP lipids interacting with the gating charges - potentially making it harder to move the voltage sensor domain and altering the channels voltage sensitivity. They also found a stable PIP binding site that reaches the D_IV S4-S5 linker, reducing the mobility of the linker and potentially competing with the C-terminal domain.

Strengths:<br /> Using multiscale simulations with course-grained simulations to capture lipid-protein interactions and the overall protein lipid fingerprint and then all-atom simulations to verify atomistic details for specific lipid-protein interactions is extremely appropriate for the question at hand. Overall, the types of simulation and their length are suitable for the questions the authors pose and a thorough set of analysis was done which illustrates the observed PIP-protein interactions.

Weaknesses:<br /> Although the set of current simulations and analysis supports the conclusions drawn nicely, the course-grained simulations have further utility than that utilized by the authors. With the 4to1 heavy atoms bead mapping in Martini 2 some detailed chemical specificity is averaged out but parameters for different PIP family members do exist - including specific PIP(4,5)P2 vs PIP(3,4)P2, and could have been explored at the course-grained level. However, performing more detailed all-atom simulation, as done in this manuscript, is always advisable to extend and/or confirm course-grained results.

Reviewer #3 (Public Review):

Summary:<br /> The authors of this manuscript identified the fossils of the newly designated species Beretella spinosa and analyzed its phylogenetic position in relation to the extinct described species and extant species. Their analysis placed the newly described species Beretella spinosa and Saccorhytus as an independent clade from the rest of the ecdysozoans. Remarkably, these species are non-vermiform, and the resulting evolutionary scenario assumes non-vermiform as early ecdysozoans.

Strengths:<br /> The study presents outstanding, novel data and provides new insights into the evolution of animal forms especially regarding their morphological diversity after the Cambrian explosion.

Weaknesses:<br /> I, as a paleontology non-expert, experienced several difficulties in reading the manuscript. This should be taken into consideration when assuming a wide range of readers including non-experts.

Reviewer #3 (Public Review):

Summary and strengths:<br /> The manuscript, "Remodelling of skeletal muscle myosin metabolic states in hibernating mammals", by Lewis et al, investigates whether myosin ATP activity may differ between states of hibernation and activity in both large and small mammals. The study interrogates (primarily) permeabilized muscle strips or myofibrils using several state-of-the-art assays, including the mant-ATP assay to investigate ATP utilization of myosin, X-ray diffraction of muscles, proteomics studies, metabolic tests, and computational simulations. The overall data suggests that ATP utilization of myosin during hibernation is different than in active conditions.

A clear strength of this study is the use of multiple animals that utilize two different states of hibernation or torpor. Two large animal hibernators (Eurasian Brown Bear, American Black Bear) represent large animal hibernators that typically undergo prolonged hibernation. Two small animal hibernators (Garden Dormouse, 13 Lined Ground Squirrel) undergo torpor with more substantial reductions in heart rate and body temperature, but whose torpor bouts are interrupted by short arousals that bring the animals back to near-summer-like metabolic conditions.

Especially interesting, the investigators analyze the impact that body temperature may have on myosin ATP utilization by performing assays at two different temperatures (8 and 20 degrees C, in 13 Lined Ground Squirrels).

The multiple assays utilized provide a more comprehensive set of methods with which to test their hypothesis that muscle myosins change their metabolic efficiency during hibernation.

Suggestions and potential weaknesses:<br /> While the samples and assays provide a robust and comprehensive coverage of metabolic needs and testing, the data is less categorical. Some of these may be dependent on sample size or statistical analysis while others may be dependent on interpretation.

(1) Statistical Analysis<br /> (1.a) The results of this study often cannot be assessed properly due to a lack of clarity in the statistical tests.<br /> For example, the results related to the large animal hibernators (Figure 1) do not describe the statistical test (in the text of the results, methods, or figure legends). (Similarly for figure 6 and Supplemental Figure 1). Further, it is not clear whether or when the analysis was performed with paired samples. As the methods described, it appears that the Eurasian Brown Bear data should be paired per animal.

(1.b) The statistical methods state that non-parametric testing was utilized "where data was unevenly distributed". Please clarify when this was used.

(1.c) While there are two different myosin isoforms, the isoform may be considered a factor. It is unclear why a one-way ANOVA is generally used for most of the mant-ATP chase data.

(1.d) While the technical replicates on studies such as the mant-ATP chase assay are well done, the total biological replicates are small. A consideration of the sample power should be included.

(1.e) An analysis of the biological vs statistical significance should be considered, especially for the mant-ATP chase data from the American Black Bear, where there appear to be shifts between the summer and winter data.

(2) Consistency of DRX/SRX data.<br /> (2.a) The investigators performed both mant-ATP chase and x-ray diffraction studies to investigate whether myosin heads are in an "on" or "off" state. The results of these two studies do not appear to be fully consistent with each other, which should not be a surprise. The recent work of Mohran et al (PMID 38103642) suggests that the mant-ATP-predicted SRX:DRX proportions are inconsistent with the position of the myosin heads. The discussion appears to lack a detailed assessment of this prior work and lack a substantive assessment contrasting the differing results of the two assays in the current study. i.e. why the current study's mant-ATP chase and x-ray diffraction results differ.

(2.b) The discussion of the current study's x-ray diffraction data relating to the I_1,1/I_1,0 ratio and how substantially different this is to the M6 results merits discussion. i.e. how can myosin both be more primed to contract during IBA versus torpor (according to intensity ratio), but also have less mass near the thick filament (M6).

(3) Possible interactions with Heat Shock Proteins<br /> Heat Shock Proteins (HSPs), such as HSP70, have been shown to be differential during torpor vs active states. A brief search of HSP and myosin reveals HPSs related to thick filament assembly and Heat Shock Cognate 70 interacting with myosin binding protein C. Especially given the author's discussion of protein stability and the potential interaction with myosin binding protein C and the SRX state, the limitation of not assessing HSPs should be discussed. (While HSP's relation to thick filament assembly might conceivably modify the interpretation of the M3 x-ray diffraction results, this reviewer acknowledges that possibility as a leap.)

Despite the above substantial concerns/weaknesses, this reviewer believes that this manuscript represents a valuable data set.

Other comments related to interpretation:<br /> (4) The authors briefly mention the study by Toepher et al [Ref 25] and that it utilizes cardiac muscles. There would benefit from increased discussion regarding the possible differences in energetics between cardiac and skeletal muscle in these states.

(5) The author's analysis of temperature is somewhat limited.<br /> (5.a) First, the authors use 20 degrees C (room temperature), not 37 degrees C, a more physiologic body temperature for large mammals. While it is true that limbs are likely at a lower temperature, 20 degrees C seems substantially outside of a normal range. Thus, temperature differences may have been minimized by the author's protocol.

(5.b) Second, the authors discuss the possibility of myosin contributing to non-shivering thermogenesis. The magnitude of this impact should be discussed. The suggestion of myosin ATP utilization also implies that there is some basal muscle tone (contraction), as the myosin ATPase utilizes ATP to release from actin, before binding and hydrolyzing again. Evidence of this tone should be discussed.

Reviewer #3 (Public Review):

Summary:<br /> Feng et al. test the hypothesis that human body size constrains the perception of object affordances, whereby only objects that are smaller than the body size will be perceived as useful and manipulable parts of the environment, whereas larger objects will be perceived as "less interesting components."

To test this idea, the study employs a multi-method approach consisting of three parts:

In the first part, human observers classify a set of 24 objects that vary systematically in size (e.g., ball, piano, airplane) based on 14 different affordances (e.g., sit, throw, grasp). Based on the average agreement of ratings across participants, the authors compute the similarity of affordance profiles between all object pairs. They report evidence for two homogenous object clusters that are separated based on their size with the boundary between clusters roughly coinciding with the average human body size. In follow-up experiments, the authors show that this boundary is larger/smaller in separate groups of participants who are instructed to imagine themselves as an elephant/cat.

In the second part, the authors ask different large language models (LLMs) to provide ratings for the same set of objects and affordances and conduct equivalent analyses on the obtained data. Some, but not all, of the models produce patterns of ratings that appear to show similar boundary effects, though less pronounced and at a different boundary size than in humans.

In the third part, the authors conduct an fMRI experiment. Human observers are presented with four different objects of different sizes and asked if these objects afford a small set of specific actions. Affordances are either congruent or incongruent with objects. Contrasting brain activity on incongruent trials against brain activity on congruent trials yields significant effects in regions within the ventral and dorsal visual stream, but only for small objects and not for large objects.

The authors interpret their findings as support for their hypothesis that human body size constrains object perception. They further conclude that this effect is cognitively penetrable, and only partly relies on sensorimotor interaction with the environment (and partly on linguistic abilities).

Strengths:<br /> The authors examine an interesting and relevant question and articulate a plausible (though somewhat underspecified) hypothesis that certainly seems worth testing. Providing more detailed insights into how object affordances shape perception would be highly desirable. Their method of analyzing similarity ratings between sets of objects seems useful and the multi-method approach is original and interesting.

Weaknesses:<br /> The study presents several shortcomings that clearly weaken the link between the obtained evidence and the drawn conclusions. Below I outline my concerns in no particular order:

(1) It is not entirely clear to me what the authors are proposing and to what extent the conducted work actually speaks to this. For example, in the introduction, the authors write that they seek to test if body size serves not merely as a reference for object manipulation but also "plays a pivotal role in shaping the representation of objects." This motivation seems rather vague motivation and it is not clear to me how it could be falsified.

Overall, the lack of theoretical precision makes it difficult to judge the appropriateness of the approaches and the persuasiveness of the obtained results. I would strongly suggest clarifying the theoretical rationale and explaining in more detail how the chosen experiments allow them to test falsifiable predictions.

(2) The authors used only a very small set of objects and affordances in their study and they do not describe in sufficient detail how these stimuli were selected. This renders the results rather exploratory and clearly limits their potential to discover general principles of human perception. Much larger sets of objects and affordances and explicit data-driven approaches for their selection would provide a more convincing approach and allow the authors to rule out that their results are just a consequence of the selected set of objects and actions.

(3) Relatedly, the authors could be more thorough in ruling out potential alternative explanations. Object size likely correlates with other variables that could shape human similarity judgments and the estimated boundary is quite broad (depending on the method, either between 80 and 150 cm or between 105 to 130 cm). More precise estimates of the boundary and more rigorous tests of alternative explanations would add a lot to strengthen the authors' interpretation.

(4) While I appreciate the manipulation of imagined body size, as a clever way to solidify the link between body size and affordance perception, I find it unfortunate that it is implemented in a between-subjects design, as this clearly leaves open the possibility of pre-existing differences between groups. I certainly disagree with the authors' statement that their findings suggest "a causal link between body size and affordance perception."

(5) The use of LLMs in the current study is not clearly motivated and I find it hard to understand what exactly the authors are trying to test through their inclusion. As it currently stands, I find it hard to discern how the presence of perceptual boundaries in LLMs could constitute evidence for affordance-based perception.

(6) Along the same lines, the fMRI study also provides little evidence to support the authors' claims. The use of congruency effects as a way of probing affordance perception is not well motivated. Importantly (and related to comment 2 above), the very small set of objects and affordances in this experiment heavily complicates any conclusions about object size being the crucial variable determining the occurrence of congruency effects.

Overall, I consider the main conclusions of the paper to be far beyond the reported data. Articulating a clearer theoretical framework with more specific hypotheses as well as conducting more principled analyses on more comprehensive data sets could help the authors obtain stronger tests of their ideas.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, the authors use the C. elegans system to explore how already-stressed neurons respond to additional mechanical stress. Exophers are large extracellular vesicles secreted by cells, which can contain protein aggregates and organelles. These can be a way of getting rid of cellular debris, but as they are endocytosed by other cells can also pass protein, lipid, and RNA to recipient cells. The authors find that when the uterus fills with eggs or otherwise expands, a nearby neuron (ALMR) is far more likely to secrete exophers. This paper highlights the importance of the mechanical environment in the behavior of neurons and may be relevant to the response of neurons exposed to traumatic injury.

Strengths:<br /> The paper has a logical flow and a compelling narrative supported by crisp and clear figures.

The evidence that egg accumulation leads to exopher production is strong. The authors use a variety of genetic and pharmacological methods to show that increasing pressure leads to more exopher production, and reducing pressure leads to lower exopher production. For example, egg-laying defective animals, which retain eggs in the uterus, produce many more exophers, and hyperactive egg-laying is accompanied by low exopher production. The authors even inject fluid into the uterus and observe the production of exophers.

Weaknesses:<br /> The main weakness of the paper is that it does not explore the molecular mechanism by which the mechanical signals are received or responded to by the neuron, but this could easily be the subject of a follow-up study.

I was intrigued by this paper, and have many questions. I list a few below, which could be addressed in this paper or which could be the subject of follow-up studies.

- Why do such a low percentage of ALMR neurons produce exophers (5-20%)? Does it have to do with the variability of the proteostress?<br /> - Why does the production of exophers lag the peak in progeny production by 24-48 hours? Especially when the injection method produces exophers right away?<br /> - As mentioned in the discussion, it would be interesting to know if PEZO-1/PIEZO is required for uterine stretching to activate exophergenesis. pezo-1 animals accumulate crushed oocytes in the uterus.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Mazeaud and colleagues pursued a small-scale screen of a targeted RNAi library to identify novel players involved in Zika (ZIKV) and dengue (DENV) virus replication. Loss-of-function of IGF2BP2 resulted in reduced titers for ZIKV of the Asian and African lineages in hepatic Huh7.5 cells, but not for either of the four DENV serotypes nor West Nile virus (WNV). The phenotype was further confirmed in two additional cell lines and using a ZIKV reporter virus. In addition, using immunoprecipitation assays the interaction between IGF2BP2 and ZIKV NS5 protein and RNA genome was detected. The work addressed the role of IGF2BP2 in the infected cell combining confocal microscopy imaging, and proteomic analysis. The approach indicated an altered distribution of IGF2BP2 in infected cells and changes in the protein interactome including disrupted association with partner mRNAs and modulation of the abundance of a specific set of protein partners in IGF2BP2 immunoprecipitated ribonucleoprotein (RNP) complexes. Finally, based on the changes in IGF2BP2 interactome and specifically the increment in the abundance of Atlastin 2, the biogenesis of ZIKV replication organelles (vRO) is investigated using a genetic system that allows virus replication-independent assembly of vRO. Electron microscopy showed that knockdown of IGF2BP2 expression reduced the number of cells with vRO.

Strengths:<br /> The role of IGF2BP2 as a proviral factor for ZIKV replication is novel.

The study follows a logical flow of experiments that altogether support the assembly of a specialized RNP complex containing IGF2BP2 and ZIKV NS5 and RNA genome.

Weaknesses:<br /> The statistical analysis should clearly indicate the number of biological replicates of experiments to support statistical significance.

The claim that IGF2BP2 knockdown impairs de novo viral organelle biogenesis and viral RNA synthesis is built upon data that show a reduction in RNA synthesis <0.5-fold as assessed using a reporter replicon, thus suggesting a limited impact of the knockdown on RNA replication.

Validation of IGF2BP2 partners that are modulated upon ZIKV infection (i.e. virus yield in knocked down cells) can be relevant especially for partners such as Atlastin 2, as the hypothesis of a role for IGF2BP2 RNP in vRO biogenesis is based on the observed increase in the abundance of Atlastin 2 in the RNP complex preciìtated from infected cells.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Rana and colleagues examined the effect of a "low impact" ampakine, an AMPA receptor allosteric modulator, on the voiding function of rats subjected to midline T9 spinal cord contusion injury. Previous studies have shown that the micturition reflex fully depends on AMPA glutaminergic signaling, and, that the glutaminergic circuits are reorganized after spinal cord injury. In chronic paraplegic rats, other circuits (no glutaminergic) become engage in the spinal reflex mechanism controlling micturition. The authors employed continuous flow cystometry and external urethral sphincter electromyography to assess bladder function and bladder-urethral sphincter coordination in naïve rats (control) and rats subjected to spinal cord injury (SCI). In the acute phase after SCI, rats exhibit larger voids with lower frequency than naïve rats. This study shows that CX1739 improves, in a dose-dependent manner, bladder function in rats with SCI. The interval between voids and the voided volume were reduced in rat with SCI when compared to controls. In summary, this is an interesting study that describes a potential treatment for patients with SCI.

Strengths:

The findings described in this manuscript are significant because neurogenic bladder predisposes patients with SCI to urinary tract infections, hydronephrosis and kidney failure. The manuscript is clearly written. The study is technically outstanding, and the conclusions are well justified by the data.

Weaknesses:

The study was conducted 5 days after spinal cord contusion when the bladder is underactive. In rats with chronic SCI, the bladder is overactive. Therefore, the therapeutic approach described here is expected to be effective only in the underactive bladder phase of SCI. The mechanism and site of action of CX1739 is not defined.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Dr. Chen et al. investigates the genes that are differentially methylated and associated with cartilage degeneration in hemophilia patients. The study demonstrates the functional mechanisms of the TNXB gene in chondrocytes and F8-/- mice. The authors first showed significant DNA methylation differences between hemophilic arthritis (HA) and osteoarthritis through genome-wide DNA methylation analysis. Subsequently, they showed a decreased expression of the differentially methylated TNXB gene in cartilage from HA patients and mice. By knocking down TNXB in vivo and in vitro, the results indicated that TNXB regulates extracellular matrix homeostasis and apoptosis by modulating p-AKT. The findings are novel and interesting, and the study presents valuable information in blood-induced arthritis research.

Strengths:<br /> The authors adopted a comprehensive approach by combining genome-wide DNA methylation analysis, in vivo and in vitro experiments using human and mouse samples to illustrate the molecular mechanisms involved in HA progression, which is crucial for developing targeted therapeutic strategies. The study identifies Tenascin XB (TNXB) as a central mediator in cartilage matrix degradation. It provides mechanistic insights into how TNXB influences cartilage matrix degradation by regulating the activation of AKT. It opens avenues for future research and potential therapeutic interventions using AKT agonists for cartilage protection in hemophilic arthropathy. The conclusions drawn from the study are clear and directly tied to the findings.

Weaknesses:<br /> (1) The study utilizes a small sample size (N=5 for both osteoarthritis and hemophilic arthropathy). A larger sample size would enhance the generalizability and statistical power of the findings.<br /> (2) The use of an animal model (F8-/- mouse) to investigate the role of TNXB may not fully capture the complexity of human hemophilic arthropathy. Differences in the biology between species may affect the translatability of the findings to human patients.<br /> (3) The study primarily focuses on TNXB as a central mediator, but it might overlook other potentially relevant factors contributing to cartilage degradation in hemophilic arthropathy. A more holistic exploration of genetic and molecular factors could provide a broader understanding of the condition.

Reviewer #3 (Public Review):

Summary:<br /> In the 'bCFS' paradigm, a monocular target gradually increases in contrast until it breaks interocular suppression by a rich monocular suppressor in the other eye. The present authors extend the bCFS paradigm by allowing the target to reduce back down in contrast until it becomes suppressed again. The main variable of interest is the contrast difference between breaking suppression and (re) entering suppression. The authors find this difference to be constant across a range of target types, even ones that differ substantially in the contrast at which they break interocular suppression (the variable conventionally measured in bCFS). They also measure how the difference changes as a function of other manipulations. Interpretation is in terms of the processing of unconscious visual content, as well as in terms of the mechanism of interocular suppression.

Strengths:<br /> Interpretation of bCFS findings is mired in controversy, and this is an ingenuous effort to move beyond the paradigm's exclusive focus on breaking suppression. The notion of using the contrast difference between breaking and entering suppression as an index of suppression depth is interesting.

Reviewer #3 (Public Review):

Summary: Richter et al. present a comprehensive anatomical analysis of the external sensory organs of the D. melanogaster larva. Extending on their previous study (Rist and Thum 2017) that analyzed the anatomy of the terminal organ, a major external taste organ of fruit fly larva, the authors examined the anatomy of the remaining head sensory organs - the dorsal organ, the ventral organ, and the labial organ-also described the sensory organs of the thoracic and abdominal segments. Using improved electron microscopy, the authors performed a three-dimensional anatomical analysis of the sensilla and adjacent ganglia to construct a complete structural and neuronal map of the external larval sensilla.

Strengths: Though the manuscript is lengthy, it is written clearly, and the presented data supports the conclusion. In addition to the classification and nomenclature of the different types of sensilla throughout the larval body, the wealth of data presented here will be valuable to the scientific community. The study offers fundamental anatomical insights, which will be helpful for future functional studies and to understand the sensory strategies of Drosophila larvae in response to the external environment. By analyzing different larval stages (L1 and L3), this work offers some insights into the developmental aspects of the larval sense organs and their corresponding sensory cells.

Weaknesses: There are no apparent weaknesses. The repetitiveness of some data and prior studies may be avoided for easy readability.

Reviewer #3 (Public Review):

Summary:<br /> The authors investigate the hypothesis that neurexins serve a crucial role as regulators of the synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body (MNTB) and the lateral superior olivary complex (LSO). It is worth mentioning that LSO neurons are an integration station of the auditory brainstem circuit displaying high reliability and temporal precision. These features are necessary for computing interaural cues to derive sound source location from comparing the intensities of sounds arriving at the two ears. In this context, the authors' findings build up according to the hypothesis first by displaying that neurexins were expressed in the MNTB at varying levels. They followed this up with the deletion of all neurexins in the MNTB through the employment of a triple knock-out (TKO). Using electrophysiological recordings in acute brainstem slices of these TKO mice, they gathered solid evidence for the role of neurexins in synaptic transmission at this glycinergic synapse primarily by ensuring tight coupling of Ca2+ channels and vesicular release sites. Additionally, the authors uncovered a connection between the deletion of neurexins and a higher number of glycinergic synapses in TKO mice, for which they provided evidence in the form of immunostainings and related it to electrophysiological data on spontaneous release. Consequently, this investigation expands our knowledge on the molecular regulation of synaptic transmission at glycinergic synapses, as well as on the auditory processing at the level of the brainstem.

Strengths:<br /> The authors demonstrate substantial results in support of the hypothesis of a critical role of neurexins for regulating glycinergic transmission in the LSO using various techniques. They provide evidence for the expression of neurexins in the MNTB and consecutively successfully generate and characterize the neurexin TKO. For their study on LSO IPSCs the authors transduced MNTB neurons by co-injection of virus-carrying Cre and ChR2 and subsequently optogenetically evoke release of glycine. As a result, they observed a significant reduction in amplitude and significantly slower rise and decay times of the IPSCs of the TKO in comparison with control mice in which MNTB neurons were only transduced with ChR2. Furthermore, they observed an increased paired pulse ratio (PPR) of LSO IPSCs in the TKO mice, indicating lower release probability. Elaborating on the hypothesis that neurexins are essential for the coupling of synaptic vesicles to Ca2+ channels, the authors show lowered Ca2+ sensitivity in the TKO mice. Additionally, they reveal convincing evidence for the connection between the increased frequency of spontaneous IPSC and the higher number of glycinergic synapses of the LSO in the TKO mice, revealed by immunolabeling against the glycinergic presynaptic markers GlyT2 or VGAT.

Weaknesses:<br /> The major concern is novelty as this work on the effects of pan-neurexin deletion in a glycinergic synapse is quite consistent with the authors' prior work on glutamatergic synapses (Luo et al., 2020). The authors might want to further work out novel aspects and strengthen the comparative perspective. Conceptually, the authors might want to be more clear about interpreting the results on the altered dependence of release on voltage-gated Ca2+ influx (Ca2+ sensitivity, coupling).

Reviewer #3 (Public Review):

This study demonstrated the application of OPM-MEG in neurodevelopment studies of somatosensory beta oscillations and connections with children as young as 2 years old. It provides a new functional neuroimaging method that has a high spatial-temporal resolution as well wearable which makes it a new useful tool for studies in young children. They have constructed a 192-channel wearable OPM-MEG system that includes field compensation coils which allow free head movement scanning with a relatively high ratio of usable trials. Beta band oscillations during somatosensory tasks are well localized and the modulation with age is found in the amplitude, connectivity, and pan-spectral burst probability. It is demonstrated that the wearable OPM-MEG could be used in children as a quite practical and easy-to-deploy neuroimaging method with performance as good as conventional MEG. With both good spatial (several millimeters) and temporal (milliseconds) resolution, it provides a novel and powerful technology for neurodevelopment research and clinical applications not limited to somatosensory areas.

The conclusions of this paper are mostly well supported by data acquired under the proper method. However, some aspects of data analysis need to be improved and extended.

(1) The colour bars selected for the pseudo-T-static pictures of beta modulation in Figures 2 and 3, which are blue/black and red/black, are not easily distinguished from the anatomical images which are grey-scale. A colour bar without black/white would make these figures better. The peak point locations are also suggested to be marked in Figure 2 and averaged locations in Figure 3 with an error bar.

(2) The data points in plots are not constant across figures. In Figures 3 and 5, they are classified into triangles and circles for children and adults, but all are circles in Figures 4 and 6.

(3) Although MEG is much less susceptible to conductivity inhomogeneity of the head than EEG, the forward modulating may still be impacted by the small head profile. Add more information about source localization accuracy and stability across ages or head size.

Reviewer #3 (Public Review):

The authors describe the role, location, and function of the MTA and MTB mating type genes in the multi-mating-type species T. thermophila. The ciliate is an important group of organisms to study the evolution of mating types, as it is one of the few groups in which more than two mating types evolved independently. In the study, the authors use deletion strains of the species to show that both mating types genes located in each allele are required in both mating individuals for successful matings to occur. They show that the proteins are localized in the cell membrane, not the cilia, and that they interact in a complex (MTRC) with a set of 6 associated (non-mating type-allelic) genes. This complex is furthermore likely to interact with a cyclin-dependent kinase complex. It is intriguing that T. thermophila has two genes that are allelic and that are both required for successful mating. This coevolved double recognition has to my knowledge not been described for any other mating-type recognition system. I am not familiar with experimental research on ciliates, but as far as I can judge, the experiments appear well performed and mostly support the interpretation of the authors with appropriate controls and statistical analyses.

The results show clearly that the mating type genes regulate non-self-recognition, however, I am not convinced that self-recognition occurs leading to the suppression of mating. An alternative explanation could be that the MTA and MTB proteins form a complex and that the two extracellular regions together interact with the MTA+MTB proteins from different mating types. This alternative hypothesis fits with the coevolution of MTA and MTB genes observed in the phylogenetic subgroups as described by Yan et al. (2021 iScience). Adding MTAxc and/or MTBxc to the cells can lead to the occupation of the external parts of the full proteins thereby inhibiting the formation of the complex, which in turn reduces non-self interactions. Self-recognition as explained in Figure 2S1 suggests an active response, which should be measurable in expression data for example. This is in my opinion not essential, but a claim of self-recognition through the MTA and MTB should not be made.

The authors discuss that T. thermophila has special mating-type proteins that are large, while those of other groups are generally small (lines 157-160 and discussion). The complex formed is very large and in the discussion, they argue that this might be due to the "highly complex process, given that there are seven mating types in all". There is no argument given why large is more complex, if this is complex, and whether more mating types require more complexity. In basidiomycete fungi, many more mating types than 7 exist, and the homeodomain genes involved in mating types are relatively small but highly diverse (Luo et al. 1994 PMID: 7914671). The mating types associated with GPCR receptors in fungi are arguably larger, but again their function is not that complex, and mating-type specific variations appear to evolve easily (Fowler et al 2004 PMID: 14643262; Seike et al. 2015 PMID: 25831518). The large protein complex formed is reminiscent of the fusion patches that develop in budding or fission yeasts. In these species, the mating type receptors are activated by ligand pheromones from the opposite mating type that induce polarity patch formation (see Sieber et al. 2023 PMID: 35148940 for a recent review). At these patches, growth (shmooing) and fusion occur, which is reminiscent (in a different order) of the tip transformation in T. thermophilia. The fusion of two cells is in all taxa a dangerous and complex event that requires the evolution of very strict regulation and the existence of a system like the MTRC and cyclin-dependent complex to regulate this process is therefore not unexpected. The existence of multiple mating types should not greatly complicate the process, as most of the machinery (except for the MTA and MTB) is identical among all mating types.

The Tetrahymena/ciliate genetics and lifecycle could be better explained. For a general audience, the system is not easy to follow. For example, the ploidy of the somatic nucleus with regards to the mating type is not clear to me. The MAC is generally considered "polyploid", but how does this work for the mating type? I assume only a single copy of the mating type locus is available in the MAC to avoid self-recognition in the cells. Is it known how the diploid origin reduces to a single mating type? This does not become apparent from Cervantes et al. 2013. Also, the explanation of co-stimulation is not completely clear (lines 49-60). Initially, direct cell-cell contact is mentioned, but later it is mentioned that "all cells become fully stimulated", even when unequal ratios are used. Is physical contact necessary? Or is this due to the "secrete mating-essential factors" (line 601)? These details are essential, for interpretation of the results and need to be explained better.

Abstract and introduction: Sexes are not mating types. In general, mating types refer to systems in which there is no obvious asymmetry between the gametes, beyond the compatibility system. When there is a physiological difference such as size or motility, sexes are used. This distinction is of importance because in many species mating types and sexes can occur together, with each sex being able to have either (when two) or multiple mating types. An example are SI in angiosperms as used as an example by the authors or mating types in filamentous fungi. See Billiard et al. 2011 [PMID: 21489122] for a good explanation and argumentation for the importance of making this distinction.

Reviewer #3 (Public Review):

Summary:

The vesicular monoamine transporter is a key component in neuronal signaling and is implicated in diseases such as Parkinson's. Understanding of monoamine processing and our ability to target that process therapeutically has been to date provided by structural modeling and extensive biochemical studies. However, structural data is required to establish these findings more firmly.

Strengths:

Dalton et al resolved a structure of VMAT2 in the presence of an important inhibitor, tetrabenazine, with the protein in detergent micelles, using cryo-EM and with the aid of protein domains fused to its N- and C-terminal ends, including one fluorescent protein that facilitated protein screening and purification. The resolution of the maps allows clear assignment of the amino acids in the core of the protein. The structure is in good agreement with a wealth of experimental and structural prediction data, and provides important insights into the binding site for tetrabenazine and selectivity relative to analogous compounds. The authors provide additional biochemical analyses that further support their findings. The comparison with AlphaFold models is enlightening.

Weaknesses:

The authors follow up their structures with molecular dynamics simulations of the tetrabenazine-bound state, and test several protonation states of acidic residues in the binding pocket, but not all possible combinations; thus, it is not clear the extent to which tetrabenazine rearrangements observed in these simulations are meaningful. Additional simulations of the substrate dopamine docked into this structure were also carried out, although it is unclear whether this "dead-end" occluded state is a relevant state for dopamine binding. The authors report release of dopamine during these simulations, but it is notable that this only occurs when all four acidic binding site residues were protonated and when an enhanced sampling approach was applied.

Reviewer #3 (Public Review):

Summary:<br /> In the present work, Zhang et al investigate the involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term drug resistance evolution that can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by the accumulation of reactive oxygen species and inhibition of DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance). However, although the authors perform several multi-disciplinary experiments, there are several caveats to the authors' proposal that ultimately do not fully support their interpretation that the observed antimicrobial resistance evolution phenotype is due to compromised DNA repair.

Strengths:<br /> The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. This is primarily supported by their observations that resistance evolution phenotypes only exist for recA deletion mutants and not other genes in the SOS response

Weaknesses:<br /> The authors do not show any direct evidence (1) that these phenotypes exist in strains harboring deletions in other DNA repair genes outside of the SOS response, (2) that DNA damage is increased, (3) that reactive oxygen species accumulate, (4) that accelerated resistance evolution can be reversed by anything other than recA complementation. The authors do not directly test alternative hypotheses. The conclusions drawn are therefore premature.

Reviewer #3 (Public Review):

Summary:<br /> Pooled optical screening has recently emerged as a powerful approach to associate complex phenotypic information from microscope images with specific genetic perturbations at the single-cell level. This is achieved by amplifying and sequencing DNA barcodes within individual cells through in-situ sequencing. This paper leverages these advances in pooled screening technology to examine the effects of gene knockdowns on high-dimensional cell morphological phenotypes beyond binary readouts.

A key challenge is how to effectively distill meaningful phenotypic dimensions from information-rich image data to connect genotype to phenotype. By screening 366 genes using CRISPRi and analyzing tens of thousands of single-cell images, this paper provides insights into genetic regulators of morphology in osteosarcoma cells. In developing this screen and analyzing its readout, the authors make several notable contributions.

First, the authors tested and optimized molecular inversion probes (MIPs) to improve rolling circle amplification and barcode imaging. Through these optimization experiments, they identified a shortened MIP design that yielded 11-fold more visible amplicons, enabling more robust barcode readout from complex images. Second, the authors address several unresolved questions regarding how to work with single-cell images at this scale. A critical aspect of this is the need to develop analysis strategies using single-cell data rather than commonly used current methodologies that condense down to an agglomerated perturbation level cell morphology information. The authors compare morphological profiling using curated feature extraction and an unsupervised deep learning approach called a β-variational autoencoder on single-cell imaging data, suggesting that the latter can capture salient aspects of variation without requiring much human input. Finally, and perhaps more importantly, the authors develop an approach, Visual Interpretation of Embeddings by constrained Walkthrough Sampling (VIEWS), towards sampling cells at the end of distributions such as a principal component dimension in a reduction of curated features or a latent space dimension extracted from an autoencoder. This allows for a rapid and efficient way of understanding extremes of morphological profiles and allows for quick interpretability of extracted morphological signal which in turn assists with downstream functional understandings of groups of genes that similarly alter a cell's morphology.

Strengths and Weaknesses:<br /> This is an interesting and rigorous paper that provides an important advance in conducting large-scale microscopy-based approaches. The methods development and computational analyses described in this paper are strong and innovative. However, the screening conducted in this paper did not identify a large number of modifiers of general U2OS cell morphology. As the authors rightly point out, several factors could contribute to the modest hit rate, including variable CRISPRi knockdown efficiency and limited phenotypic readout from just two imaging channels. Despite these limitations, the paper makes several key methodological contributions and in the opinion of this reviewer merits revision or benchmarking.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, Stubbusch and coauthors examine the foraging behavior of a marine species consuming an abundant marine polysaccharide. Laboratory experiments in a microfluidic setup are complemented with transcriptomic analyses aiming at assessing the genetic bases of the observed behavior. Bacterial cells consuming the polysaccharide form cohesive aggregates, while they start dispersing away when the byproduct of the digestion of the polysaccharide starts accumulating. Dispersing cells, tend to be attracted by the polysaccharide. Expression data show that motility genes are enriched during the dispersal phase, as expected. Counterintuitively, in the same phase, genes for transporters and digestions of polysaccharides are also highly expressed.

Strengths:<br /> The manuscript is very well written and easy to follow. The topic is interesting and timely. The genetic analyses provide a new, albeit complex, angle to the study of foraging behaviors in bacteria, adding to previous studies conducted on other species.

Weaknesses:<br /> I find this paper very descriptive and speculative. The results of the genetic analyses are quite counterintuitive; therefore, I understand the difficulty of connecting them to the observations coming from experiments in the microfluidic device. However, they could be better placed in the literature of foraging - dispersal cycles, beyond bacteria. In addition, the interpretation of the results is sometimes confusing.

Reviewer #3 (Public Review):

Summary:

The study adds to the existing data that have established that cortical development in rhesus macaque is known to recapitulate multiple facets cortical development in humans. The authors generate and analyze single cell transcriptomic data from the timecourse of embryonic neurogenesis.

Strengths:

Studies of primate developmental biology are hindered by the limited availability and limit replication. In this regard, a new dataset is useful.

The study analyzes parietal cortex, while previous studies focused on frontal and motor cortex. This may be the first analysis of macaque parietal cortex and, as such, may provide important insights into arealization, which the authors have not addressed

Reviewer #3 (Public Review):

The authors of this study have examined which cation channels specifically confer to ventral tegmental area dopaminergic neurones their autonomic (spontaneous) firing properties. Having brought evidence for the key role played by NALCN and TRPC6 channels therein, the authors aimed at measuring whether these channels play some role in so-called depression-like (but see below) behaviors triggered by chronic exposure to different stressors. Following evidence for a down-regulation of TRPC6 protein expression in ventral tegmental area dopaminergic cells of stressed animals, the authors provide evidence through viral expression protocols for a causal link between such a down-regulation and so-called depression-like behaviors. The main strength of this study lies on a comprehensive bottom-up approach ranging from patch-clamp recordings to behavioral tasks. These tasks mainly address anxiety-like behaviors and so-called depression-like behaviors (sucrose choice, forced swim test, tail suspension test). The results gathered by means of these procedures are clearcut. However, the reviewer believes that the authors should be more cautious when interpreting immobility responses to stress (forced swim, tail suspension) as "depression-like" responses. These stress models have been routinely used (and validated) in the past to detect the antidepressant properties of compounds under investigation, which by no means indicates that these are depression models. For readers interested by this debate, I suggest to read e.g. De Kloet and Molendijk (Biol. Pscyhiatry 2021).

Reviewer #3 (Public Review):

Summary:

This study uses a state-of-the-art artificial skin assay to determine the quantity of P. falciparum sporozoites expelled during feeding using mosquito infection (by standardised membrane feeding assay SMFA) using both cultured gametocytes and natural infection. Sporozoite densities in salivary glands and expelled into the skin are quantified using a well-validated molecular assay. These studies show clear positive correlations between mosquito infection levels (as determined by oocyst numbers), sporozoite numbers in salivary glands, and sporozoites expelled during feeding. This indicates potentially significant heterogeneity in infectiousness between mosquitoes with different infection loads and thus challenges the often-made assumption that all infected mosquitoes are equally infectious.

Strengths:

Very rigorously designed studies using very well validated, state-of-the-art methods for studying malaria infections in the mosquito and quantifying load of expelled sporozoites. This resulted in very high-quality data that was well-analyzed and presented. Both sources of gametocytes (cultures vs. natural infection) show consistent results further strengthening the quality of the results obtained.

Weaknesses:

As is generally the case when using SMFAs, the mosquito infections levels are often relatively high compared to wild-caught mosquitoes (e.g. Bombard et al 2020 IJP: median 3-4 ), and the strength of the observed correlations between oocyst sheet and salivary gland sporozoite load even more so between salivary gland sporozoite load and expelled sporozoite number may be dominated by results from mosquitoes with infection levels rarely observed in wild-caught mosquitoes. This could result in an overestimation of the importance of these well-observed positive relationships under natural transmission conditions.

The results obtained from these excellently designed and executed studies very well supported their conclusion - with a slight caveat regarding their application to natural transmission scenarios

This work very convincingly highlights the potential for significant heterogeneity in the infectiousness between individual P. falciparum-infected mosquitoes. Such heterogeneity needs to be further investigated and if again confirmed taken into account both when modelling malaria transmission and when evaluating the importance of low-density infections in sustaining malaria transmission.

Reviewer #3 (Public Review):

Summary:<br /> This paper by Stribling and colleagues sheds light on a decade-long P. aeruginosa outbreak of the high-risk lineage ST-621 in a US Military hospital. The origins of the outbreak date back to the late 90s and it was mainly caused by two distinct subclones SC1 and SC2. The data of this outbreak showed the emergence of antibiotic resistance to cephalosporin, carbapenems, and colistin over time highlighting the emerging risk of extensively resistant infections due to P. aeruginosa and the need for ongoing surveillance.

Strengths:<br /> This study overall is well constructed and clearly written. Since detailed information on floor plans of the building and transfers between facilities was available, the authors were able to show that these two subclones emerged in two separate buildings of the hospital. The authors support their conclusions with prospective environmental sampling in 2021 and 2022 and link the role of persistent environmental contamination to sustaining nosocomial transmission. Information on resistance genes in repeat isolates for the same patients allowed the authors to detect the emergence of resistance within patients. The conclusions have broader implications for infection control at other facilities. In particular, the paper highlights the value of real-time surveillance and environmental sampling in slowing nosocomial transmission of P. aeruginosa.

Weaknesses:<br /> My major concern is that the authors used fixed thresholds and definitions to classify the origin of an infection. As such, they were not able to give uncertainty measures around transmission routes nor quantify the relative contribution of persistent environmental contamination vs patient-to-patient transmission. The latter would allow the authors to quantify the impact of certain interventions. In addition, these results represent a specific US military facility and the transmission patterns might be specific to that facility. The study also lacked any data on antibiotic use that could have been used to relate to and discuss the temporal trends of antimicrobial resistance.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, Hajra et al have attempted to identify the role of Sirt1 and Sirt3 in regulating metabolic reprogramming and macrophage host defense. They have performed gene knockdown experiments in RAW macrophage cell lines to show that depletion of Sirt1 or Sirt3 enhances the ability of macrophages to eliminate Salmonella Typhimurium. However, in mice, inhibition of Sirt1 resulted in dissemination of the bacteria but the bacterial burden was still reduced in macrophages. They suggest that the effect they have observed is due to increased inflammation and ROS production by macrophages. They also try to establish a weak link with metabolism. They present data to show that the switch in metabolism from glycolysis to fatty acid oxidation is regulated by acetylation of Hif1a, and PDHA1.

Strengths:<br /> The strength of the manuscript is that the role of Sirtuins in host-pathogen interactions has not been previously explored in-depth making the study interesting. It is also interesting to see that depletion of either Sirt1 or Sirt3 results in a similar outcome.

Weaknesses:<br /> The major weakness of the paper is the low quality of data, making it harder to substantiate the claims. Also, there are too many pathways and mechanisms being investigated. It would have been better if the authors had focussed on either Sirt1 or Sirt3 and elucidated how it reprograms metabolism to eventually modulate host response against Salmonella Typhimurium. Experimental evidence is also lacking to prove the proposed mechanisms. For instance, they show correlative data that the knockdown of Sirt1-mediated shift in metabolism is due to HIF1a acetylation but this needs to be proven with further experiments.

Reviewer #3 (Public Review):

This study investigates subtelomeric repetitive sequences in the budding yeast Saccharomyces cerevisiae, known as Y' and X-elements. Taking advantage of yeast strain SY12 that contains only 3 chromosomes and six telomeres (normal yeast strains contain 32 telomeres) the authors are able to generate a strain completely devoid of Y'- and X-elements.

Strengths: They demonstrate that the SY12 delta XY strain displays normal growth, with stable telomeres of normal length that were transcriptionally silenced, a key finding with wide implications for telomere biology. Inactivation of telomerase in the SY12 and SY12 delta XY strains frequently resulted in survivors that had circularized all three chromosomes, hence bypassing the need for telomeres altogether. They show that survivors with fused chromosomes and so-called atypical survivors arise independently of the central recombination protein Rad52. The SY12 and SY12 delta XY yeast strains can become a useful tool for future studies of telomere biology. The conclusions of this manuscript are well supported by the data and are valuable for researchers studying telomeres.

Weaknesses: A weakness of the manuscript is the analysis of telomere transcriptional silencing. They state: "The results demonstrated a significant increase in the expression of the MPH3 and HSP32 upon Sir2 deletion, indicating that telomere silencing remains effective in the absence of X and Y'-elements". However, for the SY12 strain, their analyses indicate that the difference between the WT and sir2 strains is nonsignificant. In addition, a striking observation is that the SY12 strain (with only three chromosomes) express much less of both MPH3 and HSP32 than the parental strain BY4742 (16 chromosomes), both in the presence and absence of Sir2.

Reviewer #3 (Public Review):

Summary:<br /> In the context of the SCOURGE consortium's research, the authors conduct a GWAS meta-analysis on 4,702 hospitalized individuals of admixed American descent suffering from COVID-19. This study identified four significant genetic associations, including two loci initially discovered in Latin American cohorts. Furthermore, a trans-ethnic meta-analysis highlighted an additional novel risk locus in the CREBBP gene, underscoring the critical role of genetic diversity in understanding the pathogenesis of COVID-19.

Strengths:<br /> 1. The study identified two novel severe COVID-19 loci (BAZ2B and DDIAS) by the largest GWAS meta-analysis for COVID-19 hospitalization in admixed Americans.

2. With a trans-ethnic meta-analysis, an additional risk locus near CREBBP was identified.

Weaknesses:<br /> 1. The GWAS power is limited due to the relatively small number of cases.

2. There is no replication study for the novel severe COVID-19 loci, which may lead to false positive findings.

3. Significant differences exist in the ages between cases and controls, which could potentially introduce biased confounders. I'm curious about how the authors treated age as a covariate. For instance, did they use ten-year intervals? This needs clarification for reproducibility.

4."Those in the top PGS decile exhibited a 5.90-fold (95% CI=3.29-10.60, p=2.79x10-9) greater risk compared to individuals in the lowest decile". I would recommend comparing with the 40-60% PGS decile rather than the lowest decile, as the lowest PGS decile does not represent 'normal controls'.

5. In the field of PGS, it's common to require an independent dataset for training and testing the PGS model. Here, there seems to be an overfitting issue due to using the same subjects for both training and testing the variants.

6. The variants selected for the PGS appear arbitrary and may not leverage the GWAS findings without an independent training dataset.

7. The TWAS models were predominantly trained on European samples, and there is no replication study for the findings as well.

Reviewer #3 (Public Review):

Summary:<br /> As SMAD1/5 activities have previously been indistinguishable, these studies provide a new mouse model to finally understand unique downstream activation of SMAD1/5 target genes, a model useful for many scientific fields. Using CUT&RUN analyses with gene overlap comparisons and signaling pathway analyses, specific targets for SMAD1 versus SMAD5 were compared, identified, and interpreted. These data validate previous findings showing strong evidence that SMADs directly govern critical genes required for endometrial receptivity and decidualization, including cell adhesion and vascular development. Further, SMAD targets were overlapped with progesterone receptor binding sites to identify regions of potential synergistic regulation of implantation. The authors report strong correlations between progesterone receptor and SMAD1/5 direct targets to cooperatively promote embryo implantation. Finally, the authors validated SMAD1/5 gene regulation in primary human endometrial stromal cells. These studies provide a data-rich survey of SMAD family transcription, defining its role as a governor of early pregnancy.

Strengths:<br /> This manuscript provides a valuable survey of SMAD1/5 direct transcriptional events at the time of receptivity. As embryo implantation is controlled by extensive epithelial to stromal molecular crosstalk and hormonal regulation in space and time, the authors state a strong, descriptive narrative defining how SMAD1/5 plays a central role at the site of this molecular orchestration. The implementation of cutting-edge techniques and models and simple comparative analyses provide a straightforward, yet elegant manuscript.

Although the progesterone receptor exists as a major regulator of early pregnancy, the authors have demonstrated clear evidence that progesterone receptor with SMAD1/5 work in concert to molecularly regulate targets such as Sox17, Id2, Tgfbr2, Runx1, Foxo1 and more at embryo implantation. Additionally, the authors pinpoint other critical transcription factor motifs that work with SMADs and the progesterone receptor to promote early pregnancy transcriptional paradigms.

Weaknesses:

Although a wonderful new tool to ascertain SMAD1 versus SMAD5 downstream signaling, the importance of these factors in governing early pregnancy is not novel. Furthermore, functional validation studies are needed to confirm interactions at promoter regions. Additionally, the authors presume that all overlapped genes are shared between progesterone receptor and SMAD1/5, yet some peak representations do not overlap. Although, transcriptional activation can occur at the same time, they may not occur in the same complex. Thus, further confirmation of these transcriptional events is warranted.

Since whole murine uterus was used for these studies, the specific functions of SMAD1/5 in the stroma versus the epithelium (versus the myometrium) remain unknown. Further work is needed to delineate binding and transcriptional activation of SMAD1/5 and the progesterone receptor in the uterine compartments.

There are asynchronous gene responses in the SMAD1/5 ablated mouse model compared to the siRNA-treated human endometrial stromal cells. These differences can be confounding. Further investigation is needed to understand the meaning of these differences and as they relate to the entire SMAD transcriptome.

Reviewer #3 (Public Review):

Summary:<br /> This is an interesting paper investigating fMRI changes during sensory (visual, tactile) stimulation and absence seizures in the GAERS model. The results are potentially important for the field and do suggest that sensory stimulation may not activate brain regions normally during absence seizures. But the findings are limited by substantial methodological issues that do not enable fMRI signals related to absence seizures to be fully disentangled from fMRI signals related to the sensory stimuli.

Strengths:

Investigating fMRI brain responses to sensory stimuli during absence seizures in an animal model is a novel approach with potential to yield important insights.

Use of an awake, habituated model is a valid and potentially powerful approach.

Weaknesses:

The major difficulty with interpreting the results of this study is that the duration of the visual and tactile stimuli were 6 seconds, which is very close to the mean seizure duration per Table 1. Therefore the HRF model looking at fMRI responses to visual or auditory stimuli occurring during seizures was simultaneously weighting both seizure activity and the sensory (visual or auditory) stimuli over the same time intervals on average. The resulting maps and time courses claiming to show fMRI changes from visual or auditory stimulation during seizures will therefore in reality contain some mix of both sensory stimulation-related signals and seizure-related signals. The main claim that the sensory stimuli do not elicit the same activations during seizures as they do in the interictal period may still be true. But the attempts to localize these differences in space or time will be contaminated by the seizure related signals.

In their response to this comment the authors state that some seizures had longer than average duration, and that they attempted to model the effects of both seizures and sensory stimulation. However these factors do not mitigate the concern because the mean duration of seizures and sensory stimulation remain nearly identical, and the models used therefore will not be able to effectively separate signals related to seizures and related to sensory stimulation.

The claims that differences were observed for example between visual cortex and superior colliculus signals with visual stim during seizures vs interictal remain unconvincing due to above.

Maps shown in Figure 3 do not show clear changes in the areas claimed to be involved.

In their response the authors enlarged the cross sections. However there are still discrepancies between the images and the way they are described in the text. For example, in the Results text the authors say that comparing the interictal and ictal states revealed less activation in the somatosensory cortex during the ictal than during the interictal state, yet Figure 3 bottom row left shows greater activation in somatosensory cortex in this contrast.

Reviewer #3 (Public Review):

Summary:

This study used prolonged stimulation of a limb to examine possible plasticity in somatosensory evoked potentials induced by the stimulation. They also studied the extent that the blood brain barrier (BBB) was opened by the prolonged stimulation and whether that played a role in the plasticity. They found that there was potentiation of the amplitude and area under the curve of the evoked potential after prolonged stimulation and this was long-lasting (>5 hrs). They also implicated extravasation of serum albumin, caveolae-mediated transcytosis, and TGFb signalling, as well as neuronal activity and upregulation of PSD95. Transcriptomics was done and implicated plasticity related genes in the changes after prolonged stimulation, but not proteins associated with the BBB or inflammation. Next, they address the application to humans using a squeeze ball task. They imaged the brain and suggest that the hand activity led to an increased permeability of the vessels, suggesting modulation of the BBB.

Strengths:

The strengths of the paper are the novelty of the idea that stimulation of the limb can induce cortical plasticity in a normal condition, and it involves opening of the BBB with albumin entry. In addition, there are many datasets and both rat and human data.

Weaknesses:

The conclusions are not compelling however because of a lack of explanation of methods. The explanation of why prolonged stimulation in the rat was considered relevant to normal conditions should be as clear in the paper as it is in the rebuttal. The authors need to ensure other aspects of the rebuttal are as clear in the paper as in the rebuttal too. The only remaining concern that is significant is that it is hard to understand the figures.

Reviewer #3 (Public Review):

A brain region called the retrotrapezoid nucleus (RTN) regulates breathing in response to changes in CO2/H+, a process termed central chemoreception. A transcription factor called PHOX2B is important for RTN development and mutations in the PHOX2B gene result in a severe type of sleep apnea called Congenital Central Hypoventilation Syndrome. PHOX2B is also expressed throughout life, but its postmitotic functions remain unknown. This study shows that knockdown of PHOX2B in the RTN region in adult rats decreased expression of Task2 and Gpr4 in Nmb-expressing RTN chemoreceptors and this corresponded with a diminished ventilatory response to CO2 but did not impact baseline breathing or the hypoxic ventilatory response. These results provide novel insight regarding the postmitotic functions of PHOX2B in RTN neurons.

Main issues:<br /> 1) The experimental approach was not targeted to Nmb+ neurons and since other cells in the area also express Phox2b, conclusions should be tempered to focus on Phox2b expressing parafacial neurons NOT specifically RTN neurons

2) It is not clear whether PHOX2B is important for the transcription of pH sensing machinery, cell health, or both. If knockdown of PHOX2B knockdown results in loss of RTN neurons this is also expected to decrease Task2 and Gpr4 levels, albeit by a transcription-independent mechanism.