Reviewer #3 (Public Review):

In the present study, Chen et al. revealed the fungal composition and explored its interaction with bacteria in Caesarean section scar diverticulum (CSD) patients. Performing metagenomic and mass spectrometry analysis, they found specific fungi could alter bacterial abundance through regulating the production of several metabolites such as Goyaglycoside A and Janthitrem E, which results in disruption of bacterial composition stability. Their study drew a conclusion that abnormal fungal composition and activity are essential drivers for bacterial dysbiosis in CSD patients. However, the results are not substantial enough and there are many format errors throughout the manuscript. In addition, I have some concerns or suggestions that may help to improve this work.

Major<br /> 1. Smoke or drink conditions, as well as diseases like hypertension and diabetes are important factors that could influence the metabolism of the host, thus the authors should add them in the exclusion criteria in the Methods.<br /> 2. The sample size of this study is not large enough to draw a convincing conclusion.

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.

The approaches used by the authors are generally appropriate for the questions being asked, their comparisons are thorough, and the interpretations are generally reasonable. However, there are a number of major flaws to the comparisons used that should at least be addressed by the authors, if not overcome by modifying the methodology. Smaller concerns/comments are provided in "Recommendations for the authors."

My first major concern is about how the presence of teeth might influence both qualitative and quantitative comparisons to extant birds. The authors should discuss how the presence of teeth might facilitate or prevent feeding strategies that might be inconsistent with (for example) patterns reconstructed using finite element analysis for a comparative sample of toothless birds.

Next, the authors should discuss the potential impact that cranial kinesis might have on the functionality of the jaws - especially with regards to the mitigation of stresses experienced by the skull. Do the quantitative approaches used here to characterize the mechanics of the jaws account for kinesis in extant birds? If so, how? If not, how do the authors' account for that mechanical difference in their interpretations?

My next concern regards potential biases introduced by the approach taken to reconstruct the bohaiornithid skulls sampled here. Using elements from closely related taxa to fill out an incomplete skull during reconstruction is reasonable, but it may influence the results of subsequent shape comparisons - especially when the "donor" skull is compared to the recipient. The authors should explain how they accounted for this possibility in their methods or their interpretations.

Next, it is unclear how or where much of the data used or generated by this study are made available. I appreciate that the authors thoroughly cite the literature from which some data (e.g., extant FEA data), but all data used should be provided in the supplement. Likewise for the FEA models generated for the newly sampled taxa. The authors indicate that some R scripts are available online (Lines 787-788), but that link is currently non-functional, so I could not verify what was made available. And unless I missed it, the authors don't indicate that other data (e.g., FEA models) are also available there. Any data used in, or generated by, this paper should be made available online - including FEA models, tree files and analysis output files.

Also pertaining to the methods, in some places, the methods the authors used to analyze their data were not specified. For example, the authors mention that "all analyses of the [MA/FEA] data were performed in R" and "scripts [are] available" online (Lines 786-787), but the authors don't specify what those analyses actually are - unless that was specified elsewhere and I missed it? I know very little about FEA or MA analyses, so maybe these approaches are well understood in those circles, but I am unable to assess the approaches here without downloading and digging into the scripts.

A broader recommendation here: in several places, I found this paper difficult to follow. That's partly understandable, the authors are discussing and comparing trends across a wide variety of data types and analyses - which is certainly both challenging and commendable. But that variety of analyses has resulted in a staggering variety of acronyms that I found nearly impossible to keep track of. Minimally, I recommend that the authors redefine the most important acronyms at the start of each major subheading.

Related to that last point, in the discussion, I often found myself missing the forest for the trees, so to speak - the authors paid much attention to interpreting the results of each analytical approach for each taxon (which I appreciate), but I found it difficult to keep track of the take-home message the authors were trying to convey. I would recommend a reorganization of the discussion that follows a backbone based on the authors' key messages - for example, a section on species-level interpretations (maybe with sub-headings for each approach discussed), followed by larger-picture discussions of Bohaiornithidae and Enantiornithes more generally. The authors included a section at the end of their discussion that already provides that larger picture for Enantiornithes, but the section on "Bohaiornithid Ecology and Evolution" includes a lot of species-level comparison that I think would be better suited for species-focused sub-sections, and I think the paper would be better served by reserving this section for a bohaiornithid-level survey.

Reviewer #3 (Public Review):

Summary:<br /> In this study, the authors analyzed data from 99 individuals with implanted electrodes who were performing a word-list recall task. Because the task involves successively encoding and then recalling 25 lists in a row, they were able to measure the similarity in neural responses for items within the same list as well as items across different lists, allowing them to test hypotheses about the impact of between-list boundaries on neural responses. They find that, in addition to slow drift in responses across items, there is boundary-related structure in the medial parietal lobe such that early items in each list show similarity (for recalled items) and late items in each list show similarity (for not recalled items).

Strengths:<br /> The dataset used in this paper is substantially larger than most iEEG datasets, allowing for the detection of nuanced differences between item positions and for analyses of individual differences in boundary-related responses. There are excellent visualizations of the similarity structure between items for each region, and this work connects to a growing literature on the role of event boundaries in structuring neural responses.

Weaknesses:<br /> 1) My primary confusion in the current version of this paper is that the analyses don't seem to directly compare the two proposed models illustrated in Fig 1B, i.e. the temporal context model (with smooth drifts between items, including across lists) versus the boundary model (with similarities across all lists for items near boundaries). After examining smooth drift in the within-list analysis (Fig 2), the across-list analyses (Figs 3-5) use a model with two predictors (boundary proximity and list distance), neither of which is a smoothly-drifting context. Therefore there does not appear to be a quantitative analysis supporting the conclusion that in lateral temporal cortex "drift exhibits a relationship with elapsed time regardless of the presences of intervening boundaries" (lines 272-3).

2) The feature representation used for the neural response to each item is a gamma power time-frequency matrix. This makes it unclear what characteristics of the neural response are driving the observed similarity effects. It appears that a simple overall scaling of the response after boundaries (stronger responses to initial items during the beginning portion of the 1.6s time window) would lead to the increased cosine similarity between initial items, but wouldn't necessarily reflect meaningful differences in the neural representation or context of these items.

3) The specific form of the boundary proximity models is not well justified. For initial items, a model of e^(1-d) is used (with d being serial position), but it is not stated how the falloff scale of this model was selected (as opposed to e.g. e^((1-d)/2)). For final items, a different model of d/#items is used, which seems to have a somewhat different interpretation (about drift between boundaries, rather than an effect specific to items near a final boundary). The schematic in Fig 1B appears to show a hypothesis which is not tested, with symmetric effects at initial and final boundaries.

4) The main text description of Fig 2 only describes drift effects in lateral temporal cortex, but Fig 2 - supplement 1 shows that there is also drift and a significant subsequent memory effect in the other two ROIs as well. There is not a significant memory x drift slope interaction in these regions; are the authors arguing that the lack of this interaction (different drift rates for remembered versus forgotten items) is critical for interpreting the roles of lateral temporal cortex versus medial parietal and hippocampal regions?

5) The parameter fits for the "list distance" regressor are not shown or analyzed, though they do appear to be important for the observed similarity structure (e.g. Fig 3E). I would interpret this regressor as also being "boundary-related" in the sense that it assumes discrete changes in similarity at boundaries.

6) It is unclear to me whether the authors believe that the observed similarity after boundaries is due to an active process in which "the medial parietal lobe uses drift-resets" (line 16) to reinstate a boundary-related context, or that this similarity is simply because "the context for the first item may be the boundary itself" (lines 246-7), and therefore this effect would emerge naturally from a temporal context model that incorporates the full task structure as the "items."

Reviewer #3 (Public Review):

Summary:<br /> This is a strong manuscript by Basson and colleagues which contributes to our understanding of gender disparities in scientific publishing. The authors examine attitudes and behaviors related to manuscript submission in influential journals (specifically, Science, Nature and PNAS). The authors rightly note that much attention has been paid to gender disparities in work that is already published, but this fails to capture the unseen hurdles that occur prior to publication (which include decisions about where to publish, desk rejections, revisions and resubmissions, etc.). They conducted a survey study to address some of these components and their results are interesting:

They find that women are less likely to submit their manuscript to Science, Nature or PNAS. While both men and women feel their work would be better suited for more specialized journals, women were more likely to think their work was 'less novel or groundbreaking.'

A smaller proportion of respondents indicated that they were actively discouraged from submitting their manuscripts to these journals. In this instance, women were more likely to receive this advice than men.

Lastly, the authors also looked at self-reported acceptance and rejection rates and found that there were no gender differences in acceptance or rejection rates.

These data are helpful in developing strategies to mitigate gender disparities in influential journals.

Comments:<br /> The methods the authors used are appropriate for this study. The low response rate is common for this type of recruitment strategy. The authors provide a thoughtful interpretation of their data in the Discussion.

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.

The work carried out in this study is technically precise at all levels, molecular, cellular and microscopy. The reviewer here acknowledges how difficult is to work and develop tools and mutant lines in a non-model organism and therefore congratulates the authors in their efforts. The authors have done excellent work in this sense and all data presented seems to be solid.

Nevertheless, some of the observations, in my opinion, should be further investigated before taking the conclusion that the Hippo pathway controls cell density and a contractile behavior in the C. owczarzaki. On the hand the authors claim as main conclusions what they have partially already claimed in the previous work (Phillips et al. eLife 2022;0:e77598).

Reviewer #3 (Public Review):

Fission yeast is an important model organism and studies on fission yeast have provided many key insights into the understanding of genes and biological pathways. However, even in such a well-studied model organism, there are still many genes without known functions.

In this work, the authors took advantage of the availability of genome-wide fission yeast deletion mutants to systematically analyze the mutant phenotypes under 131 different conditions. This effort generated a genotype-phenotype dataset larger than the currently curated genotype-phenotype dataset, which is derived from studies over many decades by hundreds of fission yeast laboratories. The authors used the dataset to construct gene clusters that provide functional clues for many genes without previously known functions, including ones conserved in humans. This rich resource will surely be highly useful to the fission yeast community and beyond.

In addition, the authors also used machine learning to generate functional predictions of fission yeast genes and yield novel understandings, which are validated by experimental analysis of new ageing-related genes.

Overall, this study provides unprecedented and highly valuable resources for understanding fission yeast gene functions.

Reviewer #3 (Public Review):

This manuscript presents a comprehensive investigation into the mechanisms that explain the presence of TADs (P-TADs) in cells where cohesin has been removed. In particular, to study TADs in wildtype and cohesin depleted cells, the authors use a combination of polymer simulations to predict whole chromosome structures de novo and from Hi-C data. Interestingly, they find that those TADs that survive cohesin removal contain a switch in epigenetic marks (from compartment A to B or B to A) at the boundary. Additionally, they find that the P-TADs are needed to retain enhancer-promoter and promoter-promoter interactions.

Overall, the study is well-executed, and the evidence found provides interesting insights into genome folding and interpretations of conflicting results on the role of cohesin on TAD formation.

To strengthen their claims, the authors should compare their de-novo prediction approach to their data-driven predictions at the single cell level.

Reviewer #3 (Public Review):

In this manuscript, Chan and collaborators investigate the role of CDPK1 in regulating microneme trafficking and exocytosis in Toxoplasma gondii. Micronemes are apicomplexan-specific organelles localized at the apical end of the parasite and depending on cortical microtubules. Micronemes contain proteins that are exocytosed in a Ca²+-dependent manner and are required for T. gondii egress, motility, and host-cell invasion. In Apicomplexa, Ca²+ signaling is dependent on Ca²+-dependent protein kinases (CDPKs). CDPK1 has been demonstrated to be essential for Ca²+-stimulated micronemes exocytosis allowing parasite egress, gliding motility, and invasion. It is also known that intracellular calcium storages are mobilized following a cyclic nucleotide-mediated activation of protein kinase G. This step, occurs upstream of CDPK1 functions. However, the exact signaling pathway regulated by CDPK1 remains unknown. In this paper, the authors used phosphoproteomic analysis to identify new proteins phosphorylated by CDPK1. They demonstrated that CDPK1 activity is required for calcium-stimulated trafficking of micronemes to the apical end, depending on a complex of proteins that include HOOK and FTS, which are known to link cargo to the dynein machinery for trafficking along microtubules. Overall, the authors identified evidence for a new protein complex involved in microneme trafficking through the exocytosis process for which circumstantial evidence of its functionality is demonstrated here.

Reviewer #3 (Public Review):

In this study, the authors analyzed a unique and very stable microtubule bundle that is formed in yeast cells entering quiescence. They show that the structure is required for yeast cells to maintain viability during quiescence and that it needs to be disassembled for cell cycle re-entry. They identify different stages during the assembly process and focus on the molecular players required for microtubule bundle formation and stabilization. They identify kinetochore as well as molecular motors such as auroraB, kinesin-14, and kinesin-5 that assemble, stabilize and crosslink the microtubules of the bundle. The paper also investigates the disassembly of the structure and shows that disassembly is required for cell cycle re-entry.

The study is very comprehensive, provides quantifications to support claims, and identifies various players involved in these processes, providing mechanistic insight. It also presents various control experiments to exclude alternative explanations and support the proposed model.

It is the first molecular-level insight into how this very stable microtubule structure can be assembled, maintained, and disassembled, and how this is coordinated with cell cycle exit and re-entry. This information may be very useful when analyzing similarly stable, microtubule-based structures in other organisms such as cilia in animals, which also display cell cycle-coordinated dynamics.

Overall, this is a nicely presented study that provides important insight into the field and beyond, but there are a few points that need to be addressed regarding methods used for quantifications and data presentation.

Reviewer #3 (Public Review):

Bae et al. described the key roles of pericytes in cavernous tissues in diabetic erectile dysfunction using both mouse and human single-cell transcriptomic analysis. Erectile dysfunction (ED) is caused by dysfunction of the cavernous tissue and affects a significant proportion of men aged 40-70. The most common treatment for ED is phosphodiesterase 5 inhibitors; however, these are less effective in patients with diabetic ED. Therefore, there is an unmet need for a better understanding of the cavernous microenvironment, cell-cell communications in patients with diabetic ED, and the development of new therapeutic treatments to improve the quality of life.

Pericytes are mesenchymal-derived mural cells that directly interact with capillary endothelial cells (ECs). They play a vital role in the pathogenesis of erectile function as their interactions with ECs are essential for penile erection. Loss of pericytes has been associated with diabetic retinopathy, cancer, and Alzheimer's disease and has been investigated in relation to the permeability of cavernous blood vessels and neurovascular regeneration in the authors' previous studies. This manuscript explores the mechanisms underlying the effect of diabetes on pericyte dysfunction in ED. Additionally, the cellular landscape of cavernous tissues and cell type-specific transcriptional changes were carefully examined using both mouse and human single-cell RNA sequencing in diabetic ED. The novelty of this work lies in the identification of a newly identified pericyte (PC)-specific marker, LBH, in mouse and human cavernous tissues, which distinguishes pericytes from smooth muscle cells. LBH not only serves as a cavernous pericyte marker, but its expression level is also reduced in diabetic conditions. The LBH-interacting proteins (Cryab and Vim) were further identified in mouse cavernous pericytes, indicating that these signaling interactions are critical for maintaining normal pericyte function. Overall, this study demonstrates the novel marker of pericytes and highlights the critical role of pericytes in diabetic ED.

Reviewer #3 (Public Review):

The manuscript by Yang et al. investigated in mice how hypobaric hypoxia can modify the RBC clearance function of the spleen, a concept that is of interest. Via interpretation of their data, the authors proposed a model that hypoxia causes an increase in cellular iron levels, possibly in RPMs, leading to ferroptosis, and downregulates their erythrophagocytic capacity. However, most of the data is generated on total splenocytes/total spleen, and the conclusions are not always supported by the presented data. The model of the authors could be questioned by the paper by Youssef et al. (which the authors cite, but in an unclear context) that the ferroptosis in RPMs could be mediated by augmented erythrophagocytosis. As such, the loss of RPMs in vivo which is indeed clear in the histological section shown (and is a strong and interesting finding) can be not directly caused by hypoxia, but by enhanced RBC clearance. Such a possibility should be taken into account.

Major points:

1) The authors present data from total splenocytes and then relate the obtained data to RPMs, which are quantitatively a minor population in the spleen. Eg, labile iron is increased in the splenocytes upon HH, but the manuscript does not show that this occurs in the red pulp or RPMs. They also measure gene/protein expression changes in the total spleen and connect them to changes in macrophages, as indicated in the model Figure (Fig. 7). HO-1 and levels of Ferritin (L and H) can be attributed to the drop in RPMs in the spleen. Are any of these changes preserved cell-intrinsically in cultured macrophages? This should be shown to support the model (relates also to lines 487-88, where the authors again speculate that hypoxia decreases HO-1 which was not demonstrated). In the current stage, for example, we do not know if the labile iron increase in cultured cells and in the spleen in vivo upon hypoxia is the same phenomenon, and why labile iron is increased. To improve the manuscript, the authors should study specifically RPMs.

2) The paper uses flow cytometry, but how this method was applied is suboptimal: there are no gating strategies, no indication if single events were determined, and how cell viability was assessed, which are the parent populations when % of cells is shown on the graphs. How RBCs in the spleen could be analyzed without dedicated cell surface markers? A drop in splenic RPMs is presented as the key finding of the manuscript but Fig. 3M shows gating (suboptimal) for monocytes, not RPMs. RPMs are typically F4/80-high, CD11-low (again no gating strategy is shown for RPMs). Also, the authors used single-cell RNAseq to detect a drop in splenic macrophages upon HH, but they do not indicate in Fig. A-C which cluster of cells relates to macrophages. Cell clusters are not identified in these panels, hence the data is not interpretable).

3) The authors draw conclusions that are not supported by the data, some examples:

a) They cannot exclude eg the compensatory involvement of the liver in the RBCs clearance (the differences between HH sham and HH splenectomy is mild in Fig. 2 E, F and G)

b) Splenomegaly is typically caused by increased extramedullary erythropoiesis, not RBC retention. Why do the authors support the second possibility? Related to this, why do the authors conclude that data in Fig. 4 G,H support the model of RBC retention? A significant drop in splenic RBCs (poorly gated) was observed at 7 days, between NN and HH groups, which could actually indicate increased RBC clearance capacity = less retention.

c) Lines 452-54: there is no data for decreased phagocytosis in vivo, especially in the context of erythrophagocytosis. This should be done with stressed RBCs transfusion assays, very good examples, like from Youssef et al. or Threul et al. are available in the literature.

d) Line 475 - ferritinophagy was not shown in response to hypoxia by the manuscript, especially that NCOA4 is decreased, at least in the total spleen.

4) In a few cases, the authors show only representative dot plots or histograms, without quantification for n>1. In Fig. 4B the authors write about a significant decrease (although with n=1 no statistics could be applied here; of note, it is not clear what kind of samples were analyzed here). Another example is Fig. 6I. In this case, it is even more important as the data are conflicting the cited article and the new one: PMCID: PMC9908853 which shows that hypoxia stimulates efferocytosis. Sometimes the manuscript claim that some changes are observed, although they are not visible in representative figures (eg for M1 and M2 macrophages in Fig. 3M)

5) There are several unclear issues in methodology:

- what is the purity of primary RPMs in the culture? RPMs are quantitatively poorly represented in splenocyte single-cell suspensions. This reviewer is quite skeptical that the processing of splenocytes from approx 1 mm3 of tissue was sufficient to establish primary RPM cultures. The authors should prove that the cultured cells were indeed RPMs, not monocyte-derived macrophages or other splenic macrophage subtypes.<br /> - (around line 183) In the description of flow cytometry, there are several missing issues. In 1) it is unclear which type of samples were analyzed. In 2) it is not clear how splenocyte cell suspension was prepared.<br /> - In line 192: what does it mean: 'This step can be omitted from cell samples'?<br /> - 'TO method' is not commonly used anymore and hence it was unclear to this Reviewer. Reticulocytes should be analyzed with proper gating, using cell surface markers.<br /> - The description of 'phagocytosis of E. coli and RBCs' in the Methods section is unclear and incomplete. The Results section suggests that for the biotinylated RBCs, phagocytosis? or retention? Of RBCs was quantified in vivo, upon transfusion. However, the Methods section suggests either in vitro/ex vivo approach. It is vague what was indeed performed and how in detail. If RBC transfusion was done, this should be properly described. Of note, biotinylation of RBCs is typically done in vivo only, being a first step in RBC lifespan assay. The such assay is missing in the manuscript. Also, it is not clear if the detection of biotinylated RBCs was performed in permeablized cells (this would be required).

The authors did not substantially improve the quality of their manuscript in the revised version, at least in the case of the limitations which I have spotted. The major points which remain unclear:<br /> 1. No gating strategies for flow cytometry are provided.<br /> 2. Figure 3M still does not show a typical F4/80 vs CD11b gating, with a population of true RPMs gated.<br /> 3. In a few cases data still lack biological replicates+statistics.<br /> 4. Results from scRNA-seq are not presented more clearly (=clusters in Fig 3E are described as macrophages, but it is not explained which among the clusters are RPMs).<br /> 5. The compensatory role of liver macrophages is omitted.<br /> 6. The authors misunderstood by suggestion to perform in vivo erythrophagocytosis assay using stained RBCs. This assay quantifies the true capacity for erythrophagocytosis in RPMs or KCs in the organ, regardless of the ferroptosis that may be a subsequent consequence (please, see initial Figures in Yousseff et al. paper). Using the percentage of biotin-positive RBCs in the spleen (although this method is not well described in the Methods), the authors rather show increased RBCs clearance at 7 days following hypoxia. Hence, the model where first hypoxia increases erythrophagocytosis in RPMs, consequently leading to their ferroptosis still cannot be excluded.<br /> 7. The Methods are poorly described and unclear - the authors claimed that they have used in vivo biotinylation assay to assess the lifespan of RBCs but it is not described. Instead, the paragraph „Phagocytosis of E. coli and RBCs" suggests that RBCs were stained with biotin for phagocytic assay in culture with macrophages. Phagocytosis of E. coli is still described in the Methods although the authors opted to remove the data from the revised manuscript.<br /> Some points are unclear in the current version of the manuscript, after the addition of new data:<br /> 8. Data in Figure 4D versus 4E,F are not consistent, showing less retention versus increased retention of RBCs in the spleen (retention of senescent RBCs in the spleen should be measured anyway quantitatively, eg, with proper flow cytometry)<br /> 9. The increase of labile iron in the red pulp might not be in RPMs - especially since they seem depleted. Flow cytometry should be used to assess which cell types show increased iron levels.

Reviewer #3 (Public Review):

Summary: The manuscript by Cullinan et al., uses ANAP-tmFRET to test the hypothesis that the NTD and CTD form a complex at rest and to probe these domains for acid-induced conformational changes. They find convincing evidence that the NTD and CTD do not have a propensity to form a complex. They also report these domains are parallel to the membrane and that the NTD moves towards, and the CTD away, from the membrane upon acidification.

Strengths:<br /> The major strength of the paper is the use of tmFRET, which excels at measuring short distances and is insensitive to orientation effects. The donor-acceptor pairs here are also great choices as they are minimally disruptive to the structure being studied.

Furthermore, they conduct these measurements over several positions with the N and C tails, both between the tails and to the membrane. Finally, to support their main point, MST is conducted to measure the association of recombinant N and C peptides, finding no evidence of association or complex formation.

Weaknesses:<br /> While tmFRET is a strength, using ANAP as a donor requires the cells to be unroofed to eliminate background signal. This causes two problems. First, it removes any possible low affinity interacting proteins such as actinin (PMID 19028690). Second, the pH changes now occur to both 'extracellular' and 'intracellular' lipid planes. Thus, it is unclear if any conformational changes in the N and CTDs arise from desensitization of the receptor or protonation of specific amino acids in the N or CTDs or even protonation of certain phospholipid groups such as in phosphatidylserine. The authors do comment that prolonged extracellular acidification leads to intracellular acidification as well. But the concerns over disruption by unroofing/washing and relevance of the changes remain.

The distances calculated depend on the R0 between donor and acceptor. In turn, this depends on the donor's emission spectrum and quantum yield. The spectrum and yield of ANAP is very sensitive to local environment. It is a useful fluorophore for patch fluorometry for precisely this reason, and gating-induced conformational changes in the CTD have been reported just from changes in ANAP emission alone (PMID 29425514). Therefore, using a single R0 value for all positions (and both pHs at a single position) is inappropriate. The authors should either include this caveat and give some estimate of how big an impact changes spectrum and yield might have, or actually measure the emission spectra at all positions tested.

Overall, the writing and presentation of figures could be much improved with specific points mentioned in the recommendations for authors section.

The authors argue that the CTD is largely parallel to the plasma membrane, yet appear to base this conclusion on ANAP to membrane FRET of positions S464 and M505. Two positions is insufficient evidence to support such a claim. Some intermediate positions are needed.

Upon acidification, NTD position Q14 moves towards the plasma membrane (Figure 8B). Q14 also gets closer to C515 or doesn't change relative to 505 (Figures 7C and B) upon acidification. Yet position 505 moves away from the membrane (Figure 8D). How can the NTD move closer to the membrane, and to the CTD but yet the CTD move further from the membrane? Some comment or clarification is needed.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Nagel et al. describes studies of mouse vomeronasal sensory neuron (VSN) tuning to mouse urine samples across different sexes and strains, including wild mice, alongside mass spectrometry analysis of the same samples. The authors performed live Ca2+ imaging (CAL520 dye) of VSNs in acute vomeronasal organ (VNO) slices to determine how VSNs are tuned to pairs of stimuli that differ in their origin (e.g. male C57BL/6 versus male BALB/c urine, male C57BL/6 versus female C57BL/6, etc.). For each pair of tested odorants, the results measure the proportion of VSNs that respond to both stimuli ("generalists") or just one of the two ("specialists"), as well as metrics of tuning preference and response reliability. The authors find in most cases that generalists make up a larger proportion of responsive VSNs than specialists, but several pairwise comparisons showed a high degree of strain selectivity. Notably, the authors evaluated VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding strain-dependent differences in the representation of mouse urine. Alongside these measurements of VSN tuning, the authors report results of mass spectrometry analyses of volatiles and proteins in the same urine samples. These analyses indicated a number of molecules in each category that vary across sex and strain, and therefore represent candidate vomeronasal ligands. However, this study did not directly test whether any of these candidate molecules drives VSN activity, limiting the interpretability of these comparisons. Overall, this work provides useful information related to mouse vomeronasal chemosensation, but future work will be necessary to link the physiological measurements to the observed molecular diversity.

Strengths:<br /> A strength of the current study is its focus on characterizing the neural responses of the VNO to urine derived from wild mice. The majority of existing vomeronasal system research has relied on the use of inbred strains for both neural response recordings and investigations of candidate vomeronasal system ligands. Inbreeding in laboratory environments may alter the chemical composition of bodily secretions, thereby potentially changing the information they contain. Moreover, the more homogeneous nature of inbred strains could be critical when studying the AOS mediated social aspects. If there exist noticeable differences in the chemical composition of secretions from wild animals compared to inbred strains, this would suggest that future research must consider natural sources of candidate ligands outside of inbred strains. This work identifies some intriguing differences - worthy of further exploration - between the urine composition of wild mice versus inbred mice, as well as disparities in how the VNO responds to urine from these different sources. However, the molecular composition and VNO responsiveness to wild mouse urine was found to be highly overlapping with inbred mouse urine, supporting the continued investigation of candidate ligands found in inbred mouse urine.

Another positive aspect of this work is its use of the same set of stimuli as a previous study by the same authors (Bansal et al., 2021) in the downstream accessory olfactory bulb. The consistency in stimulus selection facilitates a comparison of information processing of sex and strain information from the sensory periphery to the brain. Although comparisons between the two connected regions are not a focus of this work, and methodological differences (e.g., Ca2+ imaging versus electrophysiology) may introduce caveats into comparisons, the support of "apples to apples" comparisons across connected circuits is critical to progress in the field.

Finally, this study directly measured VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding subtle but important differences in the representation of mouse urine in these two recipient strains. Given that there is a long history of research into strain-specific differences in social behavior, this research paves the way for future studies into how different mouse strains detect and process social chemosignals.

Weaknesses:<br /> One of the primary objectives in this study is to ascertain the extent to which the response profiles of VSNs are specific to sex and strain. The design of these Ca2+ imaging experiments uses a simple stimulus design, using two interleaved bouts of stimulation with pairs of urine (e.g. male versus female C57BL/6, male C57BL/6 versus male BALB/c) at a single dilution factor (1:100). This introduces two significant limitations: (1) the "generalist" versus "specialist" descriptors pertain only to the specific pairwise comparisons made and (2) there is no information about the sensitivity/concentration-dependence of the responses.

The functional measurements of VSN tuning to various pairs of urine stimuli are consistently presented alongside mass spectrometry-based comparisons. Although it is clear from the manuscript text that the mass spectrometry-based analysis was separated from the VSN tuning experiments/analysis, the juxtaposition of VSN tuning measurements with independent molecular diversity measurements gives the appearance to readers that these experiments were integrated (i.e., that the diversity of ligands was underlying the diversity of physiological responses). This is a hypothesis raised by the parallel studies, not a supported conclusion of the work. This data presentation style risks confusing readers.

The impact of mass spectrometry findings is limited by the fact that none of these molecules (in bulk, fractions, or monomolecular candidate ligands) were tested on VSNs. It is possible that only a very small number of these ligands activate the VNO. The list of variably expressed proteins - especially several proteins that are preferentially found in female urine - is compelling, but, again, there is no evidence presented that indicates whether or not these candidate ligands drive VSN activity. It is noteworthy that the largest class of known natural ligands for VSNs are small nonvolatiles that are found at high levels in mouse urine. These molecules were almost certainly involved in driving VSN activity in the physiology assays (both "generalist" and "specialist"), but they are absent from the molecular analysis.

Reviewer #3 (Public Review):

Summary:<br /> This study tackles the important subject of sensory driven suppression of alpha oscillations using a unique intracranial dataset in human patients. Using a model-based approach to separate changes in alpha oscillations from broadband power changes, the authors try to demonstrate that alpha suppression is spatially tuned, with similar center location as high broadband power changes, but much larger receptive field. They also point to interesting differences between low-order (V1-V3) and higher-order (dorsolateral) visual cortex. While I find some of the methodology convincing, I also find significant parts of the data analysis, statistics and their presentation incomplete. Thus, I find that some of the main claims are not sufficiently supported. If these aspects could be improved upon, this study could potentially serve as an important contribution to the literature with implications for invasive and non-invasive electrophysiological studies in humans.

Strengths:<br /> The study utilizes a unique dataset (ECOG & high-density ECOG) to elucidate an important phenomenon of visually driven alpha suppression. The central question is important and the general approach is sound. The manuscript is clearly written and the methods are generally described transparently (and with reference to the corresponding code used to generate them). The model-based approach for separating alpha from broadband power changes is especially convincing and well-motivated. The link to exogenous attention behavioral findings (figure 8) is also very interesting. Overall, the main claims are potentially important, but they need to be further substantiated (see weaknesses).

Weaknesses:<br /> I have three major concerns:<br /> 1. Low N / no single subject results/statistics: The crucial results of Figure 4,5 hang on 53 electrodes from four patients (Table 2). Almost half of these electrodes (25/53) are from a single subject. Data and statistical analysis seem to just pool all electrodes, as if these were statistically independent, and without taking into account subject-specific variability. The mean effect per each patient was not described in text or presented in figures. Therefore, it is impossible to know if the results could be skewed by a single unrepresentative patient. This is crucial for readers to be able to assess the robustness of the results. N of subjects should also be explicitly specified next to each result.

2. Separation between V1-V3 and dorsolateral electrodes: Out of 53 electrodes, 27 were doubly assigned as both V1-V3 and dorsolateral (Table 2, Figures 4,5). That means that out of 35 V1-V3 electrodes, 27 might actually be dorsolateral. This problem is exasperated by the low N. for example all the 20 electrodes in patient 8 assigned as V1-V3 might as well be dorsolateral. This double assignment didn't make sense to me and I wasn't convinced by the authors' reasoning. I think it needlessly inflates the N for comparing the two groups and casts doubts on the robustness of these analyses.

3. Alpha pRFs are larger than broadband pRFs: first, as broadband pRF models were on average better fit to the data than alpha pRF models (dark bars in Supp Fig 3. Top row), I wonder if this could entirely explain the larger Alpha pRF (i.e. worse fits lead to larger pRFs). There was no anlaysis to rule out this possibility. Second, examining closely the entire 2.4 section there wasn't any formal statistical test to back up any of the claims (not a single p-value is mentioned). It is crucial in my opinion to support each of the main claims of the paper with formal statistical testing.

While I judge these issues as crucial, I can also appreciate the considerable effort and thoughtfulness that went into this study. I think that addressing these concerns will substantially raise the confidence of the readership in the study's findings, which are potentially important and interesting.

Reviewer #3 (Public Review):

Summary:

The paper proposes an alternative to the attractor hypothesis, as an explanation for the fact that grid cell population activity patterns (within a module) span a toroidal manifold. The proposal is based on a class of models that were extensively studied in the past, in which grid cells are driven by synaptic inputs from place cells in the hippocampus. The synapses are updated according to a Hebbian plasticity rule. Combined with an adaptation mechanism, this leads to patterning of the inputs from place cells to grid cells such that the spatial activity patterns are organized as an array of localized firing fields with hexagonal order. I refer to these models below as feedforward models.

It has already been shown by Si, Kropff, and Treves in 2012 that recurrent connections between grid cells can lead to alignment of their spatial response patterns. This idea was revisited by Urdapilleta, Si, and Treves in 2017. Thus, it should already be clear that in such models, the population activity pattern spans a manifold with toroidal topology. The main new contributions in the present paper are (i) in considering some forms of recurrent connectivity that were not directly addressed before (but see comments below). (ii) in applying topological analysis to simulations of the model. (iii) in interpreting the results as a potential explanation for the observations of Gardner et al.

Strengths:

The exploration of learning in a feedforward model, when recurrent connectivity in the grid cell layer is structured in a ring topology, is interesting. The insight that this not only aligns the grid cells in a common direction but also creates a correspondence between their intrinsic coordinate (in terms of the ring-like recurrent connectivity) and their tuning on the torus is interesting as well, and the paper as a whole may influence future theoretical thinking on the mechanisms giving rise to the properties of grid cells.

Weaknesses:

1. It is not clear to me that the proposal here is fundamentally new. In Si, Kropff and Treves (2012) recurrent connectivity was dependent on the head direction tuning and thus had a ring structure. Urdapilleta, Si, and Treves considered connectivity that depends on the distance on a 2d plane.

2. The paper refers to the connectivity within the grid cell layer as an attractor. However, would this connectivity, on its own, indeed sustain persistent attractor states? This is not examined in the paper. Furthermore, is this even necessary to obtain the results in the model? Perhaps weak connections that do not produce an attractor would be sufficient to align the spatial response patterns during the learning of feedforward weights, and reproduce the results? In general, there is no exploration of how the strength of collateral interactions affects the outcome.

3. I did not understand what is learned from the local topology analysis. Given that all the grid cells are driven by an input from place cells that spans a 2d manifold, and that the activity in the grid cell network settles on a steady state that depends only on the inputs, isn't it quite obvious that the manifold of activity in the grid cell layer would have, locally, a 2d structure?

4. The modeling is all done in planar 2d environments, where the feedforward learning mechanism promotes the emergence of a hexagonal pattern in the single neuron tuning curve. This, combined with the fact that all neurons develop spatial patterns with the same spacing and orientation, implies even without any topological analysis that the emerging topology of the population activity is a torus.

However, the toroidal topology of grid cells in reality has been observed by Gardner et al also in the wagon wheel environment and in sleep, and there is substantial evidence based on pairwise correlations that it persists also in various other situations, in which the spatial response pattern is not a hexagonal firing pattern. It is not clear that the mechanism proposed in the present paper would generate toroidal topology of the population activity in more complex environments. In fact, it seems likely that it will not do so.

5. Moreover, the recent work of Gardner et al. demonstrated much more than the preservation of the topology in the different environments and in sleep: the toroidal tuning curves of individual neurons remained the same in different environments. Previous works, that analyzed pairwise correlations under hippocampal inactivation and various other manipulations, also pointed towards the same conclusion. Thus, the same population activity patterns are expressed in many different conditions. In the present model, the results of Figure 6 suggest that even across distinct rectangular environments, toroidal tuning curves will not be preserved, because there are multiple possible arrangements of the phases on the torus which emerge in different simulations.

6. In real grid cells, there is a dense and fairly uniform representation of all phases (see the toroidal tuning of grid cells measured by Gardner et al). Here the distribution of phases is not shown, but Figure 7 suggests that phases are non uniformly represented, with significant clustering around a few discrete phases. This, I believe, is also the origin for the difficulty in identifying the toroidal topology based on the transpose of the matrix M: vectors representing the spatial response patterns of individual neurons are localized near the clusters, and there are only a few of them that represent other phases. Therefore, there is no dense coverage of the toroidal manifold that would exist if all phases were represented equally. This is not just a technical issue, however: there appears to be a mismatch between the results of the model and the experimental reality, in terms of the phase coverage.

7. The manuscript makes several strong claims that incorrectly represent the relation between experimental data and attractor models, on one hand, and the present model on the other hand. For the latter, see the comments above. For the former, I provide a detailed list in the recommendations to the authors, but in short: the paper claims that attractor models induce rigidness in the neural activity which is incompatible with distortions seen in the spatial response patterns of grid cells. However, this claim seems to confuse distortions in the spatial response pattern, which are fully compatible with the attractor model, with distortions in the population activity patterns, which would be incompatible with the attractor model. The attractor model has withstood numerous tests showing that the population activity manifold is rigidly preserved across conditions - a strong prediction (which is not made, as far as I can see, by feedforward models). I am not aware of any data set where distortions of the population activity manifold have been identified, and the preservation has been demonstrated in many examples where the spatial response pattern is disrupted. This is the main point of two papers cited in the present manuscript: by Yoon et al, and Gardner et al.

8. There is also some weakness in the mathematical description of the dynamics. Mathematical equations are formulated in discrete time steps, without a clear interpretation in terms of biophysically relevant time scales. It appears that there are no terms in the dynamics associated with an intrinsic time scale of the neurons or the synapses, and this introduces a difficulty in interpreting synaptic weights as being weak or strong. As mentioned above, the nature of the recurrent dynamics within the grid cell network (whether it exhibits continuous attractor behavior) is not sufficiently clear.

In my view, the weaknesses discussed above limit the ability of the model, as it stands, to offer a compelling explanation for the toroidal topology of grid cell population activity patterns, and especially the rigidity of the manifold across environments and behavioral states. Still, the work offers an interesting way of thinking on how the toroidal topology might emerge. Perhaps with certain additional elements this may motivate new theoretical insights.

Reviewer #3 (Public Review):

Summary: This is a careful examination of the distribution of mitochondria in the basal dendrites of ferret visual cortex in a previously published volume electron microscopy dataset. The authors report that mitochondria are sparsely, as opposed to continuously distributed in the dendritic shafts, and that they tend to cluster near dendritic spines with heterogeneous orientation selectivity.

Strengths: Volume EM is the gold standard for quantification of organelle morphology. An unusual strength of this particular dataset is that the orientation selectivity of the dendritic spines was measured by calcium imaging prior to EM reconstruction. This allowed the authors to assess how spines with varying selectivity are organized relative to mitochondria, leading to an intriguing observation that they localize to heterogeneous spine clusters. The analysis is carefully performed.

Weaknesses: Using threshold distances between mitochondria and synapses as opposed to absolute distances may overlook important relationships in the data.

Reviewer #3 (Public Review):

In this study, the authors identified homozygous ZMYND12 variants in four unrelated patients. In sperm cells from these individuals, immunofluorescence revealed altered localization of DNAH1, DNALI1, WDR66, and TTC29. Axonemal localization of ZMYND12 ortholog TbTAX-1 was confirmed using the Trypanosoma brucei model. RNAi knock-down of TbTAX-1 dramatically affected flagellar motility, with a phenotype similar to ZMYND12-variant-bearing human sperm. Co-immunoprecipitation and ultrastructure expansion microscopy in T. brucei revealed TbTAX-1 to form a complex with TTC29. Comparative proteomics with samples from Trypanosoma and Ttc29 KO mice identified a third member of this complex: DNAH1. The data presented revealed that ZMYND12 is part of the same axonemal complex as TTC29 and DNAH1, which is critical for flagellum function and assembly in humans, and Trypanosoma. The manuscript is informative for the clinical and basic researchers in the field of spermatogenesis and male infertility.

Reviewer #3 (Public Review):

In "Trophic eggs affect caste determination in the ant Pogonomyrmex rugosus" Genzoni et al. probe a fundamental question in sociobiology, what are the molecular and developmental processes governing caste determination? In many social insect lineages, caste determination is a major ontogenetic milestone that establishes the discrete queen and worker life histories that make up the fundamental units of their colonies. Over the last century, mechanisms of caste determination, particularly regulators of caste during development, have remained relatively elusive. Here, Genzoni et al. discovered an unexpected role for trophic eggs in suppressing queen development - where bi-potential larvae fed trophic eggs become significantly more likely to develop into workers instead of gynes (new queens). These results are unexpected, and potentially paradigm-shifting, given that previously trophic eggs have been hypothesized to evolve to act as an additional intra-colony resource for colonies in potentially competitive environments or during specific times in colony ontogeny (colony foundation), where additional food sources independent of foraging would be beneficial. While the evidence and methods used are compelling (e.g., the sequence of reproductive vs. trophic egg deposition by single queens, which highlights that the production of trophic eggs is tightly regulated), the connective tissue linking many experiments is missing and the downstream mechanism is speculative (e.g., whether miRNA, proteins, triglycerides, glycogen levels in trophic eggs is what suppresses queen development). Overall, this research elevates the importance of trophic eggs in regulating queen and worker development but how this is achieved remains unknown.

Reviewer #3 (Public Review):

Summary:<br /> In this study, the researchers used ancient environmental DNA (aeDNA) retrieved from sediment cores, from two lakes in the Arctic, on the Yamal peninsula, in Siberia. The dating of one of the cores, showed that the sediment layers were very recent (ranging between the years 2019 - 1895). From this core they sequenced 23 libraries which were enriched for mammal mitochondrial genomes. They found a high proportion of two species that have been extinct for thousands of years, the mammoth and the woolly rhinoceros. The highest proportion of mammoth reads were found in very young layer (~81 years old) and as this initial finding does not match the temporal occurrence of the species, they confirmed the identification with several other methods. Additionally, they applied a different dating method on some samples and found that the aging of the samples was not completely congruent. The authors suggest the that the presence of these two Pleistocene megafauna in such recent sediment layers is a consequence of physical processes, specific to the study site, and that the high quality of the aeDNA recovered is a result of permafrost preservation.

Strengths:<br /> The strengths of the study are in the rigorous confirmation of the identification of the taxa with four different PCR and sequencing techniques being used, the initial enrichment panel, and then subsequent metabarcoding PCRs, and taxa specific PCR for COI and cytB. Along with the ancient DNA protocol applied, this is therefore very convincing that the DNA detected in the samples is indeed from the Pleistocene mammals. Additionally, two methods were used to age the sediment cores, and although the depth of the samples tested do not overlap, they give reasonable ages (apart from the anomalous sample) and all together these are robust results.

Weaknesses:<br /> The paper could benefit from clearer aims in the introductions because as it stands the initial aim states that the authors are looking for Arctic mammal abundances through time. However, there are no results relating to general arctic mammal biodiversity presented, which leaves the reader wondering. Perhaps the focus of the study is more on identifying and dating the Pleistocene megafauna. Additionally, it is presented as an analysis on the two taxa, but it feels like the woolly rhinoceros does not receive the same treatment as the mammoth, as there are no additional molecular results, confirmation or figures relating to DNA from this taxa.

Overall the results support that there has been some movement of DNA throughout the sediment core which may impact the dating of the last occurrence of particular extinct taxa. As highlighted, though the geological processes by which this may have arisen are specific to this particular lake and may not be broadly relevant, therefore highlighting that knowledge of each system is important to understanding DNA distribution.

Reviewer #3 (Public Review):

Neininger-Castro and colleagues developed software tools for the quantification of sarcomeres and sarcomere-precursor features in immunostained human induced pluripotent stem cell-derived cardiac myocytes (hiCMs). In the first part they used a deep-learning- based model called a U-Net to construct and train a network for binarization of immunostained cardiomyocyte images. They also wrote graphical user interface (GUI) software that will assist other labs to use this approach and made it publicly available. They did not compare their approach to existing ones, but example from one image suggests their binarization tool outperforms Otsu thresholding binarization.

In the second part they developed a software tool called sarcApp that classifies sarcomere structures in the binarized image as a Z-Line or Z-Body and assigns each to either a myofibril or to stress fibers. The tools can then automatically count and measure multiple features (33 per cell and 24 per myofibril) and report them on a per-cell, per-myofibril, and per- stress fiber basis.

To test the tools they used Blebbistatin to inhibit sarcomere assembly and showed that the sarcApp tool could capture changes in multiple features such as fewer myofibrils, fewer Z-Lines, decreased myofibril persistence, decreased Z-Line length and altered myofibril orientation in the Blebbistatin treated cells. With some changes the tool was also shown to quantify sarcomeres in titin and myomesin stained cardiomyocytes.

Finally they used sarcApp to quantify the changes in sarcomere assembly after siRNA mediated knockout of MYH7, MYH7, or MYOM. The analysis indicates that neither MYH6 nor MYH7 knockdown perturbed the assembly of Z- or M-lines, and that knockdown of MYOM perturbed the A-band/M-Line but not the Z-Line assembly according to features captured by the sarcApp tool.

Overall the authors developed and made publicly available an excellent software tool that will be very useful for labs that are interested in studying sarcomere assembly. Multiple features that are difficult to measure or count manually can be automatically measured by the software quickly and accurately.

There are however some remaining questions about these tools:<br /> 1. The binarization tool which is tailored to sarcomere image binarization appears promising but was not systematically compared with existing approaches. Example from one cell suggests it outperforms Otsu's binarization approach.<br /> 2. How robust is the tool? The tool was tested on images from one type of cardiomyocytes (hiCMs) taken from one lab using Nikon Spinning Disk confocal microscope equipped with Apo TIRF Oil 100X 1.49 NA objective or instant Structured Illumination Microscopy (iSIM), using deconvolution (Microvolution software) and in a specific magnification. It remains to be seen whether the tool would be equally effective with images taken with other microscopy systems, with other cardiomyocytes (chick or neonatal rat), with different magnifications, live imaging, etc. The authors state that this approach is also useful in other situations, but the data is not included in this manuscript.<br /> 3. The tool was developed for evaluation of sarcomere assembly. The authors show that for this application it can detect the perturbation by Blebbistatin, or knockdown of sarcomeric genes. It remains to be seen if this tool is also useful for assessment of sarcomere structure for other questions beside sarcomere assembly and in other sarcomere pathologies.

Reviewer #3 (Public Review):

Summary:<br /> How does the brain distinguish stimulus intensity reduction from response reductions due to adaptation? Ling et al study whether and how the locust olfactory system encodes stimulus intensity and repetition differently. They show that these stimulus manipulations have distinguishable effects on population dynamics.

Strengths:<br /> 1. Provides a potential strategy with which the brain can distinguish intensity decrease from adaptation. -- while both conditions reduce overall spike counts, intensity decrease can also changes which neurons are activated and adaptation only changes the response magnitude without changing the active ensemble.<br /> 2. By interleaving a non-repeated odor, they show that these changes are odor-specific and not a non-specific effect.<br /> 3. Describes how proboscis orientation response (POR) changes with stimulus repetition., Unlike the spike counts, POR increases in probability with stimulus. The data portray the variability across subjects in a clear way.

Weaknesses:<br /> 1. Behavior<br /> a. While the "learning curve" of the POR is nicely described, the behavior itself receives very little description. What are the kinematics of the movement, and do these vary with repetition? Is the POR all-or-nothing or does it vary trial to trial?

b. What are the reaction times? This can constrain what time window is relevant in the neural responses. E.g., if the reaction time is 500 ms, then only the first 500 ms of the ensemble response deserves close scrutiny. Later spikes cannot contribute.

c. The behavioral methods are lacking some key information. While references are given to previous work, the reader should not be obligated to look at other papers to answer basic questions: how was the response measured? Video tracking? Hand scored?

d. Can we be sure that this is an odor response? Although airflow out of the olfactometer is ongoing throughout the experiment, opening and closing valves usually creates pressure jumps that are likely to activate mechanosensors in the antennae.

e. What is the baseline rate of PORs in the absence of stimuli?

e.What can you say about the purpose of the POR? I lack an intuition for why a fly would wiggle the maxillary palps. This is a question that is probably impossible to answer definitively, but even a speculative explanation would help the reader better understand.

2. Physiology<br /> a. Does stimulus repetition affect "spontaneous" activity (i.e., firing in the interstimulus interval? To study this question, in Figures 2b and c, it would be valuable to display more of the pre-stimulus period, and a quantification of the stability or lability of the inter-stimulus activity.

b. When does the response change stabilize? While the authors compare repetition 1 to repetition 25, from the rasters it appears that the changes have largely stabilized after the 3rd or 4th repetition. In Figure 5, there is a clear difference between repetition 1-3 or so and the rest. Are successive repetitions more similar than more temporally-separated repetitions (e.g., is rep 13 more similar to 14 than to 17?). I was not able to judge this based on the dendrograms of Figure 5. If the responses do stabilize at it appears, it would be more informative to focus on the dynamics of the first few repetitions.

c. How do temporal dynamics change? Locust PNs have richly varied temporal dynamics, but how these may be affected is not clear. The across-population average is poorly suited to capture this feature of the activity. For example, the PNs often have an early transient response, and these appear to be timed differently across the population. These structures will be obscured in a cross population average. Looking at the rasters, it looks like the initial transient changes its timing (e.g., PN40 responses move earlier; PN33 responses move later.). Quantification of latency to first spike after stimulus may make a useful measure of the dynamics.

d. How legitimate is the link between POR and physiology? While their changes can show a nice correlation, the fact the data were taken from separate animals makes them less compelling than they would be otherwise. How feasible is it to capture POR and physiology in the same prep? This would be most helpful, but I suspect may be too technically challenging to be within scope.

Reviewer #3 (Public Review):

A major finding of this work is that loss of monocarboxylate transporter 1 (MCT1), specifically in stellate cells, can decrease fibrosis in the liver. However, the underlying mechanism whereby MCT1 influences stellate cells is not addressed. It is unclear if upstream/downstream metabolic flux within different cell types leads to fibrotic outcomes. Ultimately, the paper opens more questions than it answers: why does decreasing MCT1 expression in hepatocytes exacerbate disease, while silencing MCT1 in fibroblasts seems to alleviate collagen deposition? Mechanistic studies in isolated hepatocytes and stellate cells could enhance the work further to show the disparate pathways that mediate these opposing effects. The work highlights the complexity of cellular behavior and metabolism within a disease environment but does little to mechanistically explain it.

The observations presented are compelling and rigorous, but their impact is limited by the nearly complete lack of mechanistic insight presented in the manuscript. As also mentioned elsewhere, it is important to know whether lactate import or export (or the transport of another molecule-like ketone bodies, for example) is the decisive role of MCT1 for this phenotype. Beyond that, it would be interesting, albeit more difficult, to determine how that metabolic change leads to these fibrotic effects.

Kuppfer cells are initially analyzed and targeted. These cells may play a major role in fibrotic response. It will be interesting to determine the effects of lactate metabolism in other cells within the microenvironment, like Kuppfer cells, to gain a complete understanding of how metabolism is altered during fibrotic change.

The timing of MCT1 depletion raises concern, as this is a largely prophylactic experiment, and it remains unclear if altering MCT1 would aid in the regression of established fibrosis. Given the proposal for translation to clinical practice, this will be an important question to answer.

Reviewer #3 (Public Review):

The authors provide a detailed analysis of the sulcal and sutural imprints preserved on the natural endocast and associated cranial vault fragments of the KNM-ER3732 early Homo specimen. The analyses indicate a primitive ape-like organization of this specimen's frontal cortex. Given the geological age of around 1.9 million years, this is the earliest well-documented evidence of a primitive brain organization in African Homo.

The various points raised by the reviewers and the responses provided by the authors illustrate that paleoneurology is a research field where little consensus has been reached over the past century. This is due not only to the fragmentary preservation of most fossil endocasts, but also to the limitations of scientific inference in general, and paleoneurological inference in particular. Like any scientific hypothesis, a paleoneurological hypothesis cannot be proven, but at best be falsified, leaving a wide field of possible alternative hypotheses. Furthermore, endocranial morphology does not equate cerebral morphology. A classical example: the endocranial Broca cap is not identical to the cortical Broca area. And last but not least, taxonomy cannot resolve questions of phylogeny.

Reviewer #3 (Public Review):

Summary:

The authors discovered that the RdnE effector possesses DNase activity, and in competition, P. mirabilis having RdnE outcompetes the null strain. Additionally, they presented evidence that the RdnI immunity protein binds to RdnE, suppressing its toxicity. Interestingly, the authors demonstrated that the RdnI homolog from a different phylum (i.e., Actinomycetota) provides cross-species protection against RdnE injected from P. mirabilis, despite the limited identity between the immunity sequences. Finally, using metagenomic data from human-associated microbiomes, the authors provided bioinformatic evidence that the rdnE/rdnI gene pair is widespread and present in individual microbiomes. Overall, the discovery of broad protection by non-cognate immunity is intriguing, although not necessarily surprising in retrospect, considering the prolonged period during which Earth was a microbial battlefield/paradise.

Strengths:

The authors presented a strong rationale in the manuscript and characterized the molecular mechanism of the RdnE effector both in vitro and in the heterologous expression model. The utilization of the bacterial two-hybrid system, along with the competition assays, to study the protective action of RdnI immunity is informative. Furthermore, the authors conducted bioinformatic analyses throughout the manuscript, examining the primary sequence, predicted structural, and metagenomic levels, which significantly underscore the significance and importance of the EI pair.

Weaknesses:

1. The interaction between RdnI and RdnE appears to be complex and requires further investigation. The manuscript's data does not conclusively explain how RdnI provides a "promiscuous" immunity function, particularly concerning the RdnI mutant/chimera derivatives. The lack of protection observed in these cases might be attributed to other factors, such as a decrease in protein expression levels or misfolding of the proteins. Additionally, the transient nature of the binding interaction could be insufficient to offer effective defenses.

2. The results from the mixed population competition lack quantitative analysis. The swarm competition assays only yield binary outcomes (Yes or No), limiting the ability to obtain more detailed insights from the data.

3. The discovery of cross-species protection is solely evident in the heterologous expression-competition model. It remains uncertain whether this is an isolated occurrence or a common characteristic of RdnI immunity proteins across various scenarios. Further investigations are necessary to determine the generality of this behavior.

Comments from Reviewing Editor:

• In addition to the references provided by Reviewer #2, the first manuscript to show non-cognate binding of immunity proteins was Russell et al 2012 (PMID: 22607806).

• IdrD was shown to form a subfamily of effectors in this manuscript by Hespanhol et al 2022 PMID: 36226828 that analyzed several T6SS effectors belonging to PDDExK, and it should be cited.

Reviewer #3 (Public Review):

Summary: The authors present a thought-provoking and comprehensive re-analysis of previously published human cell genomics data that seeks to understand the relationship between the sites where the Origin Recognition Complex (ORC) binds chromatin, where the replicative helicase (Mcm2-7) is situated on chromatin, and where DNA replication actually beings (origins). The view that these should coincide is influenced by studies in yeast where ORC binds site-specifically to dedicated nucleosome-free origins where Mcm2-7 can be loaded and remains stably positioned for subsequent replication initiation. However, this is most certainly not the case in metazoans where it has already been reported that chromatin bindings sites of ORC, Mcm2-7, and origins do not necessarily overlap, likely because ORC loads the helicase in transcriptionally active regions of the genome and, since Mcm2-7 retains linear mobility (i.e., it can slide), it is displaced from its original position by other chromatin-contextualized processes (for example, see Gros et al., 2015 Mol Cell, Powell et al., 2015 EMBO J, Miotto et al., 2016 PNAS, and Prioleau et al., 2016 G&D amongst others). This study reaches a very similar conclusion: in short, they find a high degree of discordance between ORC, Mcm2-7, and origin positions in human cells.

Strengths: The strength of this work is its comprehensive and unbiased analysis of all relevant genomics datasets. To my knowledge, this is the first attempt to integrate these observations and the analyses employed were suited for the questions under consideration.

Weaknesses: The major weakness of this paper is that this comprehensive view failed to move the field forward from what was already known. Further, a substantial body of relevant prior genomics literature on the subject was neither cited nor discussed. This omission is important given that this group reaches very similar conclusions as studies published a number of years ago. Further, their study seems to present a unique opportunity to evaluate and shape our confidence in the different genomics techniques compared in this study. This, however, was also not discussed.

Reviewer #3 (Public Review):

This is an interesting work reporting ferroptosis that is involved in the tooth morphogenesis. The authors showed that Gpx4, the core anti-lipid peroxidation enzyme in ferroptosis, is upregulated in tooth development using ex vivo culture system.

Reviewer #3 (Public Review):

Summary: In this study, Peterson et al. longitudinally record and document the vocal repertoires of three Mongolian gerbil families. Using unsupervised learning techniques, they map the variability across these groups, finding that while overall statistics of, e.g., vocal emission rates and bout lengths are similar, families differed markedly in their distributions of syllable types and the transitions between these types within bouts. In addition, the large and rich data are likely to be valuable to others in the field.

Strengths:<br /> - Extensive data collection across multiple days in multiple family groups.<br /> - Thoughtful application of modern analysis techniques for analyzing vocal repertoires.<br /> - Careful examination of the statistical structure of vocal behavior, with indications that these gerbils, like naked mole rats, may differ in repertoire across families.

Weaknesses:<br /> - The work is largely descriptive, documenting behavior rather than testing a specific hypothesis.<br /> - The number of families (N=3) is somewhat limited.

Reviewer #3 (Public Review):

In this study, Zhu and authors investigate the expression and function of the clustered Protocadherins (cPcdhs) in synaptic connectivity in the mouse cortex. The cPcdhs encode a large family of cadherin-related transmembrane molecules hypothesized to regulate synaptic specificity through combinatorial expression and homophilic binding between neurons expressing matching cPcdh isoforms. But the evidence for combinatorial expression has been limited to a few cell types, and causal functions between cPcdh diversity and wiring specificity have been difficult to test experimentally. This study addresses two important but technically challenging questions in the mouse cortex: 1) Do single neurons in the cortex express different cPcdh isoform combinations? and 2) Does Pcdh isoform diversity or particular combinations among pyramidal neurons influence their connectivity patterns? Focusing on the Pcdh-gamma subcluster of 22 isoforms, the group performed 5'end-directed single-cell RNA sequencing from dissociated postnatal (P11) cortex. To address the functional role of Pcdhg diversity in cortical connectivity, they asked whether the Pcdhgs and isoform matching influence the likelihood of synaptic pairing between 2 nearby pyramidal neurons. They performed simultaneous whole-cell recordings of 6 pyramidal neurons in cortical slices, and measured paired connections by evoked monosynaptic responses. In these experiments, they measured synaptic connectivity between pyramidal neurons lacking the Pcdhgs, or overexpressing dissimilar or matching sets of Pcdhg isoforms introduced by electroporation of plasmids encoding Pcdhg cDNAs.

Overall, the study applies elegant methods that demonstrate that single cortical neurons express different combinations of Pcdh-gamma isoforms, including the upper layer Pyramidal cells that are assayed in paired recordings. The electrophysiology data demonstrate that nearby Pyramidal neurons lacking the entire Pcdhg cluster are more likely to be synaptically connected compared to the control neurons, and that overexpression of matching isoforms between pairs decreases the likelihood to be synaptically connected. These are important and compelling findings that advance the idea that the Pcdhgs are important for cortical synaptic connectivity, and that the repertoire of isoforms expressed by neurons influence their connectivity patterns potentially through a self/non-self discrimination mechanism. However, the findings are limited to probability in connectivity and do they do not support the authors' conclusions that Pcdhg isoforms regulate synaptic specificity, 'by preventing synapse formation with specific cells' or to 'unwanted partners'. Characterizations of the cellular basis of these defects are needed to determine whether they are secondary to other roles in cell positioning, axon/dendrite branching and synaptic pruning, and overall synaptic formation. Claims that Pcdh-alpha and Pcdhg C-type isoforms are not functionally required are premature, due to limitations of the experiments. Moreover, claims that 'similarity level of γ-PCDH isoforms between neurons regulate the synaptic formation' are not supported due to weak statistical analyses presented in Fig4. The overstatements should be corrected. There was also missed opportunity to clearly discuss these results in the context of other published work, including recent publications focused on the cortex.

Strengths:

- The 5' end sequencing with a Pcdhg-amplified library is a technical feat and addresses the pitfall of conventional scRNA-Seq methods due to the identical 3'sequences shared by all Pcdhg isoform and the low abundance of the variable exons. New figures with annotated cell types confirm that several pyramidal and inhibitory cortical subpopulations were captured.

-Statistical assessment of co-occurrence of isoform expression within clusters is also a strength.

- By establishing the combinatorial expression of Pcdhgs by maturing pyramidal cells, the study further substantiates the 'single neuron combinatorial code for cPcdhs' model. Although combinatorial expression is not universal (ie. serotonergic neurons), there was limited evidence. The findings that individual pyramidal neurons express ~1-3 variable Pcdhg transcripts plus the C-type transcripts aligns with single RT-PCR studies of single Purkinje cells (Esumi et al 2005; Toyoda et al 2014). They differ from the findings by Lv et al 2022, where C-type expression was lower among pyramidal neurons. OSNs also do not substantially express C-type isoforms (Mountoufaris et al 2017; Kiefer et al 2023). Differences, and the advantages of the 5'end -directed sequencing (vs. SmartSeq) could be raised in the discussion.

- Simultaneous whole-cell recordings and pairwise comparisons of pyramidal neurons is a technically outstanding approach. They assess the effects of Pcdhg OE isoform on the probability of paired connections.

- The connectivity assay between nearby pairs proved to be sensitive to quantify differences in probability in Pcdhg-cKO and overexpression mutants. The comparisons of connectivity across vertical vs lateral arrangement are also strengths. Overexpressing identical Pcdhg isoform (whether 1 or 6) reduces the probability of connectivity, but there are caveats to the interpretations (see below).

Weaknesses:

-The experiments support a role for the Pcdhgs in influencing the probability of synaptic connectivity between nearby pairs but are not sufficient evidence for synapse specificity. The cPcdhs play multiple roles in neurite arborization, synaptic density, and cell positioning. Kostadinov 2015 also showed that starburst cells lacking the Pcdhgs maintained increased % connectivity at maturity, suggesting a lack of refinement in the absence of Pcdhgs. The known roles raise questions on how these manipulations might have primary effects in these processes and then subsequently impact the probability of connectivity. Investigations of morphological aspects of pyramidal development would strengthen the study and potentially refine the findings. The authors should more clearly relate their findings to the body of cPcdh studies in the discussion.

- Pcdhg cKO-dependent effects on connectivity occur between closely spaced soma (50-100um - Figure 2E), highlighting the importance of spatial arrangement to connectivity (also noted by Tarusawa 2016). Was distance considered for the overexpression (OE) assays, and did the authors note changes in cell distribution which might diminish the connectivity? Recent work by Lv et al 2022 reported that manipulating Pcdhgs influences the dispersion of clonally-related pyramidal neurons, which also impacts the likelihood of connections. Overexpression of Pcdhgc3 increased cell dispersion and decreased the rate of connectivity between pairs. Though these papers are mentioned, they should be discussed in more detail and related to this work.

- Though the authors added suggested citations and improved the contextualization of the study, several statements do not accurately represent the cited literature. It is at the expense of crystalizing the novelty and importance of this present work. For instance, Garrett et al 2012 PMID: 22542181 was the first to describe roles for Pcdhgs in cortical pyramidal cells and dendrite arborization, and that pyramidal cell migration and survival are intact. Line 52 cited Wang et al 2002, but this was limited to gross inspection. Garrett et al is the correct citation for: 'The absence of γ-PCDH does not cause general abnormality in the development of the cerebral cortex, such as cell differentiation, migration, and survival (Wang et al., 2002).' Second, single cell cPcdh diversity is introduced very generally, as though all neuron types are expected to show combinatorial variable expression with ubiquitous C-Type expression. But those initial studies were limited to Purkinje cells (Esumi 2005 and Toyoda 2014). Profiling of serotonergic neurons and OSN reveals different patterns (citations needed for Chen 2017 PMID: 28450636; Mountofaris et al PMID: 2845063; Canzio 2023 PMID: 37347873), raising the idea that cPcdh diversity and ubiquitous C-type expression is not universal. Thus, the authors missed the opportunity to emphasize the gap regarding cPcdh diversity in the cortex.

- They have not shown rigorously and statistically that the rate of connectivity changes with% isoform matching. In Figure 4D, comparisons of % isoform matching in OE assays show a single statistical comparison between the control and 100% groups, but not between the 0%, 11% and 33% groups. Is there a significant difference between the other groups? Significant differences are claimed in the results section, but statistical tests are not provided. The regression analysis in 4E suggests a correlation between % isoform similarity and connectivity probability, but this is not sound as it is based on a mere 4 data points from 4D. The authors previously explained that they cannot evaluate the variance in these recordings as they must pool data together. However, there should be some treatment of variability, especially given the low baseline rate of connectivity. Or at the very least, they should acknowledge the limitations that prevent them from assessing this relationship. Claims in lines 230+ are not supported: ' Overall, our findings demonstrate a negative correlation between the probability of forming synaptic connections and the similarity level of γ-PCDH isoforms expressed in neuron pairs (Fig. 4E)".

-Figure 4 provides connectivity probability, but this result might be affected by overall synapse density. Did connection probability change with directionality (e.g between red to green cells, or green to red cells).

-Generally, the statistical approaches were not sufficiently described in the methods nor in the figure legends, making it difficult to assess the findings. They do not report on how they calculated FDR for connectivity data, when this is typically used for larger multivariate datasets.

- The possibility that the OE effects are driven by total Pcdhg levels, rather isoform matching, should be examined. As shown by qRT-PCR in Fig. 3, expression of individual isoforms can vary. It is reasonable that protein levels cannot be measured by IHC, although epitope tags could be considered as C-terminal tagging of cPcdhs preserves the function in mice (see Lefebvre 2008). Quantification of constant Pcdhg RNA levels by qRT-PCR or sc-RT-PCR would directly address the potential caveat that OE levels vary with isoform combinations.

-A caveat for the relative plasmid expression quantifications in Figure 3-S1 is that IHC was used to amplify the RFP-tagged isoform, and thus does not likely preserve the relationship between quantities and detection.

-Figure 1 didn't change in response to reviews to improve clarity. New panels relating to the scRNA-Seq analyses were added to supplementary data but many are central and should be included in Figure 1 (ie. S1-Fig6D). In the Results, the authors state that neuronal subpopulations generally show a combinatorial expression of some variable RNA isoforms and near ubiquitous C-type expression. But they only show data for the Layer 2/3 neuron-specific cluster in S1-Fig-6D, and so it is not clear if this pattern applies to other clusters. Fig. S1-5 show a low number of expressed isoforms per cell, but specific descriptions on whether these include C-type isoforms would be helpful. Figure 1F showing isoform profile in all neurons is not particularly meaningful. There is a lot of interest in neuron-type specific differences in cPcdh diversity, and the authors could highlight their data from S1-5 accordingly.

-The concept of co-occurrence and results should be explained within the results section, to more clearly relate this concept to data and interpretations. Explanations are now found in the methods, but this did not improve the clarity of this otherwise very interesting aspect of the study.

- The claim that C-type Pcdhgs do not functionally influence connectivity is premature. Tests were limited to PcdhgC4, which has unique properties compared to the other 2 C-type isoforms (Garrett et al 2019 PMID: 31877124; Mancia et al PMID: 36778455). The text should be corrected to limit the conclusion to PcdhgC4, and not generally to C-type. The authors should test PcdhgC3 and PcdhgC5 isoforms.

-The group generated a novel conditional Pcdh-alpha mouse allele using CRISPR methods, and state that there were no changes in synaptic connectivity in these Pcdh-alpha mutants. But this claim is premature. The Southern blots validate the targeting of the allele. But further validations are required to establish that this floxed allele can be efficiently recombined, disrupting Pcdha protein levels and function. Pcdha alleles have been validated by western blots and by demonstration of the prominent serotonergic axonal phenotype of Pcdha-KO (ie. Chen 2017 PMID: 28450636; Ing-Esteves 2018 PMID: 29439167).

-The Discussion would be strengthened by a deeper discussion of the findings to other cPcdh roles and studies, and of the limitations of the study. The idea that the Pcdhgs are influencing the rate of connectivity through a repulsion mechanism or synaptic formation (ie through negative interactions with synaptic organizers such as Nlgn - Molumby 2018, Steffen 2022) could be presented in a model, and supported by other literature.

Reviewer #3 (Public Review):

This study uses a range of methods to characterize heterogeneous neural populations within the nucleus incertus (NI). The authors focus on two major populations, expressing gsc2 and rln3a, and present solid evidence that these cells have different patterns of efferent and afferent connectivity, calcium activity and function in control of behavior. Although the study does not go as far as clarifying the role of NI in any specific neural computation or aspect of behavioral control, the findings will be valuable in support of future endeavors to do so. In particular, the authors have made two beautiful knock-in lines that recapitulate endogenous expression pattern of gsc2 and rln3a which will be a powerful tool to study the roles of the relevant NI cells. Experiments are well done and data are high quality and most claims are well supported. However, there are a few issues, detailed below, where I believe additional analysis could strengthen the paper.

• The data very clearly show different patterns of neurites for gsc2 and rln3a neurons in the IPN and the authors interpret these are being axonal arbors. However, can they rule out that some arbors might be dendritic in nature? Notably, they cite the recent Portugues lab study that confirmed that, as in other species, tegmental neurons in zebrafish extend spatially segregated axonal and dendritic arbors into IPN, and the authors speculate that these GABAergic cells might in fact be part of NI.

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.

Weaknesses:<br /> 1) The authors fail to utilize the proper controls throughout the manuscript in regard to the shWRNIP1, WT, and mutant cell lines. It is unclear why the authors failed to use the shWRNIP1WT line in the comet assay, DNA fiber assay, and the FANCD2 assays. This is a key control for i) the use of only a single shRNA (most studies will use at least 2 different shRNAs) and ii) the use of the mutant WRNIP1 lines. In several figures, the authors only show the effect of the UBZ mutant, but don't include the ATPase mutant or WT for comparison. Including these is essential.

2) The authors use the S9.6 antibody to conclude the loss of WRNIP1 causes more R-loops; however, it has been shown that this antibody detects dsRNA in addition to RNA-DNA hybrids. Accordingly, it cannot be ruled out that the increased S9.6 signal is due to increased dsRNA.

3) Multiple pieces of data do not support the conclusions. For example, Figure 1D shows shWRNIP1 to reduce damage in Aph+DRB cells compared to MRC5SV cells with Aph+DRB. This result suggests that WRNIP1 actually increases DNA damage in stressed cells with transcription blocked. Another result is seen in Figure 4a, where the number of PLA spots (presumably TRCs) increases in the shWRNIP1WT cells with Aph+RNH1 compared to Aph alone. If R-loops are required for TRC accumulation, then the RNH1 should decrease the PLA foci. This result instead suggests that WRNIP leads to increased TRCs in stressed cells with R-loops cleared by RNH1.

4) The data are mostly phenomenological and fail to yield mechanistic insight. For example, the authors state that "it remains unclear whether WRNIP1 is directly involved in the mechanisms of R-loop removal/resolution". Unfortunately, the data presented in this manuscript do not provide new insights into this unresolved question.

5) The authors only show merged images making it impossible to visualize differences in PLA foci.

Reviewer #3 (Public Review):

In this manuscript, Davidsen and collaborators introduce jAspSnFR3, a new version of aspartate biosensor derived from iGluSnFR3, that allows monitoring in real-time aspartate levels in cultured cells. A selective amino acids substitution was applied in a key region of the template to switch its specificity from glutamate to aspartate. The jAspSnFR3 does not respond to other tested metabolites and performs well, is not toxic for cultured cells, and is not affected by temperature ensuring the possibility of using this tool in tissues physiologically more relevant. The high affinity for aspartate (KD=50 uM) allowed the authors to measure fluctuations of this amino acid in the physiological range. Different strategies were used to bring aspartate to the minimal level. Finally, the authors used jAspSnFR3 to estimate the intracellular aspartate concentration. One of the highlights of the manuscript was a treatment with asparagine during glutamine starvation. Although didn't corroborate the essentiality of asparagine in glutamine depletion, the measurement of aspartate during this supplementation is a glimpse of how useful this sensor can be.

Reviewer #3 (Public Review):

Summary:<br /> The overarching goal of the authors was to understand whether emotional information conveyed through point-light biological motion can trigger automatic physiological responses, as reflected in pupil size.

Strengths:<br /> This manuscript has several noticeable strengths: it addresses an intriguing research question that fills that gap in existing literature, presents a clear and accurate presentation of the current literature, and conducts a series of experiments and control experiments with adequate sample size. Yet, it also entails several noticeable limitations - especially in the study design and statistical analyses.

Weaknesses:<br /> 1. Study design:<br /> 1.1 Dependent variable:<br /> Emotional attention is known to modulate both microsaccades and pupil size. Given the existing pupillometry data that the authors have collected, it would be both possible and valuable to determine whether the rate of microsaccades is also influenced by emotional biological motion.

1.2 Stimuli:<br /> It appears that the speed of the emotional biological motion stimuli mimics the natural pace of the emotional walker. What is the average velocity of the biological motion stimuli for each condition?

When the authors used inverted biological motion stimuli, they didn't observe any modulation in pupil size. Could there be a difference in microsaccades when comparing inverted emotional biological motion stimuli?

2. Statistical analyses<br /> 2.1 Multiple comparisons:<br /> There are many posthoc comparisons throughout the manuscript. The authors should consider correction for multiple comparisons. Take Experiment 1 for example, it is important to note that the happy over neutral BM effect and the sad over neutral BM effect are no longer significant after Bonferroni correction, which is worth noting.

2.2 The authors present the correlation between happy over sad dilation effect and the autistic traits in Experiment 1, but do not report such correlations in Experiments 2-4. Did the authors collect the Autistic Quotient measure in Experiments 2-4? It would be informative if the authors could demonstrate the reproducibility (or lack thereof) of this happy-sad index in Experiments 2-4.

2.3 The observed correlation between happy over sad dilation effect and the autistic traits in Experiment 1 seems rather weak. It could be attributed to the poor reliability of the Autistic Quotient measure or the author-constructed happy-sad index. Did the authors examine the test-retest reliability of their tasks or the Autistic Quotient measure?

2.4 Relatedly, the happy over sad dilation effect is essentially a subtraction index. Without separately presenting the pipul size correlation with happy and sad BM in supplemental figures, it becomes challenging to understand what's primarily driving the observed correlation.

2.5 For the sake of transparency, it is important to report all findings, not just the positive results, throughout the paper.

3. Structure<br /> 3.1 The Results section immediately proceeds to the one-way repeated measures ANOVA. This section could be more reader-friendly by including a brief overview of the task procedures and variables, e.g., shifting Fig. 3 to this section.

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 the entorhinal cortex. The current paper seems quite similar conceptually and in design to previous work, most notably by Park et al., 2021, Nature Neuroscience.

Below, I raise a few issues and questions on the evidence presented here for a grid-like code as the basis of navigating abstract social space or social knowledge.

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 raising the issue for future work to address the problem - or if the authors think it is a problem at all.

Data and analysis

2. Concerning the negative correlation of distance with activation in the fusiform gyrus and visual cortex: this is a slightly puzzling but potentially interesting finding. However, could this be related to reaction times? The larger the distance, the longer the reaction times, so the original finding might reflect larger activations with smaller distances.

3. Concerning the correlation of grid-like activity with behavior: is the correlation with reaction time just about how long people took (rather than a task-related neural signal)? The authors have only reported correlations with reaction time. The issue here is that the duration of reaction times also relates to the starting positions of each trial and where participants will navigate to. Considering the speed-accuracy tradeoff, could performance accuracy be negatively correlated with these grid consistency metrics? Or it could be positively correlated, which would suggest the grid signal reflects a good representation of the task.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors report the first evidence of Nav1.5 regulation by a long noncoding RNA, LncRNA-DACH1, and suggest its implication in the reduction in sodium current observed in heart failure. Since no direct interaction is observed between Nav1.5 and the LncRNA, they propose that the regulation is via dystrophin and targeting of Nav1.5 to the plasma membrane.

Strengths:

1. First evidence of Nav1.5 regulation by a long noncoding RNA.<br /> 2. Implication of LncRNA-DACH1 in heart failure and mechanisms of arrhythmias.<br /> 3. Demonstration of LncRNA-DACH1 binding to dystrophin.<br /> 4. Potential rescuing of dystrophin and Nav1.5 strategy.

Weaknesses:

1. Main concern is that the authors do not provide evidence of how LncRNA-DACH1 regulates Nav1.5 protein level. The decrease in total Nav1.5 protein by about 50% seems to be the main consequence of the LncRNA on Nav1.5, but no mechanistic information is provided as to how this occurs.<br /> 2. The fact that the total Nav1.5 protein is reduced by 50% which is similar to the reduction in the membrane reduction questions the main conclusion of the authors implicating dystrophin in the reduced Nav1.5 targeting. The reduction in membrane Nav1.5 could simply be due to the reduction in total protein.

Reviewer #3 (Public Review):

Summary:

The receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) in humans is nervous system expressed and plays an important role as an oncogene. A number of groups have been signalling ALK signalling in flies to gain mechanistic insight into its various role. In flies, ALK plays a critical role in development, particularly embryonic development and axon targeting. In addition, ALK also was also shown to regulate adult functions including sleep and memory. In this manuscript, Sukumar et al., used a suite of molecular techniques to identify downstream targets of ALK signalling. They first used targeted DamID, a technique that involves a DNA methylase to RNA polymerase II, so that GATC sites in close proximity to PolII binding sites are marked. They performed these experiments in wild-type and ALK loss of function mutants (using an Alk dominant negative ALkDN), to identify Alk responsive loci. Comparing these loci with a larval single-cell RNAseq dataset identified neuroendocrine cells as an important site of Alk action. They further combined these TaDa hits with data from RNA seq in Alk Loss and Gain of Function manipulations to identify a single novel target of Alk signalling - a neuropeptide precursor they named Sparkly (Spar) for its expression pattern. They generated a mutant allele of Spar, raised an antibody against Spar, and characterised its expression pattern and mutant behavioural phenotypes including defects in sleep and circadian function.

Strengths:

The molecular biology experiments using TaDa and RNAseq were elegant and very convincing. The authors identified a novel gene they named Spar. They also generated a mutant allele of Spar (using CrisprCas technology) and raised an antibody against Spar. These experiments are lovely, and the reagents will be useful to the community. The paper is also well written, and the figures are very nicely laid out making the manuscript a pleasure to read.

Weaknesses:

My main concerns were around the genetics and behavioural characterisation which is incomplete. The authors generated a novel allele of Spar - Spar ΔExon1 and examined sleep and circadian phenotypes of this allele. However, they have only one mutant allele of Spar, and it doesn't appear as if this mutant was outcrossed, making it very difficult to rule out off-target effects. To make this data convincing, it would be better if the authors had a second allele, perhaps they could try RNAi?

Further, the sleep and circadian characterisation could be substantially improved. In Fig 8 E-F it appears as if sleep was averaged over 30 days! This is a little bizarre. They then bin the data as day 1 - 12 and 12-30. This is not terribly helpful either. Sleep in flies, as in humans, undergoes ontogenetic changes - sleep is high in young flies, stabilises between day 3-12, and shows defects by around 3 weeks of age (cf Shaw et al., 2000 PMID 10710313). The standard in the sleep field is to average over 3 days or show one representative day. The authors should reanalyse their data as per this standard, and perhaps show data from 3-10 day old flies, and if they like from 20-30 day old flies. Further, sleep data is usually analysed and presented from lights on to lights on. This allows one to quantify important metrics of sleep consolidation including bout lengths in day and night, and sleep latency. These metrics are of great interest to the community and should be included.

The authors also claim there are defects in circadian anticipatory activity. However, these data, as presented are not solid to me. The standard in the field is to perform eduction analyses and quantify anticipatory activity e.g. using the method of Harrisingh et al. (PMID: 18003827). Further, circadian period could also be evaluated. There are several free software packages to perform these analyses so it should not be hard to do.

Reviewer #3 (Public Review):

A central step in cell division is the formation of midbody abscission that separates two daughter cells at the end of cytokinesis. The ESCRT, endosomal sorting complexes required for transport, plays a critical role in this process. Specifically, the ESCRT-III proteins are actively recruited throughout the cell at the membrane fission sites, and their oligomerization into filaments is necessary to constrict the cell membranes to the fission point. Fundamental structural elements in ESCRT-III interactome are the so-called MIT-interacting motifs (MIMs) located at the protein's C terminal portion. Recently, Sundquist and co-workers (eLife 2022) identified several cofactors interacting with ESCRT-III subunits directly implicated in abscission. Among those cofactors, they identified Calpain-7, a cysteine protease whose function is still unclear. Calpain-7 comprises two MIT domains that target ESCRT-II subunit IST1. Here, the authors use structural methods and cell assays to characterize the interactions between Calpain-7 and IST1. For the structural studies, they constructed a minimalistic system in which MT1 and MT2 domains of Calpain-7 interact with the two MIMs localized in the IST1 construct. The truncated constructs interact with high affinity, recapitulating the strength of interaction expected for the full-length constructs in the cell. Using fluorescence polarization anisotropy binding isotherms, these researchers obtained solid binding data, showing a dissociation constant of 0.09 uM for the construct containing both MIMs, ~2 uM for the second MIM domain, and 100 uM for the first MIM. These data suggest a synergistic binding mechanism between the two MIM domains. The authors expressed and purified these constructs in recombinant systems and obtained purified isotopically labeled proteins to study by NMR. To characterize the binding by NMR, the authors studied the IST1 constructs with the two MIMs in the absence and presence of Calpain-7. The IST1 construct displays a well-resolved NMR fingerprint, with most resonances assigned to specific residues. Upon addition of the Calpain-7 construct, the resonances of the residues involved in the binding either broaden beyond detection or shift significantly, which supports the fluorescence binding studies. Given the high affinity, these authors were able to crystallize these complexes and identify the binding interfaces that parallel the solution NMR studies. Mutational studies confirm the hot spots for the interactions, and the authors concluded that the MIT:MIM binding interface is responsible for the association of the full-length constructs of Calpain-7 and IST1 in the cell. Using localization experiments, the authors concluded that IST1 is responsible for recruiting Calpain-7 to midbodies, and the presence of both MIT domains of Calpain-7 and MIM domains is required for localization. Taken together, the biophysical characterization of these complexes and the cell assays led the authors to conclude that IST1 binding to Calpain-7 is necessary for its role in abscission and Nocut checkpoint maintenance.<br /> In my opinion, the research is well executed and also supported by their previous finding (see Sundquist 2022 eLife). The paper is succinct and well-written.

Reviewer #3 (Public Review):

The manuscript by Salloum et al., titled "Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating JMJD3" reports an extensive characterization of the mechanisms underlying the anti-inflammatory role of statins using different in vitro studies. Based on these approaches, the authors observed that cholesterol reduction in response to statin treatment alters mitochondrial function and they identify JMJD3 as a potential critical driver of macrophage anti-inflammatory phenotype. Overall, the study is interesting and provides new findings that could shed light on the molecular effects of statins in these cells, but a number of issues remain confusing, and the experimental design is, on some occasions, not rigorous enough to support the drawn conclusions.

Major issues:

1. Focus on JMJD3 is justified by the authors as it was among the 40 genes commonly up-regulated in macrophages exposed to statin or methyl--cyclodextrin (MCD) by RNA-Seq analysis. However, this analysis has not been presented in the manuscript and it is unclear what genes (apart from JMJD3) might play an important role in the response of these cells. A detailed characterization of both up- and down-regulated genes in these experimental conditions and a better justification for JMJD3 are required to fully support further analysis.<br /> 2. In the same line, Figures 6A and B fail to fully describe the changes found by ATAC-seq and RNA-seq. A more comprehensive analysis of these three datasets (together with previous RNA-seq studies) would help to obtain a better understanding of overlapping dysregulated genes (not only those found up-regulated) and what other epigenetic modifying factors might be involved.<br /> 3. In Figure 6C and Supplementary Figure 7, it would be noteworthy to also measure the gene expression of Kdm6a/UTX homolog Kdm6c/UTY, as it has been shown to lack demethylate H3K27me3 demethylase activity due to mutations in the catalytic site of the Jumomji-C-domain.<br /> 4. The use of rather unspecific treatments such as MG-132 (proteasome inhibitor) and GSKj4 (inhibitor of both JMJD3 and UTX) may distort the results observed and might elude their correct interpretation. To avoid this limitation, additional silencing and/or overexpression experiments are currently needed.<br /> 5. Figure 3 and Supplementary Figure 3 seem to be duplicated, please correct them. Moreover, for a better representation of these data, please include representative Seahorse profile figures of each experimental condition in these figures.<br /> 6. As stated by the authors, macrophage phenotype is much more complex than M1/M2 polarization. In this view, assessing a very limited set of genes (i.e, Il-1, IL-10, TNF, IL-6, IL-12, Arg1, Ym1, Mrc1) appears to be inappropriate. A meaningful number of markers must be added.<br /> 7. For accurate quantification of H3K27me3 global levels, please add immunoblotting against histone H3 in Supplementary Figure 1.

Reviewer #3 (Public Review):

The autocatalytic replication mechanism of misfolded Prion-like proteins (PrP) into amyloid aggregates is associated with a plethora of deleterious neurodegenerative diseases. Despite of the huge amount of research, the underlying molecular events of self-replication and identification of the toxic species are not fully understood. Many recent studies have indicated that non-fibrillar oligomeric intermediates could be more neurotoxic compared to the Prion fibrils. Various cellular factors, like the participation of other proteins and chaperone activity, also play an important role in PrP misfolding, aggregation, and neurotoxicity. The present work focuses on understanding the PrP aggregation mechanism with the identification of the associated toxic species and cellular factors. One of the significant strengths of the work is performing the aggregation assay in near-native conditions. In contrast, most in vitro studies use harsh conditions (such as high temperature, denaturant, detergent, low pH, etc.) to promote protein aggregation. The authors successfully observed the well-known seeding property of the PrP in this aggregation assay that bypasses the primary nucleation during aggregation. Moreover, the authors have shown that syntaxin 6 (Stx6), a known risk factor in prion-mediated Creutzfeldt-Jakob disease, delays fibril formation and prolongs the persistence of toxic intermediates, thus playing an anti-chaperone activity. This study will contribute to understanding the molecular mechanism of PrP aggregation and neurotoxicity. However, further studies are required to identify and characterize the toxic intermediate in the near future precisely.

Reviewer #3 (Public Review):

In this manuscript by Lu et al., the authors cloned TPC1 from Vicia faba (VfTPC1) and characterized its channel properties by patching the vacuoles isolated from VfRPC1 expressing TPC1-loss-of-function Arabidopsis mutant tpc1-2. They found that VfTPC1 displayed faster kinetics, higher voltage dependence, and less sensitivity to luminal calcium than its Arabidopsis orthologue (AtTPC1). Mutating three luminal residues (E457, E605 and D606) in AtTPC1 to the corresponding ones in VfTPC1 converted the channel into one that resembles VfTPC1: hyperactive and desensitized to luminal Ca2+. By constructing a VfTPC1 model based on the published Ca2+-bound AtTPC1-D454N (fou2) cryo-EM structures, the authors proposed a Ca2+-dependent interaction between the E605/D606 motif and a Ca2+ coordination site at the luminal entrance of the selectivity filter (D269/E637; in VfTPC1, D271/E639). Finally, they showed that vacuoles with VfTPC1 or AtTPC1- triple mutant were hyperexcitable. Overall, this is an interesting study that might have both evolutional and functional implications.

Reviewer #3 (Public Review):

The authors investigate the role of commensal microbes and molecules in the antigen presentation pathway in the development and phenotype of CD8 T cells specific for the Qa-1b-restricted peptide FL9 (QFL). The studies track both endogenous QFL-specific T cells and utilize a recently generated TCR transgenic model. The authors confirm that QFL-specific T cells in the spleen and small intestine intraepithelial lymphocyte (IEL) pool show an antigen-experienced phenotype as well as unique phenotypic and innate-like functional traits, especially among CD8+ T cells expressing Va3.2+ TCRs. They find that deficiency in the TAP transporter leads to almost complete loss of QFL-specific T cells but that loss of either Qa1 or the ERAAP aminopeptidase does not impact QFL+ T cell numbers but does cause them to maintain a more conventional, naïve-like phenotype. In germ-free (GF) mice, the QFL-specific T cells are present at similar numbers and with a similar phenotype to SPF animals, but in older animals (>18w) there is a notable loss of IEL QFL-specific cells. This drop can be avoided by neonatal colonization of GF mice with the commensal microbe Pediococcus pentosaceus but not a different commensal, Lactobacillus johnsonii, and the authors show that P. pentosaceus encodes a peptide that weakly stimulates QFL-specific T cells, while the homologous peptide from L. johnsonii does not stimulate such cells.

This study provides new insights into the way in which the differentiation, phenotype, and function of CD8+ T cells specific for Qa-1b/FL9 is regulated by peptide processing and Qa1 expression, and by interactions with the microbiota. The approaches are well designed, the data compelling, and the interpretation, for the most part, appropriate. There are a few relatively minor concerns.

1) For most of the report, the authors use a set of phenotypic traits to highlight the unique features of QFL-specific CD8+ T cells - specifically, CD44high, CD8aa+ve, CD8ab-ve. In Supp. Fig. 4, however, completely distinct phenotypic characteristics are presented, indicating that IEL QFL-specific T cells are CD5low, Thy-1low. No explanation is provided in the text about whether this is a previously reported phenotype, whether any elements of this phenotype are shared with splenic QFL T cells, what significance the authors ascribe to this phenotype (and to the fact that Qa1-deficiency leads to a more conventional Thy-1+ve, CD5+ve phenotype), and whether this altered phenotype is also seen in ERAAP-deficient mice. At least some explanation for this abrupt shift in focus and integration with prior published work is needed. On a related note, CD5 expression is measured in splenic QFL-specific CD8+ T cells from GF vs SPF mice (Supp. Fig. 9), to indicate that there is no phenotypic impact in the GF mice - but from Supp. Fig. 4, it would seem more appropriate to report CD5 expression in QFL-specific cells from the IEL, not the spleen.

2) The authors suggest the finding that QFL-specific cells from ERAAP-deficient mice have a more "conventional" phenotype indicates some form of negative selection of high-affinity clones (this result being somewhat unexpected since ERAAP loss was previously shown to increase the presentation of Qa-1b loaded with FL9, confirmed in this report). It is not clear how this argument aligns with the data presented, however, since the authors convincingly show no significant reduction in the number of QFL-specific cells in ERAAP-knockout mice (Fig. 3a), and their own data (e.g. Fig. 2a) do not suggest that CD44 expression correlates with QFL-multimer staining (as a surrogate for TCR affinity/avidity). Is there some experimental basis for suggesting that ERAAP-deficient lacks a subset of high-affinity QFL-specific cells?

3) The rationale for designing FL9 mutants, and for using these data to screen the proteomes of various commensal bacteria needs further explanation. The authors propose P4 and P6 of FL9 are likely to be "critical" but do not explain whether they predict these to be TCR or Qa-1b contact sites. Published data (e.g., PMID: 10974028) suggest that multiple residues contribute to Qa-1b binding, so while the authors find that P4A completely lost the ability to stimulate a QFL-specific hybridoma, it is unclear whether this is due to the loss of a TCR- or a Qa-1-contact site (or, possibly, both). This could easily be tested - e.g., by determining whether P4A can act as a competitive inhibitor for FL9-induced stimulation of BEko8Z (and, ideally, other Qa-1b-restricted cells, specific for distinct peptides). Without such information, it is unclear exactly what is being selected in the authors' screening strategy of commensal bacterial proteomes. This, of course, does not lessen the importance of finding the peptide from P. pentosaceus that can (albeit weakly) stimulate QFL-specific cells, and the finding that association with this microbe can sustain IEL QFL cells.

Reviewer #3 (Public Review):

In this study, Ruan et al. investigate the role of the IQCH gene in spermatogenesis, focusing on its interaction with calmodulin and its regulation of RNA-binding proteins. The authors examined sperm from a male infertility patient with an inherited IQCH mutation as well as IQCH CRISPR knockout mice. The authors found that both human and mouse sperm exhibited structural and morphogenetic defects in multiple structures, leading to reduced fertility in ICHQ-knockout male mice. Molecular analyses such as mass spectrometry and immunoprecipitation indicated that RNA-binding proteins are likely targets of IQCH, with the authors focusing on the RNA-binding protein HNRPAB as a critical regulator of testicular mRNAs. The authors used in vitro cell culture models to demonstrate an interaction between IQCH and calmodulin, in addition to showing that this interaction via the IQ motif of IQCH is required for IQCH's function in promoting HNRPAB expression. In sum, the authors concluded that IQCH promotes male fertility by binding to calmodulin and controlling HNRPAB expression to regulate the expression of essential mRNAs for spermatogenesis. These findings provide new insight into molecular mechanisms underlying spermatogenesis and how important factors for sperm morphogenesis and function are regulated.

The strengths of the study include the use of mouse and human samples, which demonstrate a likely relevance of the mouse model to humans; the use of multiple biochemical techniques to address the molecular mechanisms involved; the development of a new CRISPR mouse model; ample controls; and clearly displayed results. There are some minor weaknesses in that more background details could be provided to the reader regarding the proteins involved; some assays could benefit from more rigorous quantification; some of the mouse testis images and analyses could be improved; and larger sample sizes, especially for the male mouse breeding tests, could be increased. Overall, the claims made by the authors in this manuscript are well-supported by the data provided and there are only minor technical issues that could increase the robustness and rigor of the study.

1. More background details are needed regarding the proteins involved, in particular IQ proteins and calmodulin. The authors state that IQ proteins are not well-represented in the literature, but do not state how many IQ proteins are encoded in the genome. They also do not provide specifics regarding which calmodulins are involved, since there are at least 5 family members in mice and humans. This information could help provide more granular details about the mechanism to the reader and help place the findings in context.

2. The mouse fertility tests could be improved with more depth and rigor. There was no data regarding copulatory plug rate; data was unclear regarding how many WT females were used for the male breeding tests and how many litters were generated; the general methodology used for the breeding tests in the Methods section was not very explicitly or clearly described; the sample size of n=3 for the male breeding tests is rather small for that type of assay; and, given that ICHQ appears to be expressed in testicular interstitial cells (Fig. S10) and somewhat in other organs (Fig. S2), another important parameter of male fertility that should be addressed is reproductive hormone levels (e.g., LH, FSH, and testosterone).

3. The Western blots in Figure 6 should be rigorously quantified from multiple independent experiments so that there is stronger evidence supporting claims based on those assays.

4. Some of the mouse testis images could be improved. For example, the PNA and PLCz images in Figure S7 are difficult to interpret in that the tubules do not appear to be stage-matched, and since the authors claimed that testicular histology is unaffected in knockout testes, it should be feasible to stage-match control and knockout samples. Also, the anti-ICHQ and CaM immunofluorescence in Figure S10 would benefit from some cell-type-specific co-stains to more rigorously define their expression patterns, and they should also be stage-matched.

Reviewer #3 (Public Review):

Summary. This study sought to clarify the connection between inositol pyrophosphates (IPPs) and their regulation of phosphate homeostasis in the yeast Saccharomyces cerevisiae to answer the question of whether any of the IPPs (1-IP7, 5-IP7, and IP8) or only particular IPPs are involved in regulation. IPPs bind to SPX domains in proteins to affect their activity, and there are several key proteins in the PHO pathway that have an SPX domain, including Pho81. The authors use the latest methodology, capillary electrophoresis and mass spectrometry (CE-MS), to examine the cytosolic concentrations of PP-IPs in wild-type and strains carrying mutations in the enzymes that metabolize these compounds in rich medium and during a phosphate starvation time-course for the wild-type.

Major strengths and weaknesses. The authors have strong premises for performing these experiments: clarifying the regulatory molecule(s) in yeast and providing a unifying mechanism across eukaryotes. They use the latest methodologies and a variety of approaches including genetics, biochemistry, cell biology and protein structure to examine phosphate regulation. Their experiments are rigorous and well controlled, and the story is clearly told. The consideration of physiological levels of IPPs throughout the study was critical to interpretation of the data and a strength of the manuscript. The investigation of the structure of Pho81, its regulation by IPPs, and its interactions with Pho80 provide a vivid model for regulation.

Appraisal. The authors achieved their goal of determining the mechanistic details for phosphate regulation, revising the prior model with new insights. Additionally, they provided strong support for the idea that IP8 regulates phosphate metabolism across eukaryotes - including animals and plants in addition to fungi.

Impact. This study is likely to have broad impact because it addresses prior findings that are inconsistent with current understanding, and they provide good reasoning as to how older methods were inadequate.

Reviewer #3 (Public Review):

This study investigated cognitive mechanisms underlying approach-avoidance behavior using a novel reinforcement learning task and computational modelling. Participants could select a risky "conflict" option (latent, fluctuating probabilities of monetary reward and/or unpleasant sound [punishment]) or a safe option (separate, generally lower probability of reward). Overall, participant choices were skewed towards more rewarded options, but were also repelled by increasing probability of punishment. Individual patterns of behavior were well-captured by a reinforcement learning model that included parameters for reward and punishment sensitivity, and learning rates for reward and punishment. This is a nice replication of existing findings suggesting reward and punishment have opposing effects on behavior through dissociated sensitivity to reward versus punishment.

Interestingly, avoidance of the conflict option was predicted by self-reported task-induced anxiety. Importantly, when a subset of participants were retested over 1 week later, most behavioral tendencies and model parameters were recapitulated, suggesting the task may capture stable traits relevant to approach-avoidance decision-making.

The revised paper commendably adds important additional information and analyses to support these claims. The initial concern that not accounting for participant control over punisher intensity confounded interpretation of effects has been largely addressed in follow-up analyses and discussion.

This study complements and sits within a broad translational literature investigating interactions between reward/punishers and psychological processes in approach-avoidance decisions.

Reviewer #3 (Public Review):

The authors of this study have designed a novel screening pipeline to detect DNA motif spacing preferences between TF partners using publicly available data. They were able to recapitulate previously known composite elements, such as the AP-1/IRF4 composite elements (AICE) and predict many composite elements that are expected to be very useful to the community of researchers interested in dissecting the regulatory logic of mammalian enhancers and promoters. The authors then focus on a novel, SPICE predicted interaction between JUN and IKZF1, and show that under LPS and IL-21 treatment, JUN and IKZF1 in B cells have significant overlap in their genomic localization. Next, to know whether the two TFs physically interact, a co-immunoprecipitation experiment was performed. While JUN immunoprecipitated with an anti-IKZF1 antibody, curiously IKZF1 did not immunoprecipitate with an anti-JUN antibody. Finally, EMSA and luciferase experiments were performed to show that the two TFs bind cooperatively at an IL20 upstream probe.

Major strengths:<br /> 1. SPICE was able to recapitulate previously known composite elements, such as the AP-1/IRF4 composite elements (AICE).<br /> 2. Under LPS and IL-21 treatment, JUN and IKZF1 in B cells have significant overlap in their genomic localization. This is very good supporting evidence for the efficacy of SPICE in detecting TF partners.

Major weaknesses:<br /> 1. The authors fail to convincingly show that IKZF1 and Jun physically interact. A quantitative measurement of their interaction strength would have been ideal.<br /> 2. The super-shift experiment to show that the proteins bound to their EMSA probe were indeed IKZF1 and JUN are not very convincing and would benefit from efforts to quantify the shift (Figure 3E). Nuclear extracts from cells with single or double CRISPR knock outs of the two TFs would have been ideal.<br /> 3. There is a second band beneath the more prominent band in the EMSA experiment with recombinant IKZF1 and JUN (Figure 4C). This second band is most probably bound by IKZF1 because it becomes weaker when the IKZF1 site is mutated and is completely absent when only JUN is added. This is completely ignored by the authors. Therefore, experiments with EMSA fail to convincingly show that IKZF1 and Jun bind cooperatively. They could just as well bind independently to the two sites.

Reviewer #3 (Public Review):

Laham et al. present a manuscript investigating the function of adult-born granule cells (abGCs) projecting to the CA2 region of the hippocampus during social memory. It should be noted that no function for the general DG to CA2 projection has been proposed yet. The authors use targeted ablation, chemogenetic silencing, and in vivo ephys to demonstrate that the abGCs to CA2 projection is necessary for the retrieval of remote social memories such as the memory of one's mother. They also use in vivo ephys to show that abGCs are necessary for differential CA2 network activity, including theta-gamma coupling and sharp wave-ripples, in response to novel versus familiar social stimuli.

The question investigated is important since the function of DG to CA2 projection remained elusive a decade after its discovery. Overall, the results are interesting but focused on the social memory of the mother, and their description in the manuscript and figures is too cursory. For example, raw interaction times must be shown before their difference. The assumption that mice exhibit social preference between familiar or novel individuals such as mother and non-mother based on social memory formation, consolidation, and retrieval should be better explained throughout the manuscript. Thus, when describing the results, the authors should comment on changes in preference and how this can be interpreted as a change in social memory retrieval. Several critical experimental details such as the total time of presentation to the mother and non-mother stimulus mice are also lacking in the manuscript. The in vivo e-phys results are interesting as well but even more succinct with no proposed mechanism as to how abGCs could regulate SWR and PAC in CA2.

The manuscript is well-written with the appropriate references. The choice of the behavioral test is somewhat debatable, however. It is surprising that the authors chose to use a direct presentation test (presentation of the mother and non-mother in alternation) instead of the classical 3-chamber test which is particularly appropriate to investigate social preference. Since the authors focused exclusively on this preference, the 3-chamber test would have been more adequate in my opinion. It would greatly strengthen the results if the authors could repeat a key experiment from their investigation using such a test. In addition, the authors only impaired the mother's memory. An additional experiment showing that disruption of the abGCs to CA2 circuit impairs social memory retrieval would allow us to generalize the findings to social memories in general. As the manuscript stands, the authors can only conclude the importance of this circuit for the memory of the mother. Developmental memory implies the memory of familiar kin as well.

The in vivo ephys section (Figure 3) is interesting but even more minimalistic and it is unclear how abGCs projection to CA2 can contribute to SWR and theta-gamma PAC. In Figure 1, the authors suggest that abGCs project preferentially to PV+ neurons in CA2. At a minimum, the authors should discuss how the abGCs to PV+ neurons to CA2 pyramidal neurons circuit can facilitate SWR and theta-gamma PAC.

Finally, proposing a function for 4-6-week-old abGCs projecting to CA2 begs two questions: What are abGCs doing once they mature further, and more generally, what is the function of the DG to CA2 projection? It would be interesting for the authors to comment on these questions in the discussion.

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

The main limitation of the study is the very low number of samples analyzed, with only three replicate individuals per sex (i.e. the whole study is built on six individuals only). This provides low power to detect differential expression. Along the same line, only three species were used to evaluate the rates of non-synonymous to synonymous substitutions, which also represents a very limited dataset, in particular when trying to fit parameter-rich models such as those implemented here.

A third limitation relates to the absence of a reference genome for the species, making the use of a de novo transcriptome assembly necessary, which is likely to lead to a large number of incorrectly assembled transcripts. Of course, the production of a reference transcriptome in this non-model species is already a useful resource, but this point should at least be acknowledged somewhere in the manuscript.

Each of these shortcomings is relatively important, and together they strongly limit the scope of the conclusions that can be made, and they should at least be acknowledged more prominently. The study is valuable in spite of these limitations and the topic remains grossly understudied, so I think the study will be of interest to researchers in the field, and hopefully inspire further, more comprehensive analyses.

Reviewer #3 (Public Review):

More than 80 million people live at high altitude. This impacts health outcomes, including those related to pregnancy. Longer-lived populations at high altitudes, such as the Tibetan and Andean populations show partial protection against the negative health effects of high altitude. The paper by Yue sought to determine the mechanisms by which the placenta of Tibetans may have adapted to minimise the negative effect of high altitude on fetal growth outcomes. It compared placentas from pregnancies from Tibetans to those from the Han Chinese. It employed RNAseq profiling of different regions of the placenta and fetal membranes, with some follow-up of histological changes in umbilical cord structure and placental structure. The study also explored the contribution of fetal sex in these phenotypic outcomes.

A key strength of the study is the large sample sizes for the RNAseq analysis, the analysis of different parts of the placenta and fetal membranes, and the assessment of fetal sex differences.

A main weakness is that this study, and its conclusions, largely rely on transcriptomic changes informed by RNAseq. Changes in genes and pathways identified through bioinformatic analysis were not verified by alternate methods, such as by western blotting, which would add weight to the strength of the data and its interpretations. There is also a lack of description of patient characteristics, so the reader is unable to make their own judgments on how placental changes may link to pregnancy outcomes. Another weakness is that the histological analyses were performed on n=5 per group and were rudimentary in nature.

Reviewer #3 (Public Review):

The manuscript by Egan and coworkers investigates how Caspase-1 and Caspase-4 mediated cell death affects replication of Salmonella in human THP-1 macrophages in vitro.

Overall evaluation:

Strength of the study include the use of human cells, which exhibit notable differences (e.g., Caspase 11 vs Caspase-4/5) compared to commonly used murine models. Furthermore, the study combines inhibitors with host and bacterial genetics to elucidate mechanistic links.

The main weaknesses of the study are the inherent limitations of tissue culture models. For example, to study interaction of Salmonella with host cells in vitro, it is necessary to kill extracellular bacteria using gentamicin. However, since Salmonella-induced macrophage cell death damages the cytosolic membrane, gentamicin can reach intracellular bacteria and contribute to changes in CFU observed in tissue culture models (major point 1). This can result in tissue culture "artefacts" (i.e., observations/conclusions that cannot be recapitulated in vivo). For example, intracellular replication of Salmonella in murine macrophages requires T3SS-2 in vitro, but T3SS-2 is dispensable for replication in macrophages of the spleen in vivo (Grant et al., 2012).

Major comments:

In Figure 1: are increased CFU in WT vs CASP1-deficient THP-1 cells due to Caspase 1 restricting intracellular replication or due to Caspase-1 causing pore formation to allow gentamicin to enter the cytosol thereby restricting bacterial replication? The same question arises about Caspase-4 in Figure 2, where differences in CFU are observed only at 24h when differences in cell death also become apparent. The idea that gentamicin entering the cytosol through pores is responsible for controlling intracellular Salmonella replication is also consistent with the finding that GSDMD-mediated pore formation is required for restricting intracellular Salmonella replication (Figure 3). Similarly, the finding that inflammasome responses primarily control Salmonella replication in the cytosol could be explained by an intact SCV membrane protecting Salmonella from gentamicin (Figure 5).

Reviewer #3 (Public Review):

Strengths:

NanoPDLIM2, nanotechnologies that efficiently deliver lentivirus overcomes resistance to chemotherapy and anti-PD-1 immunotherapy. This is a new strategy for enhancing the efficiency of immune checkpoint inhibitors. This finding is important from a clinical translation perspective, but I have several minor concerns.

Weaknesses:

1. Please describe the mechanism of increased MHC class I and PD-L1 by PDLIM2.<br /> 2. Please describe the mechanism of decreased MDR1, nuclear RelA and STAT3 by PDLIM2.<br /> 3. Please determine whether PDLIM2 expression directly impacts immune cells (function and number)?<br /> 4. What is the efficiency of PDLIM2 delivery? Does delivery efficiency determine anti-tumor effect?<br /> 5. Authors used a non-immunogenic tumor model. Can you demonstrate the combination effect with PDLIM2 in immunogenic lung cancer models to determine whether the combination of PDLIM2 with anti-PD-1 Ab confers a synergistic effect without chemotherapy?<br /> 6. On page 11, % change can make one over-interpret data.<br /> 7. In Figure 5, what is the difference between 5A and 5D?<br /> 8. It is unclear whether PDLIM2 confers an additive or a synergistic effect with anti-PD-1/chemo.<br /> 9. Have the authors tested any toxicity in normal lungs?

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 sound and reasonable. This is an exciting new avenue for thinking about inference from genomic data. I have a few concerns about the presentation and then also questions about the use of empirical methylation data sets.

I think a more detailed description of demographic accuracy is warranted. For example, in L245 MSMC2 identifies the bottleneck (albeit smoothed) and only slightly overestimates recent size. In the same analysis the authors' approach with unknown mu infers a nonexistent population increase by an order of magnitude that is not mentioned.

Similarly, it seems problematic that (L556) the approach requiring estimation of site and region parameters (as would presumably be needed in most empirical systems like endangered nonmodel species mentioned in the introduction) does no better than using only SNPs. Overall, I think a more objective and perhaps quantitative comparison of approaches is warranted.

The authors simulate methylated markers at 2% (and in some places up to 20%). In many plant genomes a large proportion of cytosines are methylated (e.g. 70% in maize: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496265/). I don't know what % of these may be polymorphic, but this leads to an order of magnitude more methylated cytosines than there are SNPs. Couldn't this mean that any appreciable error in estimating methylation threatens to be of a similar order of magnitude to the SNP data? I would welcome the authors' thoughts here.

A few points of discussion about the biology of methylation might be worth including. For example, methylation can differ among cell types or cells within a tissue, yet sequencing approaches evaluate a pool of cells. This results in a reasonable fraction of sites having methylation rates not clearly 0 or 1. How does this variation affect the method? Similarly, while the authors cite literature about the stable inheritance of methylation, a sentence or so more about the time scale over which this occurs would be helpful. Finally, in some species methylated cytosines have mutation rates an order of magnitude higher than other nucleotides. The authors mention they assume independence, but how would violation of this assumption affect their inference?

Reviewer #3 (Public Review):

In this manuscript, Lewis et al. investigate the role of tetraspanins in the formation of discs - the key structure of vertebrate photoreceptors essential for light reception. Two tetraspanin proteins play a role in this process: PRPH2 and ROM1. The critical contribution of PRPH2 has been well established and loss of its function is not tolerated and results in gross anatomical pathology and degeneration in both mice and humans. However, the role of ROM1 is much less understood and has been considered somewhat redundant. This paper provides a definitive answer about the long-standing uncertainty regarding the contribution of ROM1 firmly establishing its role in outer segment morphogenesis. First, using an ingenious quantitative proteomic technique the authors show PRPH2 compensatory increase in ROM1 knockout explaining the redundancy of its function. Second, they uncover that despite this compensation, ROM1 is still needed, and its loss delays disc enclosure and results in the failure to form incisures. Third, the authors used a transgenic mouse model and show that deficits seen in ROM1 KO could be completely compensated by the overexpression of PRPH2. Finally, they analyzed yet another mouse model based on double manipulation with both ROM1 loss and expression of PRPH2 mutant unable to form dimerizing disulfide bonds further arguing that PRPH2-ROM1 interactions are not required for disc enclosure. To top it off the authors complement their in vivo studies by a series of biochemical assays done upon reconstitution of tetraspanins in transfected cultured cells as well as fractionations of native retinas. This report is timely, addresses significant questions in cell biology of photoreceptors, and pushes the field forward in a classical area of photoreceptor biology and mechanics of membrane structure as well. The manuscript is executed at the top level of technical standard, exceptionally well written, and does not leave much more to desire. It also pushes standards of the field- one such domain is the quantitative approach to analysis of the EM images which is notoriously open to alternative interpretations - yet this study does an exceptional job unbiasing this approach.

According to my expertise in photoreceptor biology, there is nothing wrong with this manuscript either technically or conceptually and I have no concerns to express.

Reviewer #3 (Public Review):

Hon et al. investigated the role of BNST CRF signaling in modulating phasic and sustained fear in male and female mice. They found that partial and full fear conditioning had similar effects in both sexes during conditioning and during recall. However, males in the partially reinforced fear conditioning group showed enhanced acoustic startle, compared to the fully reinforced fear conditioning group, an effect not seen in females. Using fiber photometry to record calcium activity in all BNST neurons, the authors show that the BNST was responsive to foot shock in both sexes and both conditioning groups. Shock response increased over the session in males in the fully conditioned fear group, an effect not observed in the partially conditioned fear group. This effect was not observed in females. Additionally, tone onset resulted in increased BNST activity in both male groups, with the tone response increasing over time in the fully conditioned fear group. This effect was less pronounced in females, with partially conditioned females exhibiting a larger BNST response. During recall in males, BNST activity was suppressed below baseline during tone presentations and was significantly greater in the partially conditioned fear group. Both female groups showed an enhanced BNST response to the tone that slowly decayed over time. Next, they knocked CRF in the BNST to examine its effect on fear conditioning, recall and anxiety-like behavior after fear. They found no effect of the knockdown in either sex or group during fear conditioning. During fear recall, BNST CRF knockdown lead to an increase in freezing in only the partially conditioned females. In the anxiety-like behavior tasks, BNST CRF knockdown lead to increased anxiolysis in the partially reinforced fear male, but not in females. Surprisingly, BNST CRF knockdown increased startle response in fully conditioned, but not partially conditioned males. An effect not observed in either female group. In a final set of experiments, the authors single photon calcium imaging to record BNST CRF cell activity during fear conditioning and recall. Approximately, 1/3 of BNST CRF cells were excited by shock in both sexes, with the rest inhibited and no differences were observed between sexes or group during fear conditioning. During recall, BNST CRF activity decreased in both sexes, an effect pronounced in male and female fully conditioned fear groups.

Overall, these data provide novel, intriguing evidence in how BNST CRF neurons may encode phasic and sustained fear differentially in males and females. The experiments were rigorous.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript Kroon et al. described two algorithms, which when combined achieve high throughput automation of "martinizing" protein structures with selected protonation states and post-translational modifications.

Strengths:<br /> A large scale protein simulation was attempted, showing strong evidence that authors' algorithms work smoothly.

The authors described the algorithms in detail and shared the open-source code under Apache 2.0 license on GitHub. This allows both reproducibility of extended usefulness within the field. These algorithms are potentially impactful if the authors can address some of the issues listed below.

Weaknesses:<br /> One major caveat of the manuscript is that the authors claim their algorithms aim to "process any type of molecule or polymer, be it linear, cyclic,<br /> branched, or dendrimeric, and mixtures thereof" and "enable researchers to prepare simulation input files for arbitrary (bio)polymers". However, the examples provided by the manuscript only support one type of biopolymer, i.e. proteins. Despite the authors' recommendation of using polyply along with martinize2/vermouth, no concrete evidence has been provided to support the authors' claim. Therefore, the manuscript must be modified to either remove these claims or include new evidence.

Method descriptions on Martinize2 and graph algorithms in SI should be core content of the manuscript. I argue that Figure S1 and Figure S2 are more important than Figure 3 (protonation state). I recommend the authors can make a workflow chart combining Figure S1 and S2 to explain Martinize2 and graph algorithms in main text.

In Figure 3 (protonation state), the figure itself and the captions are ambiguous about whether at the end the residue is simply renamed from HIS to HIP, or if hydrogen is removed from HIP to recover HIS.

In "Incorporating a Ligand small-molecule Database", the authors are calling for a community effort to build a small-molecule database. Some guidance on when the current database/algorithm combination does or does not work will help the community in contributing.

A speed comparison is needed to compare Martinize2 and Martinize.

Reviewer #3 (Public Review):

To analyze the circuit mechanisms leading to the habituation of the O-bed responses upon repeated dark flashes (DFs), the authors performed 2-photon Ca2+ imaging in larvae expressing nuclear-targeted GCaMP7f pan-neuronally panning the majority of the midbrain, hindbrain, pretectum, and thalamus. They found that while the majority of neurons across the brain depress their responsiveness during habituation, a smaller population of neurons in the dorsal regions of the brain, including the torus longitudinalis, cerebellum, and dorsal hindbrain, showed the opposite pattern, suggesting that motor-related brain regions contain non-depressed signals, and therefore likely contribute to habituation plasticity.

Further analysis using affinity propagation clustering identified 12 clusters that differed both in their adaptation to repeated DFs, as well as the shape of their response to the DF.

Next by the pharmacological screening of 1953 small molecule compounds with known targets in conjunction with the high-throughput assay, they found that 176 compounds significantly altered some aspects of measured behavior. Among them, they sought to identify the compounds that 1) have minimal effects on the naive response to DFs, but strong effects during the training and/or memory retention periods, 2) have minimal effects on other aspects of behaviors, 3) show similar behavioral effects to other compounds tested in the same molecular pathway, and identified the GABAA/C Receptor antagonists Bicuculline, Amoxapine, and Picrotoxinin (PTX). As partial antagonism of GABAAR and/or GABACR is sufficient to strongly suppress habituation but not generalized behavioral excitability, they concluded that GABA plays a very prominent role in habituation. They also identified multiple agonists of both Melatonin and Estrogen receptors, indicating that hormonal signalling may also play a prominent role in habituation response.

To integrate the results of the Ca2+ imaging experiments with the pharmacological screening results, the authors compared the Ca2+ activity patterns after treatment with vehicle, PTX, or Melatonin in the tethered larvae. The behavioral effects of PTX and Melatonin were much smaller compared with the very strong behavioral effects in freely-swimming animals, but the authors assumed that the difference was significant enough to continue further experiments. Based on the hypothesis that Melatonin and GABA cooperate during habituation, they expected PTX and Melatonin to have opposite effects. This was not the case in their results: for example, the size of the 12(Pot, M) neuron population was increased by both PTX and Melatonin, suggesting that pharmacological manipulations that affect habituation behavior manifest in complex functional alterations in the circuit, making capturing these effects by a simple difficult.

Since the 12(𝑃𝑜𝑡, 𝑀) neurons potentiate their responses and thus could act to progressively depress the responses of other neuronal classes, they examined the identity of these neurons with GABA neurons. However, GABAergic neurons in the habituating circuit are not characterized by their Adaptation Profile, suggesting that global manipulations of GABAergic signalling through PTX have complex manifestations in the functional properties of neurons.

Overall, the authors have performed an admirably large amount of work both in whole-brain neural activity imaging and pharmacological screening.

Reviewer #3 (Public Review):

In this paper, Toschi et al. performed dMRI to in vivo estimate axon diameter in the brain and demonstrated that multi-compartmental modeling (AxCaliber) is sensitive to microstructural axonal damage in rats and axon caliber increase in demyelinating lesions in MS patients, suggesting that axon diameter mapping provides a potential biomarker to bridge the gap between medical imaging contrasts and biological microstructure. In particular, authors injected ibotenic acid (IBO) and saline in the left and right rat hippocampus, respectively, and compared in vivo estimated axon diameter and ex vivo neurofilament staining in left and right fimbria. The axon size estimation was larger in the fimbria of IBO injection side, where the neurofilament intensity is higher. Correlation of axon size estimation and neurofilament intensity was observed in both injection sides. Further, higher axon diameter estimation was observed in normal appearing white matter (NAWM) of MS patients, compared with the healthy subjects. The axon size estimation increased in hypointense lesions of T1 weighted contrast, but not in isointense lesions. Through the comparison of dMRI-estimated axon size and histology-based fluorescence intensity, authors indirectly validated the sensitivity of axon diameter mapping to the tissue microstructure in the rat brain, and further explored the axon size change in the brain of MS patients. However, the dMRI protocol and biophysical modeling in this study were not fully optimized to maximize the sensitivity to axon size estimation, and the dMRI-estimated axon size (4.4-5.4 micron) was much larger than values reported in previous histological studies (0.5-3 micron) [Barazany et al., Brain 2009]. Finally, although the modified AxCaliber model incorporated two fiber bundles in different directions, the fiber dispersion in each bundle was not considered (c.f. fiber dispersion ~20-30 degree in corpus callosum), potentially leading to overestimated axon diameter.

The conclusions in this study are supported by experimental results. However, the dMRI protocol and biophysical model could be further optimized and validated:<br /> 1. To in vivo estimate the axon diameter ~1 micron using dMRI, strong diffusion weighting (b-value) should be applied to maximize the signal decay due to intra-axonal restricted diffusion and minimize the signal contribution of extra-cellular hindered diffusion. However, authors only applied maximal b-value = 4000 s/mm2, much smaller than values ~15,000-20,000 s/mm2 in previous studies [Assaf et al., MRM 2008; Huang et al., BSAF 2020, 225:1277]. The use of low diffusion weighting in this study leads to a lower bound ~4-6 micron for accurate diameter estimation, the so-called resolution limit in [Nilsson et al., NMR Biomed 2017, 30:e3711]. In other words, the estimated axon diameter is potentially overestimated and related with the imaging protocol and image quality, confounding the biological interpretation.<br /> 2. In this study, the positive correlation of dMRI-estimated axon size and neurofilament fluorescence intensity is indeed an encouraging result, and yet this validation is indirect since it relies on the positive correlation between neurofilament intensity and axon diameter in histology.<br /> 3. Authors did not consider the fiber dispersion in the proposed dMRI model. This can lead to overestimated axon diameter, even in the highly aligned WM, such as corpus callosum with ~20-30 degree dispersion in histology [Ronen et al., BSAF 2014, 219:1773; Leergaard et all, PLoS One 2010, 5(1), e8595] and MRI [Dhital et al., NeuroImage 2019, 189, 543; Novikov et al., NeuroImage 2018, 174:518].

Reviewer #3 (Public Review):

This study examined the changes in fear response, as measured by the flight initiation distances (FID), of birds living in urban areas. The authors examined the FIDs of birds during the pandemic (COVID-19 lockdown restrictions) compared to FIDs measured before the pandemic (mostly in 2018 & 2019). The main study justification was that human presence changed drastically during the pandemic lockdowns and the change in human presence might have influenced the fear response of birds as a result of changing the "landscape of fear". Human presence was quantified using a 'stringency' index (government-mandated restrictions). Urban areas were selected from within five different cities, which included four European cities (Czech Republic - Prague, Finland - Rovaniemi, Hungary - Budapest, Poland - Poznan), and one city in the global south (Australia - Melbourne). Using 6369 flight initiation distances across 147 different bird species, the authors found that FIDs were not significantly different before the pandemic versus during the pandemic, nor was the variation in FID explained by the level of 'stringency'.

Major strengths: There are several strengths to this study that allows for understanding the variety of factors that influence a bird's response to fear (measured as flight initiation distances). This study also demonstrates that FIDs are highly variable between species and regions.<br /> Specifically,<br /> 1) One of the major strengths of this paper is the focus on birds living in urban areas, a habitat type that is hypothesized to have changed drastically in the 'landscape of fear' experienced by animals during the pandemic lockdown restrictions (due to the presumed decrease in human presence and densities). Maintaining the focus on urban birds allowed for a deeper examination of the effect of human behaviour changes on bird behaviour in urban habitats, which are at the interface of human-wildlife interactions.<br /> 2) This study accounted for several variables that are predicted to influence flight initiation distances in birds including species, genus, region (country), variability between years, pandemic year (pre- versus during), the strictness of government-mandated lockdown measures, and ecological factors such as the human observer starting distance, flock size, species-specific body size, ambient air temperature (also a proxy of the timing during the breeding season), time of day, date of data collection (timing within the regional [Europe or Australia] breeding season), and categorization of urban site type (e.g. park, cemetery, city centre).<br /> 3) This study examined FIDs in two years previous to the pandemic (mostly 2018 and 2019, one site was 2014) which would account for some of the within- and between-year FID variation exhibited prior to the pandemic.<br /> 4) This study uses strong statistical approaches (mixed effect models) which allows for repeat sampling, and a post hoc analysis testing for a phylogenetic signal.

Major weaknesses: The authors used government 'stringency' as a proxy for human presence and densities, however, this may not have been an accurate measure of actual human presence at the study sites and during measurements of FIDs. Furthermore, although the authors accounted for many factors that are predicted to influence fear response and FIDs in birds, there are several other factors that may have contributed to the high level of variation and patterns in FIDS observed during this study, thus resulting in the authors' conclusion that FIDs did not vary between pre- and during pandemic years.<br /> Specifically,<br /> 1) The authors used "government stringency" as a measure of change in human activity, which makes the assumption that the higher the level of 'stringency', the fewer humans in urban areas where birds are living. However, the association between "stringency" and actual human presence at the study sites was not measured, nor was 'stringency' compared to other measures of human presence such as human mobility.<br /> 2) There was considerable variation in FID measurements, which can be seen in the figures, indicating that most of the variation in FID was not accounted for in the authors' models. Factors that may have contributed to variation in FIDs that were not accounted for in this study are as follows:<br /> a. The authors accounted for the date of data collection using the 'day' since the start of the general region's breeding season (Europe: Day 1 = 1 April; Australia: Day 1 = 15 August). Using 'day' since the breeding season started probably was an attempt to quantify the effect of the breeding stage (e.g. territory establishment, nest young, fledgling) on FIDs. However, breeding stages vary both within- and between species, as well as between sub-regions (e.g. Finland vs. Hungary). As different species respond to predation or human presence differently depending on the stage during their breeding cycle, more specificity in the breeding cycle stage may allow for explaining the observed variation and patterns in FID.<br /> b. Variation in species-specific FIDs may also vary with habitat features within urban sites, such as the proximity of trees and other protective structures (e.g. perches and cover), the openness of the area, and the level of stressors present (e.g. noise pollution, distance to roads). Perhaps accounting for this habitat heterogeneity would account for the FID variation measured in this study.<br /> c. The authors accounted for species and genus within their models, however, FIDs may vary with other species-specific (or even specific populations of a species) characteristics such as whether the species/population is neophobic versus neophilic, precocial versus altricial, and the level of behavioural plasticity exhibited. These variables were not accounted for in the analysis.<br /> d. Three different methods of measuring the distances between flight and the observer location were used, and FIDs were only measured once per bird, such that there were no measures of repeatability for a test subject. Thus, variation surrounding the measurement of FIDs would have contributed to the variation in FIDs seen during this study.<br /> 3) The sample design of this study may have influenced the FID variability associated with specific species, and specific populations of species. A different number of species were sampled across the time periods of interest; 68 species were sampled before the pandemic versus 135 species after the pandemic. However, the authors do not appear to have directly compared the FIDs for the same species before the pandemic compared to during the pandemic (e.g. the FIDs of Eurasian blackbirds before the pandemic versus during the pandemic). Furthermore, within the same country-city, it is unclear whether the species observed before the pandemic were observed at the same location (e.g. same habitat type such as the same park) during the pandemic. As a species' FID response may be influenced by population characteristics and features specific to each site (e.g. habitat openness), these factors may have influenced the variability in FID measurements in this study.<br /> 4) The models in this study accounted for many factors predicted to affect FIDs (see the section on major strengths), however, the number of fixed and random factors are large in number compared to the total sample size (N =6369), such that models may have been over-extended.

Overarching main conclusion<br /> Overall, this study examines factors influencing FIDs in a variety of bird species and concludes that FIDs did not differ during the pandemic lockdowns compared to before the pandemic (2019 and earlier). Furthermore, FIDs were not influenced by the strictness of government-mandated restrictions. Although the authors accounted for many factors influencing the measurement of FIDs in birds, the authors did not achieve their aim of disentangling the effects of pandemic-specific ecological effects from ecological effects unrelated to the pandemic (such as habitat heterogeneity). Their findings indicate that FIDs are highly variable both within- and between- species, but do not strongly support the conclusion that FIDs did not change in urban species during the pandemic lockdown. Therefore, this study is of limited impact on our understanding of how a drastic change in human behaviour may impact bird behaviour in urban habitats. Overall, the study demonstrates the challenges in using FIDs as a general fear response in birds, even during a pandemic lockdown when fewer humans are presumably present, and this study illustrates the large degree of variation in FIDs in response to a human observer.

Reviewer #3 (Public Review):

A big open question in evolutionary biology is how single cells become multicellular organisms, capable of adaptation as a collective. Many cells form groups, but adaptation at the level of the group tends to be inefficient (especially in comparison to cells). Theoretically, it has been proposed that groups formed by clonal development (cells remain attached to each other after division) can more readily lead to group-level adaptation than groups coming together through the aggregation of different cells post-division. To evaluate empirically the plausibility of this hypothesis, the authors compared adaptation in two lines of yeast that differ only in a couple of mutations determining their mechanism of group formation. Ace2 mutants develop through staying together, and Floc mutants through aggregation. They performed a form of size selection (through settling) as a way to select for multicellularity (this selection regime has been used before to obtain multicellular phenotypes). This selective regime has two components: growing (largely due to differences between cells) and settling (largely due to differences between groups). Thus, the authors assume that increases in fitness through growth are due mostly to adaptation at the single-cell level, whereas increases in fitness through settling are mostly due to adaptation at the multicellular level. They find that adaptation in clonal groups is mostly through settling and that aggregative groups adapt more through growth (despite getting bigger).

Overall this assumption makes sense (especially in a positive way) but growth, in this case, is also selecting against groups in the snowflake case and less strongly so in the floc case in which cells aggregate and disaggregate with some probability, and therefore cells can keep growing. That is, in addition to assortment the result is somewhat expected because there is less of a trade-off between growth and settling in floc: having a higher density in floc probably leads to higher aggregation and indirectly benefits settling, whereas in the clonal case, larger groups mean that a larger proportion of cells is not growing.

The main result of the paper holds true: clonal development favors multicellular adaptation relative to aggregative multicellularity, but the reason is not exclusively a difference in the distribution of variation, but a difference in the trade-off between single cell and multicellular traits.

In the second part of the paper, the authors beautifully show that the mechanisms of group formation affect evolutionary processes. Clonal aggregation leads to a decrease in the effective population size (because the descendants of mutants are likely to be in the same group, and therefore be selected together). This result shows that the mode of development can affect evolution!

Reviewer #3 (Public Review):

Summary:<br /> Smith-Magenis syndrome (SMS) is associated with obesity and is caused by deletion or mutations in one copy of the Rai1 gene which encodes a transcriptional regulator. Previous studies have shown that Bdnf gene expression is reduced in the hypothalamus of Rai1 heterozygous mice. This manuscript by Javed et al. further links SMS-associated obesity with reduced Bdnf gene expression in the PVH.

Strengths:<br /> The authors show that deletion of the Rai1 gene in all BDNF-expressing cells or just in the PVH BDNF neurons postnatally caused obesity. Interestingly, mutant mice displayed sexual dimorphism in the cause for the obesity phenotype. Overall, the data are well presented and convincing except the data from LM22A-4.

Weaknesses:<br /> 1. The most serious concern is about data from LM22A-4 administration experiments (Figure 5 and associated supplemental figures). A rigorous study has demonstrated that LM22A-4 does not activate TrkB (Boltaev et al., Science Signaling, 2017), which is consistent with unpublished results from many labs in the neurotrophin field. It is tricky to interpret body weight data from pharmacological studies because compounds always have some side effects, which can reduce body weight non-specifically.

2. The resolution of all figures are poor, and thus I could not judge the quality of the micrographs.

3. Citation of the literature is not precise. The study by An et al. (2015) shows that deletion of the Bdnf gene in the PVH leads to obesity due to increased food intake and reduced energy expenditure (not just hyperphagic obesity; Line 72). Furthermore, the study by Unger et al. (2017) carried out Bdnf deletion in the VMH and DMH using AAV-Cre and did not discuss SF1 neurons at all (Line 354). The two studies by Yang et al. (Mol Endocrinol, 2016) and Kamitakahara et al. (Mol Metab, 2015) did use SF1-Cre to delete the Bdnf gene and did not observe any obesity phenotype.

4. Animal number is not described in many figure legends.

Reviewer #3 (Public Review):

This paper considers a challenging motor control task - the critical stability task (CST) - that can be performed equally well by humans and macaque monkeys. This task is of considerable interest since it is rich enough to potentially yield important novel insights into the neural basis of behavior in more complex tasks that point-to-point reaching. Yet it is also simple enough to allow parallel investigation in humans and monkeys, and is also easily amenable to computational modeling. The paper makes a compelling argument for the importance of this type of parallel investigation and the suitability of the CST for doing so.

Behavior in monkeys and in human subjects suggests that behavior seems to cluster into different regimes that seem to either oscillate about the center of the screen, or drift more slowly in one direction. The authors show that these two behavioral regimes can be reliably reproduced by instructing human participants to either maintain the cursor in the center of the screen (position control objective), or keep the cursor still anywhere in the screen (velocity control objective) - as opposed to the usual 'instruction' to just not let the cursor leave the screen. A computational model based on optimal feedback control can similarly reproduce the two control regimes when the costs are varied

Overall, this is a creative study that successfully leverages experiments in humans and computational modeling to gain insight into the nature of individual differences in behavior across monkeys (and people). The approach does work and successfully solves the core problem the authors set out to address. I do think that more comprehensive approaches might be possible that might involve, e.g. using a richer set of behavioral features to classify behavior, fitting a parametric class of control objectives rather than assuming a binary classification, and exploring the reliability of the inference process in more detail.

In addition, the authors do fully establish that varying control objectives is the only way to obtain the different behavioral phenotypes observed. It may, for instance, be possible that some other underlying differences (e.g. the sensitivity to effort costs or the extent of signal-dependent noise) might also lead to a similar range of behaviors as varying the position versus velocity costs.

Specific Comments:<br /> The simulations convincingly show that varying the control objective via the cost function can reproduce the different observed behavioral regimes. However, in principle, the differences in behavior among the monkeys and among the humans in Experiment 1 might not necessarily be due to difference in other aspects of the model. For instance, for a fixed cost function, differences in motor execution noise might perhaps lead the model to favor a position-like strategy or a velocity-like strategy. Or differences in the relative effort cost might alter the behavioral phenotype. Given that the narrative is about inferring control objectives, it seems important to rule out more systematically that some other factor might not potentially dictate each individual's style of performing the task. One approach to rule this out might be to try to formally fit the parameters of the model (or at least a subset of them) under a fixed cost function (e.g. velocity-based), and check whether the model might still recover the different regimes of behavior when parameters *other than the cost function* are varied.

The approach to the classification problem is somewhat ad hoc and based on fairly simplistic, hand-picked features (RMS position and RMS velocity). I do wonder whether a more comprehensive set of behavioral features might enable a clearer separation between strategies, or might even reveal that the uninstructed subjects were doing something qualitatively different still from the instructed groups. Different control objectives ought to predict meaningfully different control policies - that is, different ways of updating hand position based on current state of the cursor and hand - e.g. the hand/cursor gain, which does clearly differ across instructed strategies. Would it be possible to distinguish control strategies more accurately based on this level of analysis, rather than based on gross task metrics? Might this point to possible experimental interventions (e.g. target jumps) that might validate the inferred objective?

It seems that the classification problem cannot be solved perfectly, at least on a single-trial level. Although it works out that the classification can recover which participants were given which instructions, it's not clear how robust this classification is. It should be straightforward to estimate the reliability of the strategy classification by simulating participants and deriving a "confusion matrix", i.e. calculating how often e.g. data generated under a velocity-control objective gets mis-classified as following a position-control objective. It's not clear how this kind of metric relates to the decision confidence outputted by the classifier.

The problem of inferring the control objective is framed as a dichotomy between position control and velocity control. In reality, however, it may be a continuum of possible objectives, based on the relative cost for position and velocity. How would the problem differ if the cost function is framed as estimating a parameter, rather than as a classification problem?

Reviewer #3 (Public Review):

This manuscript describes the use of scRNA-seq to decipher the cellular heterogeneity, molecular dynamics and signaling interactions during fibrocartilaginous enthesis formation. They delineate the enthesis growth and the temporal atlas from embryonic stage to postnatal stage by scRNA-seq, compared the development pattern of enthesis origins with tendon and articular cartilage, then demonstrated the cellular complexity and heterogeneity of postnatal enthesis growth and revealed the molecular dynamics and signaling networks during enthesis formation.

This manuscript used appropriate and validated methodology in line with current state-of-the-art, and the conclusions of this paper are mostly well supported by data, more in vitro or in vivo experiments are encouraged to verify the key molecular dynamics and signaling networks revealed by scRNA-seq during enthesis formation.

This manuscript facilitates better understand of the enthesis development, which will benefit the important field of enthesis research.

Reviewer #3 (Public Review):

The major claim from the paper is the dependence of two factors that determine the polymerization of MreB from a Gram-positive, thermophilic bacteria 1) The role of nucleotide hydrolysis in driving the polymerization. 2) Lipid bilayer as a facilitator/scaffold that is required for hydrolysis-dependent polymerization. These two conclusions are contrasting with what has been known until now for the MreB proteins that have been characterized in vitro. The experiments performed in the paper do not completely justify these claims as elaborated below.

Major comments:

1. No observation of filaments in the absence of lipid monolayer can also be accounted due to the higher critical concentration of polymerization for MreBGS in that condition. It is seen that all the negative staining without lipid monolayer condition has been performed at a concentration of 0.05 mg/mL. It is important to check for polymerization of the MreBGS at higher concentration ranges as well, in order to conclusively state the requirement of lipids for polymerization.

2. The absence of filaments for the non-hydrolysable conditions in the lipid layer could also be because the filaments that might have formed are not binding to the planar lipid layer, and not necessarily because of their inability to polymerize.

3. Given the ATPase activity measurements, it is not very convincing that ATP rather than ADP will be present in the structure. The ATP should have been hydrolysed to ADP within the structure. The structure is now suggestive that MreB is not capable of hydrolysis, which is contradictory to the ATP hydrolysis data.

Reviewer #3 (Public Review):

In this study, Ye et al investigated how a peptide that binds to the transmembrane (TM) domain of the T cell receptor (TCR) subunits affects TCR activation. The objective was to test the allosteric relaxation model of TCR activation. To this end, the authors leveraged their previously established strategy of designing TM-targeting peptides and studied how such peptide alters the TCR activation and downstream signaling cascades in Jurkat T cells. The authors found that the TM-targeting peptide inhibited phosphorylation of the TCR submits, phosphorylation of downstream signaling proteins such as ZAP70, and calcium influx in T cells. Using immunoprecipitation experiments, the authors proposed that the peptide binds into the membrane gap between CD3 and CD3 subunits in the TCR complex. The authors conclude that their data support the allosteric TCR activation model, in which allosteric changes in the TM bundle in the TCR complex determine the receptor signaling.

The use of pH-responsive TM-targeting peptides, which the authors previously developed, is a novel aspect of this study. Those peptides can be quite powerful for understanding molecular mechanisms of receptor signaling, such as the allosteric activation model as tested in this study. The manuscript contains several interesting approaches and observations, but there are concerns about the experimental design and interpretation of the results. More importantly, the authors' primary conclusion that the allosteric changes in the TM bundles determine TCR activation is not fully supported by the data presented. For example:

1. The authors provided confocal fluorescence images showing the colocalization of fluorescently labeled peptides and TCR subunits. Based on the data, they concluded that "PITCR is able to bind to TCR". This is misleading, because given the spatial resolution of the imaging technique, "colocalization" does not indicate binding or interaction between molecules. Because the peptide binding to the TM region is the pillar of the primary finding of this study, direct evidence supporting the peptide-TM binding or interaction is essential.<br /> 2. In calcium response experiments, the authors compared calcium influx (indicated by Indo-1 ratio) under different cell activation conditions (Figure 2). There are some concerns about how the authors interpreted the data: (1) The calcium plots from OKT3 activation in A-C panels are inconsistent. The plot in (A) showed a calcium peak after activation, which is not present in the plots shown in (B) and (C). There is no explanation or discussion on this inconsistency. (2) What is more concerning is that this prominent calcium peak in (A) was used to draw the conclusion that the designer peptide inhibitor effectively reduces calcium response. However, inconsistent with that conclusion, the calcium plots are indistinguishable for the three conditions: with PITCR (peptide inhibitor), with PITCRG41P (negative control that should not affect TCR activation), or no peptide. All three plots have similar magnetite and fluctuations. This does not support the authors' conclusion that the PITCR (peptide inhibitor) reduces calcium response in T cells.<br /> 3. Different types of T cells were used for separate measurements: E6-1 Jurkat T cells were used for calcium influx experiments, J. OT.hCD8+ Jurkat cells were used for CD69 measurements, and primary murine CD4+ T cells were used for colocalization imaging experiments. Rationales for the choices of cells in different measurements are also unclear. This is different from the common practice where different cell types are used in repeated experiments to test the generality of a finding. Here, they were used for different experiments, and findings were lumped together as "T cells", without further evidence/discussion on how translatable the findings from different cell types are.<br /> 4. The authors set out to test the model that TCR activation by pMHC occurs through allosteric changes in the TM region, but in most experiments, they activated Jurkat T cells by anti-CD3 antibody, not by antigen peptides. The anti-CD3 antibody activates TCR signaling through clustering. It is unclear whether TCR activation by anti-CD3 leads to the same allosteric changes in the TM region as activation by pMHC.

As such, the main claim of the paper, namely that the designer peptide affects TCR signaling by disrupting the allosteric changes in the TM region, remains insufficiently supported by the data presented.

Reviewer #3 (Public Review):

Here the authors use high-parameter flow cytometry to address expression patterns of inhibitory receptors and concordant functional responses in CD8+ T cells from people living with HIV (PLWH) during early vs. long-term ART treatment in order to understand the potential evolution of exhausted T cells in HIV infection. High-dimensional bioinformatic analysis is employed to uncover different subsets of CD8+ T cells expressing TIM-3, TIGIT, PD1, LAG3, and CD39. Stimulation assays were further conducted to assess polyclonal T cell responses (superantigen) or HIV-gag-specific CD8+ T cells, and whether the responding cells displayed inhibitory receptors. Finally, inhibitory receptor blockade was used (focusing on TIGIT and TIM-3 only) to examine the potential reversal of exhaustion. The authors found that CD107a+ degranulating central memory T cells apparently were sensitive to TIGIT blockade, yielding increased responses in cells from ART-treated PLWH.

Methods and Results Major Strengths: Sample size and data density. Longitudinal samples from long-term treated PLWH. Mechanistic studies to assess inhibitory receptor blockade.

Methods and Results Major Weaknesses: Lack of clarity on flow cytometric analysis and statistical methodology, including correction for multiple comparisons. Clustering density in tSNE analysis is unjustified, leading to potentially spurious outcomes. Insufficient raw flow cytometry data presented on inhibitory receptor expression in the various contexts of the study to allow determination of whether the subsequent bioinformatic analysis was merited due to the very low expression of 3/5 markers examined. Unclear whether differences observed are biologically meaningful (despite statistical differences). Finally, although the longitudinal samples are a distinct strength of the study, changes over time within individuals are unfortunately not assessed.

Aims and conclusions: The authors do find differences between the cohorts as described in the manuscript; however, the biological relevance of the findings is questionable due to an absence of direct studies on the cell populations found to be different. The use of unbiased clustering analysis is both a strength and a weakness. Specifically, the algorithm uncovers potential cell clusters that might be missed; however, the clustering program requires pre-set inputs on the expected number of clusters to be found, leading to possible irrelevant subsets being identified. The conclusions of the study are appropriately limited in scope.

Impact: There have been numerous studies of CD8+ T cell inhibitory receptor expression and T cell exhaustion in the context of HIV infection. It is well-accepted that T-cell exhaustion is a hallmark of progressive infection. This study contributes to the current knowledge in this area specifically through the examination of very long-term ART-treated PLWH. Unfortunately, it is not clear that several of the examined inhibitory receptors could be adequately detected, limiting the interpretation of the findings. Finally, it is unclear that this study justifies the potential use of TIGIT blockade to improve T cell function given the unclear biological relevance of the differential populations of CD8+ T cells observed.

Reviewer #3 (Public Review):

Henault et al. address the important open question of whether hybridization could trigger TE mobilization. To do this they analysed MA lines derived from crosses of Saccharomyces paradoxus and Saccharomyces cerevisiae using long-read sequencing. These MA lines were already analysed in a previous publication using Illumina short-read data but the novelty of this work is the long-read sequencing data, which may reveal previously missed information. It is an interesting message of this study that hybridization between the two species did not lead to much TE activity. Due to this low activity, the authors performed an additional TE activity assay in vivo to measure transposition rates in hybrid backgrounds. The study is well written and I cannot spot any major problems. The study provides some important messages (like the influence of the genotype and mitochondrial DNA on transposition rates).

Major comments<br /> - What I miss the most in this work is the perspective of the host defence against TEs in Saccharmoces. Based on such a mechanistic perspective, why do the authors think that hybridization could lead to a TE reactivation? For example, in Drosophila small RNAs important for the defence against a TE, are solely maternally transmitted. Hybrid offspring will thus solely have small-RNAs complementary to the TEs of the mother but not to the TEs of the father, therefore a reactivation of the paternal TEs may be expected. I was thus wondering, what is the situation in yeast. Why would we expect an upregulation of TEs? Without such a mechanistic explanation the hypothesis that TEs should be upregulated in hybrids is a bit vague, based on a hunch.

Reviewer #3 (Public Review):

In this manuscript, Gustison et al., describe the development of an automated whole-brain mapping pipeline, including the first 3D histological atlas of the prairie vole, and then use that pipeline to quantify Fos immunohistochemistry as a measure of neural activity during mating and pair bonding in male and female prairie voles. Prairie voles have become a useful animal model for examining the neural bases of social bonding due to their socially monogamous mating strategy. Prior studies have focused on identifying the role of a few neuromodulators (oxytocin, vasopressin, dopamine) acting in a limited number of brain regions. The authors use this unbiased approach to determine which areas become activated during mating, cohabitation, and pair bonding in both sexes to identify 68 brain regions clustered in seven brain-wide neuronal circuits that are activated over the course of pair bonding. This is an important study because i) it generates a valuable tool and analysis pipeline for other investigators in the prairie vole research community and ii) it highlights the potential involvement of many brain regions in regulating sexual behavior, social engagement, and pair bonding that have not been previously investigated.

Strengths of the study include the unbiased assessment of neural activity using the automated whole brain activity mapped onto the 3D histological atlas. The design of the behavioral aspect of the study is also a strength. Brains were collected at baseline and 2.5, 6 and 22 hrs after cohabitation with either a sibling or opposite-sex partner. These times were strategically chosen to correspond to milestones in pair bond development. Behavior was also quantified during epochs over the 22 hr period providing useful information on the progression of behaviors (e.g. mating) during pair bonding and relating Fos activation to specific behaviors (e.g. sex vs bonding). The sibling co-housed group provided an important control, enabling the identification of areas specifically activated by sex and bond formation. The analyses of the data were rigorous, resulting in convincing conclusions. While there was nothing particularly surprising in terms of the structures that were identified to be active during the mating and cohabitation, the statistical analysis revealed interesting relationships in terms of interactions of the various clusters, and also some level of synchrony in brain activation between partners. Furthermore, ejaculation was found to be the strongest predictor of Fos activation in both males and females. The sex differences identified in the study were subtle and less than the authors expected, which is interesting.

While the study provides a potentially useful tool and approach that may be of general use to the prairie vole community and identifies in an anatomically precise manner areas that may be important for mating or pair bond formation, there are some weaknesses as well. The study is largely descriptive. It is impossible to determine whether the activated areas are simply involved in sex or in the pair bond process itself. In other words, the authors did not use the Fos data to inform functional testing of circuits in pair bonding or mating behaviors. However, that is likely beyond the scope of this paper in which the goal was more to describe the automated, unbiased approach. This weakness is offset by the value of the comprehensive and detailed analysis of the Fos activation data providing temporal and precise anatomical relationships between brain clusters and in relation to behavior. The manuscript concludes with some speculative interpretations of the data, but these speculations may be valuable for guiding future investigations.

Reviewer #3 (Public Review):

Summary:<br /> The study by Karaś et al. reveals how multi-protein systems can evolve into single-protein equivalents, shedding light on the molecular events enabling gene loss during evolution. This work is valuable for researchers in evolutionary fields and offers potential applications in protein and organism engineering. While the findings lack broader appeal and societal implications, the evidence presented supports the proposed molecular mechanism. Using computational methods and biochemical analysis, the authors traced the evolutionary simplification of bacterial small heat shock proteins, linking specific mutations to functional changes. The study's strength lies in its vertical approach, identifying functional residues, but it does not introduce new techniques, limiting its novelty and significance.

Strengths:<br /> 1) Experimental Approach<br /> The research question was clearly outlined and the author's approach to answering it was systematic. In particular, their model system was highly suitable to address the research question. The authors employed appropriate experimental and computational techniques, and their 'vertical approach' was beneficial in that it allowed them to discover functional residues in the sHsp system which may not have been possible otherwise. Overall, their approach to this study was solid.

2) Reproducibility<br /> The results were presented well. The number of experimental repeats was suitable, as well as their analysis of the data. The values for standard deviation were reasonable, and their results using the alternative ancestors for the substrate aggregation assays helped support the robustness of their observations.

Weaknesses:<br /> During the mutational experiments, the authors examined seven potential substitutions identified through ASR and measured their impact on protein disaggregation activity. Positions 66 and 109 exhibited a significant decrease in luciferase refolding stimulation. To explore the combined effect of these mutations, the authors created the double mutant AncA0. However, predicting the most impactful combination of mutations due to epistatic effects is challenging. A more effective strategy would be to test various combinations of mutations to identify the double mutant with the greatest decrease in luciferase refolding stimulation and/or alternatively perform a co-evolutionary study to try to understand any epistatic effects between the mutations.

Reviewer #3 (Public Review):

Summary:<br /> This study aimed to understand the neural correlates of memory recall over short (1-day) and long (14-days) intervals in children (5-7 years old) relative to young adults. The results show that children recall less than young adults and that this is accompanied by less activation (relative to young adults) in brain networks associated with memory retrieval.

Strengths:<br /> This paper is one of few investigating long-term memory (multiple days) in a developmental population, an important gap in the field. Also, the authors apply a representational similarity analysis to understand how specific memories evolve over time. This analysis shows how the specificity of memories decreases over time in children relative to adults. This is an interesting finding.

Weaknesses:<br /> Overall, these results are consistent with what we already know: recall is worse in children relative to adults (e.g., Cycowicz et al., 2001) and children activate memory retrieval networks to a lesser extent than adults (Bauer et al, 2017).

It seems that the reduced activation in memory recall networks is likely associated with less depth of memory encoding in children due to inattentiveness, reduced motivation, and documented differences in memory strategies. In regards to this, there was consideration of IQ, sex, and handedness but these were not included as covariates as they were not significant although I note p<.16 suggests there was some level of association nonetheless. Also, IQ is measured differently for the children and adults so it's not clear these can be directly contrasted. The authors suggest the instructed elaborative encoding strategy is effective for children and adults but the reference in support of this (Craik & Tulving, 1975) does not seem to support this point.

Reviewer #3 (Public Review):

Overall, this is a strong manuscript that uses multiple current techniques to provide specific mechanistic insight into prior discoveries of the contributions of the Bcl11b transcription factor to mossy fiber synapses of dentate gyrus granule cells. The authors employ an adult deletion of Bcl11b via Tamoxifen-inducible Cre and use immunohistochemical, electron microscopy, and electrophysiological studies of synaptic plasticity, together with viral rescue of C1ql2, a direct transcriptional target of Bcl11b or Nrxn3, to construct a molecular cascade downstream of Bcl11b for DG mossy fiber synapse development. They find that C1ql2 re-expression in Bcl11b cKOs can rescue the synaptic vesicle docking phenotype and the impairments in MF-LTP of these mutants. They also show that C1ql2 knockdown in DG neurons can phenocopy the vesicle docking and plasticity phenotypes of the Bcl11b cKO. They also use artificial synapse formation assays to suggest that C1ql2 functions together with a specific Nrxn3 splice isoform in mediating MF axon development, extending these data with a C1ql2-K262E mutant that purports to specifically disrupt interactions with Nrxn3. All of the molecules involved in this cascade are disease-associated and this study provides an excellent blueprint for uncovering downstream mediators of transcription factor disruption. Together this makes this work of great interest to the field. Strengths are the sophisticated use of viral replacement and multi-level phenotypic analysis while weaknesses include the linkage of C1ql2 with a specific Nrxn3 splice variant in mediating these effects.

Here is an appraisal of the main claims and conclusions:

1. C1ql2 is a downstream target of Bcl11b which mediates the synaptic vesicle recruitment and synaptic plasticity phenotypes seen in these cKOs. This is supported by the clear rescue phenotypes of synapse anatomy (Fig.2) and MF synaptic plasticity (Fig.3). One weakness here is the absence of a control assessing over-expression phenotypes of C1ql2. It's clear from Fig.1D that viral rescue is often greater than WT expression (totally expected). In the case where you are trying to suppress a LoF phenotype, it is important to make sure that enhanced expression of C1ql2 in a WT background does not cause your rescue phenotype. A strong overexpression phenotype in WT would weaken the claim that C1ql2 is the main mediator of the Bcl11b phenotype for MF synapse phenotypes.

2. Knockdown of C1ql2 via 4 shRNAs is sufficient to produce the synaptic vesicle recruitment and MF-LTP phenotypes. This is supported by clear effects in the shRNA-C1ql2 groups as compared to nonsense-EGFP controls. One concern (particularly given the use of 4 distinct shRNAs) is the potential for off-target effects, which is best controlled for by a rescue experiment with RNA-insensitive C1ql2 cDNA as opposed to nonsense sequences, which may not elicit the same off-target effects.

3. C1ql2 interacts with Nrxn3(25b+) to facilitate MF terminal SV clustering. This claim is theoretically supported by the HEK cell artificial synapse formation assay (Fig.5), the inability of the K262-C1ql2 mutation to rescue the Bcl11b phenotype (Fig.6), and the altered localization of C1ql2 in the Nrxn1-3 deletion mice (Fig.7). Each of these lines of experimental evidence has caveats that should be acknowledged and addressed. Given the hypothesis that C1ql2 and Nrxn3b(25b) are expressed in DG neurons and work together, the heterologous co-culture experiment seems strange. Up till now, the authors are looking at pre-synaptic function of C1ql2 since they are re-expressing it in DGNs. The phenotypes they are seeing are also pre-synaptic and/or consistent with pre-synaptic dysfunction. In Fig.5, they are testing whether C1ql2 can induce pre-synaptic differentiation in trans, i.e. theoretically being released from the 293 cells "post-synaptically". But the post-synaptic ligands (Nlgn1 and and GluKs) are not present in the 293 cells, so a heterologous synapse assay doesn't really make sense here. The effect that the authors are seeing likely reflects the fact that C1ql2 and Nrxn3 do bind to each other, so C1ql2 is acting as an artificial post-synaptic ligand, in that it can cluster Nrxn3 which in turn clusters synaptic vesicles. But this does not test the model that the authors propose (i.e. C1ql2 and Nrxn3 are both expressed in MF terminals). Perhaps a heterologous assay where GluK2 is put into HEK cells and the C1ql2 and Nrxn3 are simultaneously or individually manipulated in DG neurons?

4. K262-C1ql2 mutation blocks the normal rescue through a Nrxn3(25b) mechanism (Fig.6). The strength of this experiment rests upon the specificity of this mutation for disrupting Nrxn3b binding (presynaptic) as opposed to any of the known postsynaptic C1ql2 ligands such as GluK2. While this is not relevant for interpreting the heterologous assay (Fig.5), it is relevant for the in vivo phenotypes in Fig.6. Similar approaches as employed in this paper can test whether binding to other known postsynaptic targets is altered by this point mutation.

5. Altered localization of C1ql2 in Nrxn1-3 cKOs. These data are presented to suggest that Nrx3(25b) is important for localizing C1ql2 to the SL of CA3. Weaknesses of this data include both the lack of Nrxn specificity in the triple a/b KOs as well as the profound effects of Nrxn LoF on the total levels of C1ql2 protein. Some measure that isn't biased by this large difference in C1ql2 levels should be attempted (something like in Fig.1F).

Reviewer #3 (Public Review):

This study demonstrates that from fish to mammals CIB2/3 is required for hearing, revealing the high degree of conservation of CIB2/3 function in vertebrate sensory hair cells. The modeling data reveal how CIB2/3 may affect the conductance of the TMC1/2 channels that mediate mechanotransduction, which is the process of converting mechanical energy into an electrical signal in sensory receptors. This work will likely impact future studies of how mechanotransduction varies in different hair cell types.

One caveat is that the experiments with the mouse mutants are confirmatory in nature with regard to a previous study by Wang et al., and the authors use lower resolution tools in terms of function and morphological changes. Another is that the modeling data is not supported by electrophysiological experiments, however, as mentioned above, future experiments may address this weakness.

Reviewer #3 (Public Review):

Summary:<br /> Using ex vivo electrophysiology and morphological analysis, Boi et al. investigate the electrophysiological and morphological properties of serotonergic and dopaminergic subpopulations in the dorsal raphe nucleus (DRN). They performed labor-intensive and rigorous electrophysiology with posthoc immunohistochemistry and neuronal reconstruction to delineate the two major cell classes in the DRN: DRN-DA and DRN-5HT, named according to their primary neurotransmitter machinery. They find that the dopaminergic (DRN-DA) and serotonergic (DRN-5HT) neurons are electrophysiologically and morphologically distinct, and are altered following striatal injection of the toxin 6-OHDA. However, these alterations were largely prevented in DRN-5HT neurons by pre-treatment with desipramine. These findings suggest an important interplay between catecholaminergic systems in healthy and parkinsonian conditions, as well as a relationship between neuronal structure and function.

Strengths:<br /> A large, well-validated dataset that will be a resource for others.<br /> Complementary electrophysiological and anatomical characterizations.<br /> Conclusions are justified by the data.<br /> Relevant for basic scientists interested in DRN cell types and physiology.<br /> Relevant for those interested in serotonin and/or DRN neurons in Parkinson's Disease.

Weaknesses:<br /> Given the scope of the author's questions and hypotheses, I did not identify any major weaknesses.

Reviewer #3 (Public Review):

Ghasemahmad et al. examined behavioral and neurochemical responses of male and female mice to vocalizations associated with mating and restraint. The authors made two significant and exciting discoveries. They revealed that the affective content of vocalizations modulated both behavioral responses and the release of acetylcholine (ACh) and dopamine (DA) but not serotonin (5-HIAA) in the basolateral amygdala (BLA) of male and female mice. Moreover, the results show sex-based differences in behavioral responses to vocalizations associated with mating. The authors conclude that behavior and neurochemical responses in male and female mice are experience-dependent and are altered by vocalizations associated with restraint and mating. The findings suggest that ACh and DA release may shape behavioral responses to context-dependent vocalizations. The study has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the BLA while an animal listens to social vocalizations; however, multiple concerns must be addressed to substantiate their conclusions.

Major concerns:

1. The authors normalized all neurochemical data to the background level obtained from a single pre-stimulus sample immediately preceding playback. The percentage change from the background level was calculated based on a formula, and the underlying concentrations were not reported. The authors should report the sample and background concentrations to make the results and analyses more transparent. The authors stated that NE and 5-HT had low recovery from the mouse brain and hence could not be tracked in the experiment. The authors could be more specific here by relating the concentrations to ACh, DA, and 5-HIAA included in the analyses.

2. For the EXP group, the authors stated that each animal underwent 90-min sessions on two consecutive days that provided mating and restraint experiences. Did the authors record mating or copulation during these experiments? If yes, what was the frequency of copulation? What other behaviors were recorded during these experiences? Did the experiment encompass other courtship behaviors along with mating experiences? Was the female mouse in estrus during the experience sessions?

3. For the mating playback, the authors stated that the mating stimulus blocks contained five exemplars of vocal sequences emitted during mating interactions. The authors should clarify whether the vocal sequences were emitted while animals were mating/copulating or when the male and female mice were inside the test box. If the latter was the case, it might be better to call the playback "courtship playback" instead of "mating playback".

4. Since most differences that the authors reported in Figure 3 were observed in Stim 1 and not in Stim 2, it might be better to perform a temporal analysis - looking at behaviors and neurochemicals over time instead of dividing them into two 10-minute bins. The temporal analysis will provide a more accurate representation of changes in behavior and neurochemicals over time.

5. In Figures 2 and 3, the authors show the correlation between Flinching behavior and ACh concentration. The authors should report correlations between concentrations of all neurochemicals (not just ACh) and all behaviors recorded (not just Flinching), even if they are insignificant. The analyses performed for the stim 1 data should also be performed on the stim 2 data. Reporting these findings would benefit the field.

6. The mice used in the study were between p90 - p180. Although CBA/CaJ mice display normal hearing, sexual behaviors, and social behaviors for at least 1 year (Ohlemiller, Dahl, and Gagnon, JARO 11: 605-623, 2010), the age of the mice covers a range of 90 days. It would strengthen the authors' argument that the affective content of vocalizations modulated both behavioral responses and the release of acetylcholine (ACh) and dopamine (DA) but not serotonin (5-HIAA) in the basolateral amygdala (BLA) of male and female mice if there were no correlations between the magnitude of the neural responses and age.

7. The authors reported neurochemical levels estimated as the animals listened to the sounds played back. What about the sustained effects of changes in neurochemicals? Are there any potential long-term effects of social vocalizations on behavior and neurochemical levels? The authors might consider discussing long-term effects.

8. Histology from a single recording was shown in supplementary figure 1. It would benefit the readers if additional histology was shown for all the animals, not just the colored schematics summarizing the recording probe locations. Further explanation of the track location is also needed to help the readers. Make it clear for the readers which dextran-fluorescein labeling image is associated with which track in the schematic.

9. The authors did not control for the sounds being played back with a speaker. This control may be necessary since the effects are more pronounced in Stim 1 than in Stim 2. Playing white noise rather than restraint or courtship vocalizations would be an excellent control. However, the authors could perform a permutation analysis and computationally break the relationship between what sound is playing and the neurochemical data. This control would allow the authors to show that the actual neurochemical levels are above or below chance.

10. The authors indicated that each animal's post-vocalization session was also recorded. No data in the manuscript related to the post-vocalization playback period was included. This omission was a missed opportunity to show that the neurochemical levels returned to baseline, and the results were not dependent on the normalization process described in major concern #1. The data should be included in the manuscript and analyzed. It would add further support for the model described in Figure 6.

11. The authors could use a predictive model, such as a binary classifier trained on the CSF sampling data, to predict the type of vocalizations played back. The predictive model could support the conclusions and provide additional support for the model in Figure 6.

Reviewer #3 (Public Review):

Using the zebrafish model, this paper by Kraus A. et al., described the anti-virus response in the Olfactory bulb (OB) neurons and microglia. This paper used the behavioral test, neuron calcium imaging, and single-cell transcriptomic analysis. Importantly, this paper discovered that following IHNV infection, the OB neuron increased Pacap expression, which likely protects the neuron cells and mediates the anti-viral defense response. Overall, the findings presented in this paper are quite interesting.

Major strength:<br /> (1) The author demonstrated for the first time that zebrafish OSN neurons sense the IHNV viruses and transmit the viral signal to OB neurons. The zebrafish can be used as a new system to investigate the viral-neuron interaction and understand the mechanisms of how the neurons in the CNS to viral infection through the peripheral chemosensory system.

(2) This paper generated the first zebrafish OB sc-RNA sequencing data. The sc-RNA sequencing data generated in this paper will also help other zebrafish researchers who study the OB neurons.

Major weakness:<br /> The experiment results presented in this paper are not well-integrated. For example, it is unclear how the behavioral phenotype is connected to the neuronal calcium phenotype. It is also unclear how the behavioral or neuronal calcium imaging results is connected to the scRNA sequencing result.

Reviewer #3 (Public Review):

This manuscript aims to exploit experimental measurements of the extracellular voltages produced by colliding action potentials to adjust a simplified model of action potential propagation that is then used to predict the extracellular fields at axon terminals. The overall rationale is that when solving the cable equation (which forms the substrate for models of action potential propagation in axons), the solution for a cable with a closed end can be obtained by a technique of superposition: a spatially reflected solution is added to that for an infinite cable and this ensures by symmetry that no axial current flows at the closed boundary. By this method, the authors calculate the expected extracellular fields for axon terminals in different situations. These fields are of potential interest because, according to the authors, their magnitude can be larger than that of a propagating action potential and may be involved in ephaptic signalling. The authors perform direct measurements of colliding action potentials, in the earthworm giant axon, to parameterise and test their model.

Although simplified models can be useful and the trick of exploiting the collision condition is interesting, I believe there are several significant problems with the rationale, presentation, and application, such that the validity and potential utility of the approach is not established.

Simplified model vs. Hogdkin and Huxley<br /> The authors employ a simplified model that incorporates a two-state membrane (in essence resting and excited states) and adds a recovery mechanism. This generates a propagating wave of excitation and key observables such as propagation speed and action potential width (in space) can be adjusted using a small number of parameters. However, even if a Hodgkin-Huxley model does contain a much larger number of parameters that may be less easy to adjust directly, the basic formalism is known to be accurate and typical modifications of the kinetic parameters are very well understood, even if no direct characterisations already exist or cannot be obtained. I am therefore unconvinced by the utility of abandoning the Hodgkin-Huxley version.

In several places in the manuscript, the simplified model fits the data well whereas the Hodgkin-Huxley model deviates strongly (e.g. Fig. 3CD). This is unsatisfying because it seems unlikely that the phenomenon could not be modelled accurately using the HH formulation. If the authors really wish to assert that it is "not suitable to predict the effects caused by AP [collision]" (p9) they need to provide a good deal more analysis to establish the mechanism of failure.

(In)applicability of the superposition principle<br /> The reflecting boundary at the terminal is implemented using the symmetry of the collision of action potentials. However, at a closed cable there is no reflecting boundary in the extracellular space and this implied assumption is particularly inappropriate where the extracellular field is one objective of the modelling, as here. I believe this assumption is not problematic for the calculation of the intracellular voltage, because extracellular voltage gradients can usually be neglected, but the authors need to explain how the issue was dealt with for the calculation of the extracellular fields of terminals. I assume they were calculated from the membrane currents of one-half of the collision solution, but this does not seem to be explained. It might be worth showing a spatial profile of the calculated field.

Missing demonstrations<br /> Central analytical results are stated rather brusquely, notably equations (3) and (4) and the relation between them. These merit an expanded explanation at the least. A better explanation of the need for the collision measurements in parameterising the models should also be provided.

Adjusted parameters<br /> I am uncomfortable that the parameters adjusted to fit the model are the membrane capacitance and intracellular resistance. These have a physical reality and could easily be measured or estimated quite accurately. With a variation of more than 20-fold reported between the different models in Appendix 2 we can be sure that some of the models are based upon quite unrealistic physical assumptions, which in turn undermines confidence in their generality.

p8 the values of both the extracellular (100 Ohm m) and intracellular resistivity (1 Ohm m) appear to be in error, especially the former.

(In)applicability to axon terminals<br /> The rationale of the application of the collision formalism to axon terminals is somewhat undermined by the fact that they tend not to be excitable. There is experimental evidence for this in the Calyx of Held and the cerebellar pinceau. The solution found via collision is therefore not directly applicable in these cases.

Comparison with experimental data<br /> More effort should be made to compare the modelling with the extracellular terminal fields that have been reported in the literature.

Choice of term "annihilation"<br /> The term annihilation does not seem wholly appropriate to me. The dictionary definitions are something along the lines of complete destruction by an external force or mutual destruction, for example of an electron and a positron. I don't think either applies exactly here. I suggest retaining the notion of collision which is well understood in this context.

Reviewer #3 (Public Review):

The authors utilize chimpanzee-human hybrid cell lines to assess cis-regulatory evolution. These hybrid cell lines offer a well-controlled environment, enabling clear differentiation between cis-regulatory effects and environmental or other trans effects.<br /> In their research, Wang et al. expand the range of chimpanzee-human hybrid cell lines to encompass six new developmental cell types derived from all three germ layers. This expansion allows them to discern cell type-specific cis-regulatory changes between species from more pleiotropic ones. Although the study investigates only two iPSC clones, the RNA- and ATAC-seq data produced for this paper is a valuable resource.

The authors begin their analysis by examining the relationship between allele-specific expression (ASE) as a measure of species divergence and cell type specificity. They find that cell-type-specific genes exhibit more divergent expression. By integrating this data with measures of constraint within human populations, the authors conclude that the increased divergence of tissue-specific genes is, at least in part, attributable to positive selection. A similar pattern emerges when assessing allele-specific chromatin accessibility (ASCA) as a measure of divergence of cis-regulatory elements (CREs) in the same cell lines.

By correlating these two measures, the authors identify 95 CRE-gene pairs where tissue-specific ASE aligns with tissue-specific ASCA. Among these pairs, the authors select two genes of interest for further investigation. Notably, the authors employ an intriguing machine-learning approach in which they compare the inferred chromatin state of the human sequence with that of the chimpanzee sequence to pinpoint putatively causal variants.

Overall, this study delves into the examination of gene expression and chromatin accessibility within hybrid cell lines, showcasing how this data can be leveraged to identify potential causal sequence differences underlying between-species expression changes.

I have three major concerns regarding this study:

1. The only evidence that the cells are indeed differentiated in the right direction is the expression of one prominent marker gene per cell type. Especially for the comparison of conservation between the differentiated cell types, it would be beneficial to describe the cell type diversity and the differentiation success in more detail.

2. Check for a potential confounding effect of sequence similarity on the power to detect ASE or ASCA.

3. In the last part the authors showcase 2 examples for which the log2 fold changes in chromatin state scores as inferred by the machine learning model Sei are used. This is an interesting and creative approach, however, more sanity checks on this application are necessary.

Reviewer #3 (Public Review):

The manuscript by Bimai et al describes a structural and functional characterization of an anaerobic ribonucleotide reductase (RNR) enzyme from the human microbe, P. copri. More specifically, the authors aimed to characterize the mechanism by how (d)ATP modulates nucleotide reduction in this anaerobic RNR, using a combination of enzyme kinetics, binding thermodynamics, and cryo-EM structural determination. One of the principal findings of this paper is the ordering of a NxN 'flap' in the presence of ATP that promotes RNR catalysis and the disordering of both this flap and the glycyl radical domain (GRD) when the inhibitory effector, dATP, binds. The latter is correlated with a loss of substrate binding, which is the likely mechanism for dATP inhibition. It is important to note that the GRD is remote (>30 Ang) from the binding site of the dATP molecule, suggesting long-range communication of the structural (dis)ordering. The authors also present evidence for a shift in oligomerization in the presence of dATP. The work does provide evidence for new insights/views into the subtle differences of nucleotide modulation (allostery) of RNR through long-range interactions.

The strengths of the work are the impressive, in-depth structural analysis of the various regulated forms of PcRNR by (d)ATP using cryo-EM. The authors present seven different models in total, with striking differences in oligomerization and (dis)ordering of select structural features, including the GRD that is integral to catalysis. The authors present several, complementary biochemical experiments (ITC, MST, EPR, kinetics) aimed at resolving the binding and regulatory mechanism of the enzyme by various nucleotides. The authors present a good breadth of the literature in which the focus of allosteric regulation of RNRs has been on the aerobic orthologues.

Given the resolution of some of the structures in the remote regions that appear to be of importance, the rigor of the work could have been improved by complementing this experimental studies with molecular dynamics (MD) simulations to reveal the dynamics of the GRD and loops/flaps at the active site. The biochemical data supporting the loss of substrate binding with dATP association is compelling, but the binding studies of the (d)ATP regulatory molecules are not; the authors noted less-than-unity binding stoichiometries for the effectors. Also, the work would benefit from additional support for oligomerization changes using an additional biochemical/biophysical approach.

Overall, the authors have mostly achieved their overall aims of the manuscript. With focused modifications, including additional control experiments, the manuscript should be a welcomed addition to the RNR field.

Reviewer #3 (Public Review):

Light harvesting (LH) associated with photosynthesis, photoprotection, and the formation of useful pigment-protein complexes are all major functions of carotenoid (Car) pigments. However, the connections between quinone exchange, prokaryotic reaction center (RC)-LH complex formation, and Car depletion in the LH are not entirely understood. This article examined the native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in the filamentous anoxygenic phototroph Roseiflexus castenholzii. The authors show with a high degree of detail using crystallography and Cryo-EM complemented with biophysical techniques important results of a new conformation of a LH. They could assigned the amino acid sequences of subunit X and two hypothetical proteins, Y and Z, that formed the quinone channel and maintained the RC-LH connections. This study identifies a new architectural basis for the regulation of bacterial RC-LH complex and quinone exchange by Cars assembly, which is distinct from the well known purple bacteria. These findings represent a significant advancement of diversity and development of bacterial photosynthetic machinery.

Reviewer #3 (Public Review):

The present paper uncovers evidence of the coordination of two brain areas involved in a two-step learning process in birdsong plasticity. Indeed, songbirds can modify their song based on an error-correction mechanism that involves a motor bias expressed by a basal ganglia-thalamo-cortical loop. After training (hundreds or a few thousands of renditions), the motor bias necessary to correct vocal errors becomes independent of the BG-thalamo-cortical loop and is transferred into the long-term motor program stored in a primary motor network. Current understanding claims that the output nucleus of the BG-thalamo-cortical loop, LMAN, trains the primary motor networks (in area RA) to drive the learning transfer. However, no clear evidence for such entrainment was available until now. In the present study, the authors elegantly show that correlations in trial-by-trial fluctuations in the premotor activity in LMAN and RA are present spontaneously (in multi-unit electrophysiological recordings) and are increased during a lab-induced plasticity protocol. The change in correlation is specific to the syllable that undergoes plasticity. Moreover, perturbing LMAN activity through low-intensity and spatially broad electrical stimulation of LMAN during the premotor window prevents behavioral adaptation. Altogether, their results convincingly show that the entrainment of RA neural populations by LMAN neurons is present during baseline, strengthened during plasticity in a syllable-specific manner, and necessary for song plasticity.

This study thus provides important validation of the current model for the 2-step learning process underlying song learning and plasticity, where a BG-thalamo-cortical network drive motor bias to correct vocal errors based on a reinforcement learning mechanism, while the song motor engram is updated slowly through the adjustment of song-related activity in the primary motor areas. Beyond the songbird field, these results will be of importance to all studying sensorimotor learning and adaptation, and more broadly the formation of memory through a two-step learning process.

The authors present the context for their hypothesis clearly, state their hypothesis precisely, and conduct a thorough investigation of the posed question. The conclusions are well supported by data.

In particular, the statistical evaluation of the covariance of LMAN and RA activity in the premotor window is adequate and the interpretation of the results is therefore well backed by their analysis. The methods used here to assess covariation between LMAN and RA activity during singing set the ground for future studies looking at the coordination between brain areas during behavior.

Reviewer #3 (Public Review):

Jie Yang et al. investigated the transgenerational behavioral modification of a high-sugar diet (HSD) in Drosophila and revealed the underlying molecular and neural mechanisms. It has been reported that HSD exposure decreases sweet sensitivity in gustatory sensory neurons, resulting in reduced sugar response (Proboscis extension reflex, PER) in flies. The current study reports that this effect can be transmitted across generations through the maternal germline. Furthermore, the authors show that H3K27me3 modification is enhanced in the first-generation progenies of HSD-treated flies (F1), and genetical or pharmacological disruption of PCL-PRC2 complex blocks the behavioral change and restores the sweet sensitivity in the Gr5a+ sweet sensory neurons. The authors further analyze the differentially expressed genes in the F1 flies. Among H3K27me3 hypermethylated regions, they focus on homeobox genes and find a transcription factor Caudal (Cad), which shows decreased expression in the F1 flies. Knocking down Cad in Gr5a+ neurons results in decreased PER response to sucrose.

Transgenerational changes in physiology and metabolism have been broadly studied, while inherited changes at the behavioral level are much less investigated. This work provides convincing evidence for transgenerational modification of feeding behavior and digs out the underlying molecular and neural mechanisms. However, there still are several concerns that need to be clarified.

1) The epigenetic regulator PCR2 has been found to play an essential role in the 7d-HSD-induced modification of the PER response. In this study, it's important to clarify for the transgenerational change, whether epigenetic modification is required in the flies exposed to HSD (F0), the progenies (F1), or both. It would be very helpful for better interpretation if the procedures of HSD treatment in RNAi experiments and the drug treatments were stated in more detail. In addition, the F0 flies should be examined as the control.<br /> 2) The information on the drug treatment period is also missing for imaging experiments (Fig.4C). Moreover, the response curve is very different from those recorded in the same neurons in previous studies. What's the reason? Please also provide a representative image showing which part of the Gr5a neurons is recorded.<br /> 3) It's unclear whether the decreased Cad expression upon HSD treatment specifically occurred in Gr5a+ neurons or a lot of cells. If the change in gene expression is significant in the qPCR test, it should occur in a large number of cells, most likely including different types of gustatory sensory neurons. If lower cad expression led to lower neural response and thereby lower behavioral response, how to specifically decrease the PER response to sucrose but not to other tastes? --whether HSD-induced desensitization is specific to sucrose in the offspring?<br /> 4) In Fig.2D, data are sorted for genomic regions showing an up-regulated modification of H3K27me. It's unclear whether similar sorting was performed in panel C. This needs to be clarified.

Reviewer #3 (Public Review):

The authors aim to gain a more comprehensive understanding of the role of FIKK4.1 in parasite biology. To achieve this, they used a novel approach termed PerTurboID that allows them to map changes in the conformational and interaction environment of proteins that are in close proximity of the tagged gene of interest. Here the authors focus on two proteins KHARP and PTP4 who are known targets of FIKK4.1 and assessed the impact of the genetic disruption of the kinase on the interaction environment of these proteins. The experimental strategy identifies a range of changes that indicate that changes go beyond the direct targets of FIKK4.1 and therefore creates new insights of interaction networks that are regulated by this specific kinase.

The strength of this approach is not only that it can identify new interaction networks relating to FIKK4.1 but that serves as a proof of concept that can be used for a wide range of applications in parasite biology. At the same time as the authors have noted themselves the extent of the biotin pulse is important and most likely needs to be calibrated for every specific application. In addition, this approach is only suitable for proteins that can be tagged without impacting their function.

The authors present very convincing evidence that the PerTurboID is suitable to study FIKK kinases in parasites and have used this to shed new light on how FIKK4.1 is involved directly or indirectly in a wider range of biological activities in the parasite.

The main impact of this work is that it provides a wider understanding of the relationship between a specific kinase and structural as well as biological consequences. The methodology is also very powerful and will have a wide range of applications.

Reviewer #3 (Public Review):

Summary:<br /> The authors present a biophysically detailed model of the basolateral amygdala (BLA) that is capable of fear learning through a depression-dominated spike-timing dependent plasticity (STDP) mechanism. Furthermore, the model also replicates experimentally measured rhythmic signatures of baseline amygdala activity and changes of these signatures during and after fear learning. The authors furthermore carefully dissect the contributions of the three different types of interneurons (parvalbumin-positive (PV), somatostatin-positive (SOM), and vaso-active peptide-positive (VIP) interneurons) in regulating network activity to allow for the association between conditioned and unconditioned stimuli.

Strengths:<br /> The biophysical detail of the model allows the authors to go beyond a simple modelling of the fear learning process in terms of spiking activity of the principal cells and to link the associative learning to several oscillatory rhythms in the BLA, namely high and low theta and gamma rhythms. This provides an understanding of the generation and function of these rhythms in the baseline amygdala circuit as well as of the functional consequences of alterations of these rhythms during and after the fear learning process. This offers a new and uniquely detailed insight into the mechanistic level.

Weaknesses:<br /> The main weakness of the approach is the lack of experimental data from the BLA to constrain the biophysical models. This forces the authors to use models based on other brain regions and leaves open the question of whether the model really faithfully represents the basolateral amygdala circuitry. Furthermore, the authors chose to use model neurons without a representation of the morphology. However, given that PV and SOM cells are known to preferentially target different parts of pyramidal cells and given that the model relies on a strong inhibition form SOM to silence pyramidal cells, the question arises whether SOM inhibition at the apical dendrite in a model representing pyramidal cell morphology would still be sufficient to provide enough inhibition to silence pyramidal firing. Lastly, the fear learning relies on the presentation of the unconditioned stimulus over a long period of time (40 seconds). The authors justify this long-lasting input as reflecting not only the stimulus itself but as a memory of the US that is present over this extended time period. However, the experimental evidence for this presented in the paper is only very weak.

The authors achieved the aim of constructing a biophysically detailed model of the BLA not only capable of fear learning but also showing spectral signatures seen in vivo. The presented results support the conclusions with the exception of a potential alternative circuit mechanism demonstrating fear learning based on a classical Hebbian (i.e. non-depression-dominated) plasticity rule, which would not require the intricate interplay between the inhibitory interneurons. This alternative circuit is mentioned but a more detailed comparison between it and the proposed circuitry is warranted.

The presented model demonstrates how the complex interplay between different types of interneurons is able to precisely control neural activity to enable learning to happen. Furthermore, the presented work shows this interactive control of activity by the interneurons gives rise to specific oscillatory signatures. Since the three types of interneurons considered here are found throughout the brain, the findings will likely have a big impact on other studies of interneuron function and learning in general.

Reviewer #3 (Public Review):

In the present study, Iversen et al investigate the effect of middle cerebral artery occlusion (MCAo) on penumbral capillary blood flow in rat brains. Using Laser Speckle Contrast imaging and two-photon microscopy, they found that during MCAo the red blood cell dynamics become chaotic in penumbral capillaries despite an apparent constant residual blood flow. They further conclude that these disturbances would cause decreases in steady-state cerebral metabolic rate of oxygen (CMRO2), and tissue oxygen tension (PtO2) using a post hoc biophysical model for oxygen extraction. Interestingly, the authors present data excluding a role for pericytes in altering capillary blood flow. From this observation, the study raises potentially interesting questions on the origin of the disturbance but fails to address them by not investigating the upstream arteriolar behavior. Increased vasomotion, palpability, or intermittent vasospasm may trigger capillary blood flow disturbances without necessarily impacting residual blood flow resting as measured by Laser Speckle Contrast imaging. Furthermore, the data are very poorly presented, here are some examples:<br /> Fig 1b is incorrectly labeled and, assuming this is the "first" 1f panel, the scale bar shows 500 µm while the legend says 200.<br /> Fig 1d is poorly convincing as pink or grey, as detailed in the legend, are not visible. It also looks like there is a second core and penumbra on the more rostral left part of the brain.<br /> Line 219 time is misspelled.<br /> Fig 2, what does "percent of alle capillaries" on the y axes mean? 2d is presented before 2c in the text.<br /> What is the rationale for presenting the statistics from Fig 3 in Fig 4? Panels 4e and 4f are not discussed. The reference in the Fig 4 legend is not formatted.<br /> Fig 6 is presented before Fig 5.<br /> The overall lack of a central hypothesis combined with the aforementioned weaknesses prevents the study from achieving its proposed goal "to characterize microvascular flow disturbances in penumbral tissue in a rat model of acute ischemic stroke".

Reviewer #3 (Public Review):

Summary:<br /> Bidirectional transsynaptic signaling via cell adhesion molecules and cell surface receptors contributes to the remarkable specificity of synaptic connectivity in the brain. Zaman et al., investigate how the receptor tyrosine kinase Kit and its trans-cellular kit ligand regulate molecular layer interneuron (MLI)- Purkinje cell (PC) connectivity in the cerebellum. Presynaptic Kit is specific for MLIs, and forms a trans-synaptic complex with Kit ligand in postsynaptic PC cells. The authors begin by generating Kit cKOs via an EUCOMM allele to enable cell-type specific Kit deletion. They cross this Kit cKO to the MLI-specific driver Pax2-Cre and conduct validation via Kit IHC and immunoblotting. Using this system to examine the functional consequences of presynaptic MLI Kit deletion onto postsynaptic PC cells, they record spontaneous and miniature synaptic currents from PC cells and find a selective reduction in IPSC frequency. Deletion of Kit ligand from postsynaptic PC cells also results in reduced IPSC frequency, together supporting that this trans-synaptic complex regulates GABAergic synaptic formation or maturation. The authors then show that sparse Kit ligand overexpression in PCs decreases neighboring uninfected control sIPSCs in a potentially competitive manner.

Strengths:<br /> Overall, the study addresses an important open question, the data largely support the authors' conclusions, the experiments appear well-performed, and the manuscript is well-written. I just have a few suggestions to help shore up the author's interpretations and improve the study.

Weaknesses:<br /> The strong decrease in sIPSC frequency and amplitude in control uninfected cells in Figure 4 is surprising and puzzling. The competition model proposed is one possibility, and I think the authors need to do additional experiments to help support or refute this model. The authors can conduct similar synaptic staining experiments as in Fig S4 but in their sparse infection paradigm, comparing synapses on infected and uninfected cells. Additional electrophysiological parameters in the sparse injection paradigm, such as mIPSCs or evoked IPSCs, would also help support their conclusions.

The authors should validate KL overexpression and increased cell surface levels using their virus to support their overexpression conclusions.

Reviewer #3 (Public Review):

This manuscript provides a more or less quantitative analysis of protein synthesis in lymphocytes. I have no issue with the data as presented, as I'm sure all measurements have been expertly done. I see no need for additional experimental work, although it would be helpful if the authors could comment on the possibility of measuring the rate of synthesis of a defined protein, say a histone, in cells prior to and after activation. The conclusion the authors leave us with is the idea that the rates of protein synthesis recorded here are incompatible with observed rates of T cell division in vivo. Indeed, in the final paragraph of the discussion, the authors note the mismatch between what they consider a requirement for cell division, and the observed rates of protein synthesis. They then invoke unconventional mechanisms to make up for the shortfall, without -in this reviewer's opinion- discussing in adequate detail the technical limitations of the methodology used.

A key question is the broad interest, novelty, and extension of current knowledge, in comparison with Argüello's (reference 27) 'SunRise' method. It would be helpful for the authors to stake out a clear position as to the similarities and differences with reference 27: what have we learned that is new? The authors could cite reference 27 in the introduction of their manuscript, given the similarity in approach. That said, the findings reported here will generate further discussion.

The manuscript would increase in impact if the authors were to clearly define why a particular measurement is important and then show the actual experiment/result. As an example, it would be helpful to explain to the non-expert why the distinction between monosomes, polysomes, and stalled versions of the same is important, and then explain the rationale of the actual experiment: how can these distinctions be made with confidence, and what are confounding variables? The initial use of human cells, later abandoned in favor of the OT-1 in vitro and in vivo models, requires contextualization. If the goal is to address the relationship between rates of translation and cell division of antigen-activated T cells in vivo, then a lot of the work on the human model and the in vitro experiments becomes more of a distraction, unless properly contextualized. Is there any reason to assume that antigen-specific activation in vivo will impact translation differently than the use of the PMA/ionomycin/IL2 cocktail? The way the work is presented leaves me with the impression that everything that was done is included, regardless of whether it goes to the core of the question(s) of interest.

It would be helpful if the authors made explicit some of the assumptions that underlie their quantitative comparisons. Likewise, the authors should discuss the limitations of their methods and provide alternative interpretations where possible, even if they consider them less/not plausible, with justification. As they themselves note, improvements in the RPM protocols raised the increase in translating ribosomes upon activation from 10-fold to 15-fold. Who's to say that is the best achievable result? What about the reliability/optimization of the other measurements?

The composition of the set of proteins produced upon activation will differ from cell to cell (CD4, CD8, B, resting vs. dividing). Even if analyses are performed on fixed cells, the ability of the monoclonal anti-puromycin antibody to penetrate the matrix of the various fixed cell types may not be equal for all of them, depending on protein composition, susceptibility to fixation etc. Is it possible for puromycin to occupy the ribosome's A site and terminate translation without forming a covalent bond with the nascent chain? This could affect the staining with anti-puromycin antibodies and also underestimate the number of nascent chains.

I believe that the concept of FACS-based quantitation also requires an explanation for the non-expert. For the FACS plots shown, the differences between the highest and lowest RPM scores for cells that divided and that have a similar CFSE score is at least 10-fold. Does that mean that divided cells can differ by that margin in terms of the number of nascent chains present? If I make the assumption that cells stimulated with PMA/ionomycin/IL2 respond more or less synchronously, why would there be a 10-fold difference in absolute fluorescence intensity (anti=puromycin) for randomly chosen cells with similar CFSE values? While the use of MFI values is standard practice in cytofluorimetry, the authors should devote some comments to such variation at the population level.

It is assumed that for cells to complete division, they must have produced a full and complete copy of their proteome and only then divide. What if cells can proceed to divide even when expressing a subset of the proteome of departure (=the threshold set required for initiation of division), only to complete synthesis of the 'missing ' portion once cell division is complete? Would this obviate the requirement for an unusual mechanism of protein acquisition (trogocytosis; other)?

Translation is estimated to proceed at a rate of ~6 amino acids per second, but surely there is variability in this number attributable to inaccuracies of the methods used, in addition to biological variability. Were these so-called standard values determined for a range of different tissues? It stands to reason that there might be variation depending on the availability of initiation/elongation factors, NTPs, aminoacyl tRNAs etc. What is the margin of error in calculating chain elongation rates based on the results shown here?

Reviewer #3 (Public Review):

The authors introduce two new concepts for antimicrobial resistance borrowed from pharmacology, "variant vulnerability" (how susceptible a particular resistance gene variant is across a class of drugs) and "drug applicability" (how useful a particular drug is against multiple allelic variants). They group both terms under an umbrella term "drugability". They demonstrate these features for an important class of antibiotics, the beta-lactams, and allelic variants of TEM-1 beta-lactamase.

The strength of the result is in its conceptual advance and that the concepts seem to work for beta-lactam resistance. However, I do not necessarily see the advance of lumping both terms under "drugability", as this adds an extra layer of complication in my opinion.

I also think that the utility of the terms could be more comprehensively demonstrated by using examples across different antibiotic classes and/or resistance genes. For instance, another good model with published data might have been trimethoprim resistance, which arises through point mutations in the folA gene (although, clinical resistance tends to be instead conferred by a suite of horizontally acquired dihydrofolate reductase genes, which are not so closely related as the TEM variants explored here).

The impact of the work on the field depends on a more comprehensive demonstration of the applicability of these new concepts to other drugs.

Reviewer #3 (Public Review):

This study performs in vivo recordings of neurons in the mouse superior colliculus and their afferents from the retina, retinal ganglion cells (RGCs). Building on a preparation they previously published, this study adds the use of optogenetic identification of inhibitory neurons (aka optotagging) to compare RGC connectivity to excitatory and inhibitory neurons in SC. Using this approach, the authors characterize connection probability, strength, and response correlation between RGCs and their target neurons in SC, finding several differences from what is observed in the retina-thalamus-visual cortex pathway. As such, this may be a useful dataset for efforts to understand retinocollicular connectivity and computations.

Reviewer #3 (Public Review):

This manuscript describes the development of CRISPR knockouts for gh, fsh and tsh in the fast-aging Nothobranchius furzeri grz strain. CRISPR knockouts have been published before, and the strength of the paper is that here, the authors include a novel, easy and fast way of rescuing the loss of function in the entire body by electroporation in muscle. This offers flexibility in timing and dosage, and leads to intriguing results regarding the role of these hormones in growth and fertility. Finally they also add a conditional doxycycline-dependent overexpression model that would allow even more control over the modalities of the rescue. The phenotypes of the knockouts were not the key message of the paper and remained at times only superficially described. The doxycycline-dependent overexpression was only minimally validated, and here it is not yet clear how robust this system is in terms of overexpression levels, timing, and reversibility.

Overall this study brings a new set of tools in the killifish toolbox that can have much wider applications and will be appreciated also in other teleost models.

Reviewer #3 (Public Review):

The present study used novel data logging devices to record the foraging behavior of wandering albatrosses. Specifically, the authors showed how localized winds and wave heights influence their ability to take off from the sea surface, which is the most expensive behavior they engage in while foraging. There is no better platform for this initial work because these birds are so large, the equipment they can carry without creating significant impact is tremendous.

The results were impressive, presented well, and the paper was generally written in an accessible way to readers with less knowledge. The authors provide a convincing set of results that support the aims and conclusions. The theory and application could be used to inform our understanding of flight behavior in other seabirds.

Although the idea of taking off from the sea surface may sound trivial, it is essential to understand that albatrosses and other soaring seabirds have wings that are adapted for soaring (i.e. long and narrow). The trade off, however, is that powered flight through wing flapping is energetically expensive because the wings have a shallow amplitude and generate less power compared to a shorter, wider wing. Thus, wind is everything and this study shows how wind facilitates the ability of the birds to gain flight using wind and waves. Awesome!

Reviewer #3 (Public Review):

In this work, the authors tried to profile time-dependent changes in gene and protein expression during BMP-induced amnion differentiation from hPSCs. The authors depicted a GATA3 - TFAP2A - ISL1/HAND1 order of amniotic gene activation, which provides a more detailed temporary trajectory of amnion differentiation compared to previous works. As a primary goal of this study, the above temporal gene/protein activation order is amply supported by experimental data. However, the mechanistic insights on amniotic fate decision, as well as the transcriptomic analysis comparing amnion-like cells from this work and other works remain limited. While this work allows us to see more details of amnion differentiation and understand how different transcription factors were turned on in a sequence and might be useful for benchmarking the identity of amnion in ex utero cultured human embryos/embryoids, it provides limited insights on how amnion cells might diverge from primitive streak / mesoderm-like cells, despite some transcriptional similarity they shared, during early development.

Reviewer #3 (Public Review):

In this manuscript by Berrocal and coworkers, the authors do a deep dive into the transcriptional regulation of the eve gene in both an endogenous and ectopic background. The idea is that by looking at eve expression under non-native conditions, one might infer how enhancers control transcriptional bursting. The main conclusion is that eve enhancers have not evolved to have specific behaviors in the eve stripes, but rather the same rates in the telegraph model are utilized as control rates even under ectopic or 'de novo' conditions. For example, they achieve ectopic expression (outside of the canonical eve stripes) through a BAC construct where the binding sites for the TF Giant are disrupted along with one of the eve enhancers. Perhaps the most general conclusion is that burst duration is largely constant throughout at ~ 1 - 2 min. This conclusion is consistent with work in human cell lines that enhancers mostly control frequency and that burst duration is largely conserved across genes, pointing to an underlying mechanistic basis that has yet to be determined.

Reviewer #3 (Public Review):

Strengths:

On the positive side, I thought the use of ChatGPT to score the sentiment of text was novel and interesting, and I was largely convinced by the parts of the methods which illustrate that the AI provides broadly similar sentiment and politeness scores to humans who were asked to rank a sub-set of the reviews. The paper is mostly clear and well-written, and tackles a question of importance and broad interest (i.e. the potential for bias in the peer review process, and the objectivity of peer review).

Weaknesses:

The sample size and scope of the paper are a bit limited, and I have concerns covering diverse aspects including statistical/inferential issues, missing references, and suggestions for other material that could be included that would greatly increase the usefulness of the paper. A major limitation is that the paper focuses on published papers, and thus is a biased sample of all the reviews that were written, which prevents the paper properly answering the questions that it sets out to answer (e.g. is peer review repeatable, fair and objective).

Reviewer #3 (Public Review):

This paper reports a considerable technical achievement: the optogenetic activation of single retinal ganglion cells in vivo in monkeys. As clearly specified in the paper, this is an important step towards causal tests of the role of specific ganglion cell types in visual perception. Yet this methodological advance is not described currently in sufficient detail to replicate or evaluate. The paper could be improved substantially by including additional methodological details. Some specific suggestions follow.

The start of the results needs a paragraph or more to outline how you got to Figure 1. Figure 1 itself lacks scale bars, and it is unclear, for example, that the ganglion cells targeted are in the foveal slope.

The text mentions the potential difficulties targeting ganglion cells at larger eccentricities where the soma density increases. If this is something that you have tried it would be nice to include some of that data (whether or not selective activation was possible). Related to this point, it would be helpful to include a summary of the ganglion cell density in monkey retina.

Related to the point in the previous paragraph - do you have any experiments in which you systematically moved the stimulation spot away from the target ganglion cell to directly test the dependence of stimulation on distance? This would be a valuable addition to the paper.

The activity in Figure 1 recovers from activation very slowly - much more slowly than the light response of these cells, and much more slowly than the activity elicited in most optogenetic studies. Can you quantify this time course and comment on why it might be so slow?

Traces from non-targeted cells should be shown in Figure 1 along with those of targeted cells.

Reviewer #3 (Public Review):

a) Important findings<br /> - This study confirms that Gr28 subfamily members are expressed in distinct sets of taste neurons in Drosophila larvae, supporting previous findings (e.g., Kwon et al., 2011).<br /> - Neurons expressing different members of the Gr28 family exhibit distinct behavioral responses when chemically activated with capsaicin.<br /> - Silencing experiments reveal that neurons expressing Gr28bc are necessary for larval avoidance of four bitter compounds, whereas neurons expressing Gr28be are necessary for avoiding lobeline and caffeine.<br /> - Inserting either Gr28ba or Gr28bc into the GR28 mutant line restored larval avoidance of denatonium.<br /> - Calcium imaging experiments show that Gr28ba and Gr28bc are involved in sensing denatonium, while none of the GR28 family members are involved in detecting quinine.

b) Caveats<br /> - The authors did not acknowledge that neurons expressing members of the GR28 family also express other Gr family members, which could potentially contribute to the detection and behavioral responses to the tested bitter compounds.<br /> - Gal4 lines from various studies exhibit varying expression patterns, highlighting the necessity for improved reagents. These findings also suggest the importance of employing different Gal4 lines for each receptor to validate the results of the current study.<br /> - Activating or silencing neurons pertains to the function of the neurons rather than the receptors.<br /> - Inconsistency is observed in the use of different reagents across the experiments. Specifically, all six Gal4 lines were utilized in the Chemical Activation experiments, while only two lines were employed in the silencing experiments.<br /> - The Alphafold structure prediction is exciting but lacks conclusive evidence.

Reviewer #3 (Public Review):

Leeds et al. employ elegant in vitro experiments and sophisticated numerical modeling to investigate the ability of mechanical coupling to coordinate the growth of individual microtubules within microtubule bundles, specifically k-fibers. While individual microtubules naturally polymerize at varying rates, their growth must be tightly regulated to function as a cohesive unit during chromosome segregation. Although this coordination could potentially be achieved biochemically through selective binding of polymerases and depolymerases, the authors demonstrate, using a novel dual laser trap assay, that mechanical coupling alone can also coordinate the growth of in vitro microtubule pairs.

By reanalyzing recordings of single microtubules growing under constant force (data from their own previous work), the authors investigate the stochastic kinetics of pausing and show that pausing is suppressed by tension. Using a constant shared load, the authors then show that filament growth is tightly coordinated when pairs of microtubules are mechanically coupled by a material with sufficient stiffness. In addition, the authors develop a theoretical model to describe both the natural variability and force dependence of growth, using no freely adjustable parameters. Simulations based on this model, which accounts for stochastic force-dependent pausing and intrinsic variability in microtubule growth rate, fit the dual-trap data well.

Overall, this study illuminates the potential of mechanical coupling in coordinating microtubule growth and offers a framework for modeling k-fibers under shared loads. The research exhibits meticulous technical rigor and is presented with exceptional clarity. It provides compelling evidence that a minimal, reconstituted biological system can exhibit complex behavior. As it currently stands, the paper is highly informative and valuable to the field.

To provide further clarity regarding the implications of their study, the authors may wish to address the following points in more detail:

- Considering the authors' understanding of the quantitative relationship between forces, microtubule growth, and coordination, is the dual trap assay necessary to demonstrate this coordination? What advantages does the (semi)experimental system offer compared to a purely in silico treatment?

- What are the limitations of studying a system comprising only two individual microtubules? How might the presence of crosslinkers, which are typically present in vivo between microtubules, influence their behavior in this context?

- How dependent are the results on the chosen segmentation algorithm? Can the distributions of pause and run durations truly be fitted by "simple" Gaussians, as indicated in Figure S5-2? Given the inherent limitations in accurately measuring short durations and the application of threshold durations, it is likely that the first bins in the histograms underestimate events. Cumulative plots could potentially address this issue.

Reviewer #3 (Public Review):

The authors investigated the initial steps involved in angiogenesis. Using appropriate experimental tools they associated engineered vasculature models with a strong mathematical analysis. The study provides a dynamic view of the early steps involved in angiogenesis. It shows significant fluctuations in the onset of angiogenesis that suggest transitions between order and disorder in cell organization. The data obtained strongly support the hypothesis and support the conclusion of the study. This work brings new insights into the comprehension of the complex processes involved in the onset of angiogenesis and it provides a strong model to predict how VEGF will activate the delta-NOTCH signaling. Nevertheless, it would be important to describe in more detail how the current study can be used for a better understanding of the angiogenesis process in physiological and in pathological situations.

Reviewer #3 (Public Review):

Prior work from the Kaverina lab and others had determined that beta-cells build a microtubule network that differs from the canonical radial organization typical in most mammalian cell types and that this organization facilitates the regulated secretion of insulin-containing secretory granules (IGs). In this manuscript, the authors tested the hypothesis that kinesin-driven microtubule sliding is an underlying mechanism that establishes a sub-membranous microtubule array that regulates IG secretion. They employed knock-down and dominant-negative strategies to convincingly show microtubule sliding does, in fact, drive the assembly of the sub-membranous microtubule band. They also used live cell imaging assays to demonstrate that kinesin-mediated microtubule sliding in beta-cells is triggered by extracellular high glucose. Overall, this is an interesting and important study that relates microtubule dynamics to an important physiological process. The experiments were rigorous and well-controlled.

Reviewer #3 (Public Review):

Summary:<br /> 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.

Major comments:<br /> 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.

Reviewer #3 (Public Review):

Summary:<br /> Variants in the UBA5 gene are associated with rare developmental and epileptic encephalopathy, DEE44. This research developed a system to assess in vivo and in vitro genotype-phenotype relationships between UBA5 allele series by humanized UBA5 fly models and biochemical activity assays. This study provides a basis for evaluating current and future individuals afflicted with this rare disease.

Strengths:<br /> The authors developed a method to measure the enzymatic reaction activity of UBA5 mutants over time by applying the UbiReal method, which can monitor each reaction step of ubiquitination in real time using fluorescence polarization. They also classified fruit fly carrying humanized UBA5 variants into groups based on phenotype. They found a correlation between biochemical UBA5 activity and phenotype severity.

Weaknesses:<br /> In the case of human DEE44, compound heterozygotes with both loss-of-function and hypomorphic forms (e.g., p.Ala371Thr, p.Asp389Gly, p.Asp389Tyr) may cause disease states. The presented models have failed to evaluate such cases.

Reviewer #3 (Public Review):

The work presented in the manuscript tries to identify tRNA modifications present in Mycobacterium tuberculosis (Mtb) using reverse transcription-derived error signatures with tRNA-seq. The study identified enzyme homologs and correlates them with presence of respective tRNA modifications in Mtb. The study used several chemical treatments (IAA and alkali treatment) to further enhance the reverse transcription signals and confirms the presence of modifications in the bases. tRNA modifications by two enzymes TruB and MnmA were established by doing tRNA-seq of respective deletion mutants. Ultimately, authors show that MnmA-dependent tRNA modification is important for intracellular growth of Mtb. Overall, this report identifies multiple tRNA modifications and discuss their implication in Mtb infection.

Reviewer #3 (Public Review):

In this study, the authors sought to test the hypothesis that blocking triglyceride storage in adipose tissue by knockout of DGAT1 and DGAT2 in adipocytes would lead to ectopic lipid deposition, lipodystrophy, and impaired glucose homeostasis. Surprisingly, the authors found the opposite result, with DGAT1/2 DKO in adipocytes leading to increased energy expenditure, minimal ectopic lipid deposition, and improved glucose homeostasis with HFD feeding. These metabolic improvements were largely attributed to increased beiging of the white fat and increased brown adipose tissue activity. This study provides an interesting new paradigm whereby impairing fat storage, the major function of adipose tissue, does not lead to severe metabolic disease, but rather improves it. The authors provide a comprehensive assessment of the metabolism of these DKO mice under chow and HFD conditions, which support their claims. The study lacks in mechanistic insight, which would strengthen the study, but does not detract from the authors' major conclusions.

Reviewer #3 (Public Review):

In this study, Sun et al examine the role of the splicing factor SRSF1 in spermatogenesis in mice. Alternative splicing is important for spermatogenic development, but its regulation and major developmental roles during spermatogenesis are not well understood. The authors set out to better define both SRSF1 function in testes and the contribution of alternative splicing. They collect several large 'omics datasets to define SRSF1 targets in testis, including RNA interactions by CLIP-seq in whole testis, protein interactions by IP-mass spec in whole testis, and RNA sequencing to detect expression levels and splice variants. They also examine the phenotype of germline conditional knockouts (cKO) for Srsf1, using the early-acting Vasa-Cre, and find a severe depletion of germ cells starting at 7 days post partum (dpp) and culminating with a lack of germ cells (Sertoli Cell Only Syndrome) by adulthood. They detect differences in gene expression as well as differences in splicing between control and knockout, including 9 genes that are downregulated, experience alternative splicing, and whose transcripts are also bound by SRSF1, and identify the Tial1/Tiar transcript as one of these targets. They conclude that SRSF1 is required for homing and self-renewal of spermatogonial stem cells, at least in part through its regulation of Tial1/Tiar splicing.

Strengths of the paper include detailed phenotyping of the Srsf1 cKO, which convincingly supports the Sertoli Cell Only phenotype, establishes the timing of the first appearance of the spermatogonial defect, and provides new insight into the role of splicing factors and SRSF1 specifically in spermatogenesis. Another strength is the generation of CLIP-seq, IP-MS, and RNA-seq datasets which will be a useful resource for the field of germ cell development. Major weaknesses include a lack of robust support for two major claims: first, there is inadequate support for the claim of defects in either "homing" or "self-renewal" of spermatogonia in the cKO, and second, there is inadequate support for the claim that altered splicing of the Tial1 transcript mediates the effect of SRSF1 loss. A moderate weakness is the superficial discussion of the CLIP, RNA-seq, and IP-MS datasets, limiting their otherwise high utility for other researchers. Overall, the paper as it stands will have a moderate impact on the field of male reproductive biology. Specific points that should be addressed to improve support for the claims are below.

Major comments

1) In Fig 1D, it appears that SRSF1 is expressed most strongly in spermatogonia by immunofluorescence, but this is inconsistent with the sharp rise in expression detected by RT-qPCR at 20 days post partum (dpp) (Fig. 1B), which is when round spermatids are first added; this discrepancy should be explained or addressed.

2) It is important to provide a more comprehensive basic description of the CLIP-seq datasets beyond what is shown in the tracks shown in Fig. 2B. This would allow a better assessment of the data quality and would also provide information about the transcriptome-wide patterns of SRSF1 binding. No information or quality metrics are provided about the libraries, and it is not stated how replicates are handled to maximize the robustness of the analysis. The distribution of peaks across exons, introns, and other genomic elements should also be shown.

3) The claim that SRSF1 is required for "homing and self-renewal" of SSCs is made in multiple places in the manuscript. However, neither homing nor self-renewal is ever directly tested. A single image is shown in Fig. 5E of a spermatogonium at 5dpp that does not appropriately sit on the basal membrane, potentially indicating a homing defect, but this is not quantified or followed up. There is good evidence for depletion of spermatogonia starting at 7 dpp, but no further explanation of how homing and/or self-renewal fit into the phenotype.

4) In Fig. 6A (lines 258-260) very few genes downregulated in the cKO are bound by SRSF1 and undergo abnormal splicing. The small handful that falls into this overlap could simply be noise. A much larger fraction of differentially spliced genes are CLIP-seq targets (~33%), which is potentially interesting, but this set of genes is not explored.

5) The background gene set for Gene Ontology analyses is not specified. If these were done with the whole transcriptome as background, one would expect enrichment of spermatogenesis genes simply because they are expressed in testes. The more appropriate set of genes to use as background in these analyses is the total set of genes that are expressed in testis.

6) In general, the model is over-claimed: aside from interactions by IP-MS, little is demonstrated in this study about how SRSF1 affects alternative splicing in spermatogenesis, or how alternative splicing of TIAL1 specifically would result in the phenotype shown. It is not clear why Tial1/Tiar is selected as a candidate mediator of SRSF1 function from among the nine genes that are downregulated in the cKO, are bound by SRSF1, and undergo abnormal splicing. Although TIAL1 levels are reduced in cKO testes by Western blot (Fig. 7J), this could be due just be due to a depletion of germ cells from whole testis. The reported splicing difference for Tial1 seems very subtle and the ratio of isoforms does not look different in the Western blot image.

Reviewer #3 (Public Review):<br /> <br /> Tutor et al. present their work on Kelch13/K13 from Plasmodium falciparum, the causative agent of malaria. This protein is involved in resistance against artemisinin (ART), one of the most commonly used drugs to treat malaria. Despite having identified the mutation in K13 that leads to resistance to ART, the exact molecular mechanism, function of K13, and impact of the K13 mutations still need to be elucidated. This is where the authors step in to investigate the relationship between endocytosis and K13, as well as the impact of depleting the protein using knock-sideway (KS). Using light microscopy, the authors demonstrate how K13-YFP forms a pore associated with fluorescently labeled dextran, which is taken up into tubules that move toward the digestive vacuole. This tubule formation is not sensitive to jasplakinolide (JAS) treatment. Using electron microscopy, they show that K13 is localized at the dark contrast border of the cytostome, and knocking down K13 leads to the disruption of the cytostome structure. Upon removal of K13, the structure changes, and the opening enlarges. The impact of KS induction on the cytostome was quantified using TEM and tomography. The authors also provide reconstructions of the cytostome in both induced and non-induced parasites. Finally, they measure the impact of KS on haem degradation. These data provide clear information on the function of K13 in cytostome formation and the implication of this structure in endocytosis for Plasmodium falciparum.

The conclusions of this paper are well supported by the data, but some data analysis should be clarified and extended, and some complementary experiments would further strengthen the authors' claims.

Reviewer #3 (Public Review):

The authors report a study in which they use intracranial recordings to dissociate subjectively aware and subjectively unaware stimuli, focusing mainly on prefrontal cortex. Although this paper reports some interesting findings (the videos are very nice and informative!) the interpretation of the data is unfortunately problematic for several reasons. I will detail my main comments below. If the authors address these comments well, I believe the paper may provide an interesting contribution to further specifying the neural mechanisms important for conscious access (in line with Gaillard et al., Plos Biology 2009).

The main problem with the interpretation of the data is that the authors have NOT used a so-called "no-report paradigm". The idea of no report paradigms is that subjects passively view a certain stimulus without the instruction to "do something with it", e.g., detect the stimulus, immediately or later in time. Because of the confusion of this term, specifically being related to the "act of reporting", some have argued we should use the term no-cognition paradigm instead (Block, TiCS, 2019, see also Pitts et al., Phil Trans B 2018). The crucial aspect is that, in these types of paradigms, the critical stimulus should be task-irrelevant and thus not be associated with any task (immediately or later). Because in this experiment subjects were instructed to detect the gratings when cued 600 ms later in time, the stimuli are task relevant, they have to be reported about later and therefore trigger all kinds of (known and potentially unknown) cognitive processes at the moment the stimuli are detected in real-time (so stimulus-locked). You could argue that the setup of this delayed response task excludes some very specific report related processes (e.g., the preparation of an eye-movement), which is good, however this is usually not considered the main issue. For example when comparing masked versus unmasked stimuli (Gaillard et al., 2009 Plos Biology), these conditions usually also both contain responses but these response related processes are "averaged out" in the specific contrasts (unmasked > masked). In this paper, RT differences between conditions (that are present in this dataset) are taken care of by using this delayed response in this paper, which is a nice feature for that and is not the case for the above example set-up.

Given the task instructions, and this being merely a delayed-response task, it is to be expected that prefrontal cortex shows stronger activity for subjectively aware versus subjectively unaware stimuli. Unfortunately, given the nature of this task, the novelty of the findings is severely reduced. The authors cannot claim that prefrontal cortex is associated with "visual awareness", or what people have called phenomenal consciousness (this is the goal of using no-cognition paradigms). The only conclusion that can be drawn is that prefrontal cortex activity is associated with accessing sensory input: and hence conscious access. This less novel observation has been shown many times before and there is also little disagreement about this issue between different theories of consciousness (e.g., global workspace theory and local recurrency theories both agree on this).

The best solution at this point seems to rewrite the paper entirely in light of this. My advice would be to state in the introduction that the authors investigate conscious access using iEEG and then not refer too much to no-cognition paradigm or maybe highlight some different strategies about using task-irrelevant stimuli (see Canales-Johnson et al., Plos Biology 2023; Hesse et al., eLife 2020; Hatamimajoumerd et al Curr Bio 2022; Alilovic et al., Plos Biology 2023; Pitts et al., Frontiers 2014; Dwarakanth et al., Neuron 2023 and more). Obviously, the authors should then also not claim that their results solve debates about theories regarding visual awareness (in the "no-cognition" sense, or phenomenal consciousness), for example in relation to the debate about the "front or the back of the brain", because the data do not inform that discussion. Basically, the authors can just discuss their results in detail (related to timing, frequency, synchronization etc) and relate the different signatures that they have observed to conscious access.

I think the authors have to discuss the Gaillard et al PLOS Biology 2009 paper in much more detail. Gaillard et al also report a study related to conscious access contrasting unmasked and masked stimuli using iEEG. In this paper they also report ERP, time frequency and phase synchronization results (and even Granger causality). Because of the similarities in approach, I think it would be important to directly compare the results presented in that paper with results presented here and highlight the commonalities and discrepancies in the Discussion.

In the Gaillard paper they report a figure plotting the percentage of significant frontal electrodes across time (figure 4A) in which it can be seen that significant electrodes emerge after approximately 250 ms in PFC as well. It would be great if the authors could make a similar figure to compare results. In the current paper there are much more frontal electrode contacts than in the Gaillard paper, so that is interesting in itself.

In my opinion, some of the most interesting results are not highlighted: the findings that subjectively unaware stimuli show increased activations in the prefrontal cortex as compared to stimulus absent trials (e.g., Figure 4D). Previous work has shown PFC activations to masked stimuli (e.g., van Gaal et al., J Neuroscience 2008, 2010; Lau and Passigngham J Neurosci 2007) as well as PFC activations to subjectively unaware stimuli (e.g., King, Pescetelli, and Dehaene, Neuron 2016) and this is a very nice illustration of that with methods having more detailed spatial precision. Although potentially interesting, I wonder about the objective detection performance of the stimuli in this task. So please report objective detection performance for the patients and the healthy subjects, using signal detection theoretic d'. This gives the reader an idea of how good subjects were in detecting the presence/absence of the gratings. Likely, this reveals far above chance detection performance and in that case I would interpret these findings as "PFC activation to stimuli indicated as subjectively unaware" and not unconscious stimuli. See Stein et al., Plos Biology 2021 for a direct comparison of subjectively and objectively unaware stimuli.

In Figure 7 of the paper the authors want to make the case that the contrast does not differ between subjectively aware stimuli and subjectively unaware stimuli. However so far they've done the majority of their analyses across subjects, and for this analysis the authors only performed within-subject tests, which is not a fair comparison imo. Because several P values are very close to significance I anticipate that a test across subjects will clearly show that the contrast level of the subjectively aware stimuli is higher than of the subjectively unaware stimuli, at the group level. A solution to this would be to subselect trials from one condition (NA) to match the contrast of the other condition (NU), and thereby create two conditions that are matched in contrast levels of the stimuli included. Then do all the analyses on the matched conditions.

Related, Figure 7B is confusing and the results are puzzling. Why is there such a strong below chance decoding on the diagonal? (also even before stimulus onset) Please clarify the goal and approach of this analysis and also discuss/explain better what they mean.

I was somewhat surprised by several statements in the paper and it felt that the authors may not be aware of several intricacies in the field of consciousness. For example a statement like the following "Consciousness, as a high-level cognitive function of the brain, should have some similar effects as other cognitive functions on behavior (for example, saccadic reaction time). With this question in mind, we carefully searched the literature about the relationship between consciousness and behavior; surprisingly, we failed to find any relevant literature." This is rather problematic for at least two reasons. First, not everyone would agree that consciousness is a high-level cognitive function and second there are many papers arguing for a certain relationship between consciousness and behavior (Dehaene and Naccache, 2001 Cognition; van Gaal et al., 2012, Frontiers in Neuroscience; Block 1995, BBS; Lamme, Frontiers in Psychology, 2020; Seth, 2008 and many more). Further, the explanation for the reaction time differences in this specific case is likely related to the fact that subjects' confidence in that decision is much higher in the aware trials than in the unaware trials, hence the speeded response for the first. This is a phenomenon that is often observed if one explores the "confidence literature". Although the authors have not measured confidence I would not make too much out of this RT difference.

I would be interested in a lateralized analysis, in which the authors compare the PFC responses and connectivity profiles using PLV as a factor of stimulus location (thus comparing electrodes contralateral to the presented stimulus and electrodes ipsilateral to the presented stimulus). If possible this may give interesting insights in the mechanism of global ignition (global broadcasting), supposing that for contralateral electrodes information does not have to cross from one hemisphere to another, whereas for ipsilateral electrodes that is the case (which may take time). Gaillard et al refer to this issue as well in their paper, and this issue is sometimes discussed regarding to Global workspace theory. This would add novelty to the findings of the paper in my opinion.

Reviewer #3 (Public Review):

The authors report on the nature of interventions that were applied to aid and improve engagement in cervical screening, brought about by the SARS CoV Pandemic in Sweden.

I appreciate that the impact of these interventions, given that they are recent, will take some time to quantify but the description (and reach) of the policy changes that occurred in a short amount of time is of significant interest to the screening community. The piece on HPV Even Faster is particularly novel; I am not aware of another example of where this has been enacted within a routine programme.

The authors make reference to (15) where the reader can find greater details relating to the population who received the offer of self sampling (and the nature of the device). However I was a little confused (in this stand alone piece) as to who the self sampling group constituted exactly. Did this group not include pregnant women, women invited for first screen or women on non routine recall?

The authors state that "the most likely explanation for the large increase in population coverage seen is that the sending of self-sampling kits resulted in improved attendance in particular among previously non-attending women" - why is this written as speculation at this stage (?) is it not possible to attribute directly the contribution made by self sampling, or is this in hand?

While self sampling is certainly an option that can support uptake and enfranchisement in cervical screening - its overall performance is fundamentally contingent on the number of women who then comply with follow up should the HPV test be positive; it is not simply about who returns the sample. It would have been of interest to see the proportion of women who did comply with follow up.

Reviewer #3 (Public Review):

'Collateral sensitivity' occurs when drug-resistance mutations render a drug target more sensitive to inhibition by another drug, which has been previously described in some detail for malaria parasite dihydroorotate dehydrogenase (DHODH - see refs 36, 46, and 47, for example). Although it has been suggested that combinations of such drugs could potentially suppress the emergence of resistance, cross-resistance-associated mutation (or copy-number variation, CNV) could render such combination strategies ineffective. In the current study, the authors assess a new pairing of DHODH-targeting drugs. Cross-resistant parasites with DHODH mutation or CNV arise following either sequential or combined drug selection, suggesting that the drug combination described would likely fail to effectively suppress the emergence of resistance.

The strength of the study is that it describes, for a particular drug combination, different mutations associated either with collateral sensitivity or with cross-resistance, and the authors conclude that "combination treatment with DSM265 and TCMDC-125334 failed to suppress resistance". They go on to say that this "brings into question the usefulness of pursuing further DHODH inhibitors." More specific interpretations and implications of the study are as follows:<br /> a. Other combinations may also fail but there may be combinations that can effectively suppress resistance. A more exhaustive analysis of mutational space will likely be required to determine which combinations if any, would be predicted to succeed in a clinical setting.<br /> b. It was previously reported that "a combination of [DHODH] wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. ... Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate" (Ref 46). Also, "selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites" (Ref 36). It is possible that these previously tested combinations would also yield cross-resistant mutants if selected further.<br /> c. Although increased DHODH copy number "confers only moderately reduced susceptibility" to the drug used for selection and although these clones were not assessed here for cross-resistance, it seems likely that CNV may represent a general mechanism that could undermine other collateral resistance strategies.

Reviewer #3 (Public Review):

The authors described their extensive single-cell analysis of Candida undergoing (sub-inhibitory) antibiotic treatment versus no treatment. To do so, the authors used a microfluidics platform they had previously developed, and they optimized, characterized, and validated it for this particular application. Their findings included: (a) the transcription of untreated cells is driven mostly by cell cycle phase, (b) treated cells can be clustered into several major groups and a few outlier groups that the authors termed comets, (c) cells undergoing FCZ treatment can adopt one of two different states (possibly bistability). I found the results interesting and the approach to be sound, and much of the results confirmed my prior expectations. The authors provide a detailed depiction of what is going on in the transcriptome during sub-inhibitory treatment, although this did not always lead to a mechanistic explanation. The clinical relevance was unclear to me beyond a proof of concept application for single-cell transcriptomics. In my opinion, an interesting follow-up would be to follow the transcriptional trajectory of lineages undergoing antimicrobial switching (on and off). The main issues I identified were the author's use of the term tolerance versus resistance, interpretation of "comets", clustering approach, description of fitness, and comparison between time points.

Reviewer #3 (Public Review):

Increased LRRK2 kinase activity is known to confer Parkinson's disease risk. While much is known about disease-causing LRRK2 mutations that increase LRRK2 kinase activity, the normal cellular mechanisms of LRRK2 activation are less well understood. Rab GTPases are known to play a role in LRRK2 activation and to be substrates for the kinase activity of LRRK2. However, much of the data on Rabs in LRRK2 activation comes from over-expression studies and the contributions of endogenously expressed Rabs to LRRK2 activation are less clear. To address this problem, Bondar and colleagues tested the impact of systematically depleting candidate Rab GTPases on LRRK2 activity as measured by its ability to phosphorylate Rab10 in the human A549 type 2 pneumocyte cell line. This resulted in the identification of a major role for Rab12 in controlling LRRK2 activity towards Rab10 in this model system. Follow-up studies show that this role for Rab12 is of particular importance for the phosphorylation of Rab10 by LRRK2 at damaged lysosomes. Increases in LRRK2 activity in cells harboring disease-causing mutants of LRRK2 and VPS35 also depend (at least partially) on Rab12. Confidence in the role of Rab12 in supporting LRRK2 activity is strengthened by parallel experiments showing that either siRNA-mediated depletion of Rab12 or CRISPR-mediated Rab12 KO both have similar effects on LRRK2 activity. Collectively, these results demonstrate a novel role for Rab12 in supporting LRRK2 activation in A549 cells. It is likely that this effect is generalizable to other cell types. However, this remains to be established. It is also likely that lysosomes are the subcellular site where Rab12-dependent activation of LRRK2 occurs. Independent validation of these conclusions with additional experiments would strengthen this conclusion and help to address some concerns that much of the data supporting a lysosome localization for Rab12-dependent activation of LRRK2 comes from a single method (LysoIP). Furthermore, there is a discrepancy between panel 4A versus 4D in the effect of LLoMe-induced lysosome damage on LRRK2 recruitment to lysosomes that will need to be addressed to strengthen confidence in conclusions about lysosomes as sites of LRRK2 activation by Rab12.

Reviewer #3 (Public Review):

This manuscript by Bellegarda et al. examined the in vivo dynamic behavior of the Reissner fiber and its interactions with cilia and sensory neurons in the central canal of zebrafish larvae. The authors accomplished this by performing live imaging with a transgenic reporter zebrafish line in which the fiber is GFP-tagged and by finely tracking the movement of the fiber. Interestingly, they discovered that the fiber undergoes a dynamic vibratory-like movement along the dorsoventral axis. The authors then utilized a pulsed laser to precisely cut the fiber, which frequently resulted in a fast retraction behavior and a loss of calcium activity in sensory neurons in the central canal called CSF-CNs. Mechanical modeling of the elastic properties of the fiber indicated that the fiber is a soft elastic rod with graded tension along the rostrocaudal axis. Finally, by performing live imaging of motile cilia and the fiber in the central canal, they found that the two interact in close proximity and that cilia motility is affected when the fiber was cut. The authors concluded that the Reissner fiber is a dynamic structure under tension that interacts with sensory neurons and cilia in the central canal.

Strengths:<br /> 1. The study utilizes state-of-the-art microscopy techniques and beautiful transgenic zebrafish tools to characterize the in vivo behavior of the Reissner fiber and found that it exhibits surprising dynamic movements along the dorsal-ventral axis. This observation has important implications for the physiology and function of the Reissner fiber.

2. By performing a series of clever laser cutting experiments, the authors reveal that the Reissner fiber is under tension in the central canal of zebrafish. This finding provides direct experimental evidence to support the hypothesis that the Reissner fiber functions in a biomechanical manner during spinal cord development and body axis straightening.

3. By developing a mechanical model of the Reissner fiber and its retraction behavior, the authors estimate the elastic properties of the fiber and found that it is more akin to an elastic polymer rather than a stiff rod. This is a useful finding that illuminates the biophysical properties of the fiber.

4. Through calcium and cilia imaging studies, the authors demonstrate that the Reissner fiber likely interacts with motile cilia and regulates the activity of ciliated sensory neurons (CSF-CNs). The authors propose a model in which fiber-cilia interactions may occur via weak interactions or frictional forces. This model is plausible and opens several new doors for additional investigation.

Weaknesses:<br /> 1. All the live imaging experiments appear to be performed with animals paralyzed via the injection of a chemical agent (bungarotoxin). Does paralysis and/or bungarotoxin negatively impact the behavior of the Reissner fiber? Some data from non-paralyzed animals would ameliorate this concern.

2. Although the authors convincingly demonstrate that the Reissner fiber is under graded tension, it remains unclear what is the relevance and function of tension on this structure. The photoablation data presented do not delineate between the relevance of the fiber being intact or tension on the fiber as cutting the fiber impacts both. Is fiber tension required for body straightening? At the site of fiber photoablation, does a spinal curvature develop? If cultured, do the ablated animals exhibit a scoliotic phenotype?

3. One of the most potentially impactful conclusions of the paper is that the Reissner fiber interacts with cilia, but the evidence is insufficient to support this. Although some motile cilia are near the fiber (Figure 3A), many cilia are not near the fiber. The provided images and videos do not clearly demonstrate that cilia physically contact or influence the behavior of the Reissner fiber. Further, the data is lacking to conclude that the Reissner fiber directly impacts cilia motility as they did not observe an overall statistically significant difference before and after ablation (Supplemental Figure 1A). Higher magnification, higher resolution, higher acquisition rate and/or colocalization analyses of fiber-cilia interactions could alleviate this concern.

4. Similarly, how does the Reissner fiber interact with CSF-CN sensory neurons? The authors suggest that the fiber interacts with CSF-CN sensory neurons by modulating their spontaneous calcium activity via weak interactions or frictional forces from motile ciliated ependymal radial glial cells. While the calcium imaging data of the CSF-CNs is convincing and sound, the exact nature of the fiber-neuron interaction is unclear. Do cilia or apical extensions on CSF-CN sensory neurons sense the fiber or forces through a mechanosensing or chemosensing mechanism? There is some additional confusion as the authors appear to focus their cilia experiments on ependymal radial glial cells in section 4, rather than CSF-CNs. The addition of an illustrative cartoon would add clarity.

Overall, the conclusions of the study are well supported by the data presented. However, the strength of the conclusions could be enhanced by additional controls, alternative experimental approaches and clarifications.

This manuscript is an important contribution to the fields of spinal cord development and body axis development, which are fundamental questions in neurobiology, developmental biology, and musculoskeletal biology. In recent years, the Reissner fiber and motile cilia function have been linked to cerebrospinal fluid flow signaling and body straightening, but the precise form and function of the fiber remain unclear. This study provides new insight into the dynamic and biophysical properties of the Reissner fiber in vivo in zebrafish and proposes a model in which the fiber interacts with cilia and sensory neurons. This study provides novel insight into the cellular mechanisms that underlie the pathogenesis of disorders such as idiopathic scoliosis.

Reviewer #3 (Public Review):

CaMKII is a multimeric kinase of great biologic interest due to its crucial roles in long-term memory, cardiac pacemaking, and fertilization. CaMKII subunits organize into holoenzymes comprised of 12-14 subunits, adopting a donut-like, double-ringed structure. In this manuscript, Lucic et al challenge two models in the CaMKII field, which are somewhat related. The first is a longstanding topic in the field about whether the autophosphorylation of a crucial residue, Thr286, can be phosphorylated between intact holoenzymes (inter-holoenzyme phosphorylation). The second is a more recent biochemical finding, which tested the long-running theory that CaMKII exchanges subunits between holoenzymes to create mixed oligomers. These two models are connected by the idea that subunit exchange could facilitate phosphorylation between subunits of different holoenzymes by allowing subunits to integrate into a different holoenzyme and driving transphosphorylation within the CaMKII ring. Here, the authors attempt to show that one intact holoenzyme phosphorylates another intact holoenzyme at Thr286. The authors also provide evidence suggesting that subunit exchange is not occurring under their conditions, and therefore not driving this phosphorylation event. The authors propose a model where instead of exchanging subunits, two holoenzymes interact via their kinase domains to enable transphosphorylation at Thr286 without integrating into the holoenzyme structure. In order for the authors to successfully convince readers of all three facets of this new model, they need to provide evidence that 1) transphosphorylation at Thr286 happens when subunit exchange is blocked, 2) subunit exchange does not occur under their conditions, and 3) there are interactions between kinases of different holoenzymes that lead to productive autophosphorylation at Thr286.

Strengths:<br /> The authors have designed and performed a battery of cleverly designed and orthogonal experiments to test these models. Using mutagenesis, they mixed a kinase-dead mutant with an active kinase to ask whether transphosphorylation occurs. They observe phosphorylation of the kinase-dead variant in this experiment, which indicates that the active kinase must have phosphorylated it. A few key questions arise here: 1) whether this phosphorylation occurred within a single CaMKII holoenzyme ring (which is the canonical mechanism for Thr286 phosphorylation), 2) whether the phosphorylation occurred between two separate holoenzyme rings, and 3) why was this not observed in previous literature? To address questions 1 and 2, the authors implemented an innovative strategy introducing a genetically-encoded photocrosslinker in the oligomerization domain, which when crosslinked using UV light, should lock the holoenzyme in place. The rate of phosphorylation was the same when comparing uncrosslinked and crosslinked CaMKII variants, indicating that phosphorylation is occurring between holoenzymes, rather than through a subunit exchange mechanism that would require some type of disassembly and reassembly (presumably blocked by crosslinking). The 3rd question remains as to why this has not been previously observed, as it has not been for lack of effort. The authors mention low temperature and low concentration as culprits, however, Bradshaw et al, JBC v. 277, 2002 carry out a series of careful experiments that indicated that autophosphorylation at T286 is not concentration-dependent (meaning that the majority of phosphorylation occurs via intra-holoenzyme), and this is done over a concentration and temperature range. It is possible that due to the mutants used in the current manuscript, it allows for the different behavior of the kinase-dead domains, which will have an empty nucleotide-binding pocket. Further studies will need to elucidate these details, and importantly, understand what physiological conditions facilitate this mechanism.

The most convincing data that subunit exchange does not occur is from the crosslinking mass spectrometry experiment. The authors created mixtures of 'light' and 'heavy' CaMKII holoenzymes, either activated or not and then used a Lys-Lys crosslinker (DSS) to trap the enzyme in its final state. The results of this experiment indicate that subunit exchange is not occurring under their conditions. A caveat here is that there are not many lysines at hub-hub interfaces, which is the crux of this experiment. If there is no subunit exchange under their conditions, how does transphosphorylation occur between holoenzymes? The authors show very nice mass photometry data indicating that there are populations of 24-mers, which corresponds to a double-holoenzyme. Paired with the data from their crosslinking mass spectrometry which shows crosslinks between kinase domains of different holoenzymes, this indicates that perhaps kinases between holoenzymes do interact, and they do so in a competent manner to allow transphosphorylation to occur.

Weaknesses:<br /> The authors should be commended for performing three orthogonal experiments to test whether CaMKII holoenzymes exchange subunits to form heterooligomers. However, there are technical issues that dampen the strength of the results shown here. For simplicity, let's consider that CaMKII holoenzymes are comprised of two stacked hexameric rings. It has been proposed that the stable unit of CaMKII assembly and perhaps also disassembly and subunit exchange is a vertical dimer unit (comprised of one subunit from each hexameric ring). In the UV crosslinking data shown in this paper, the authors have a significant number of monomers, some crosslinked dimers (of which there are two populations), and fewer higher-order oligomers. To effectively block subunit exchange, robust crosslinking into hexamers is necessary, which the authors have not done. Incomplete crosslinking results in smaller species that can still exchange (and/or dissociate), confounding the results of this experiment. In addition, Figure 3 shows a trapping experiment, where if the exchange was occurring, there would be an oligomeric band in Lane 8, which is visible and highlighted with a blue arrow by the authors. This result is explained by nonspecific UV effects, however by eye it is not clear if there is an equivalent band in lane 10. The overall issue here is inefficient crosslinking.

The authors also employ a single-molecule TIRF experiment to further interrogate subunit exchange. Upon inspection of the TIRF images, it is not clear that the authors are achieving single molecule resolution (there are evident overlapping and distorted particles). The analysis employed here is Pearson's correlation coefficient, which is not sufficient for single molecule analysis and would not account for particle overlap, particles that are too bright, and/or particles that are too dim. For example, an alternative explanation for the authors' results is that activation results in aggregation (high correlation), and subsequent EGTA treatment leads to dissociation at these low concentrations (low correlation). However, further experimentation and analysis are necessary.

Taken together, the authors have provided important food for thought regarding inter-holoenzyme phosphorylation and subunit exchange. However, given the shortcomings discussed here, it remains unclear exactly what mechanisms are at play within and between CaMKII holoenzymes once activated.

Reviewer #3 (Public Review):

Joechner and their co-authors performed an extensive analysis of two existing datasets from sleeping children aged between 5 to 18 years. By identifying discrete events of slow oscillations (SOs) and (fast) sleep spindles they examined not only the developmental changes of these distinct sleep grapho-elements. They also took a closer look at their interplay, e.g., to what extend sleep spindles are co-occurring with slow oscillation up-states, as this coupling is thought to underlie sleep-dependent memory consolidation.

The authors found that both sleep spindles and slow oscillation undergo a change across the young age, e.g., while sleep spindles increased in frequency approaching the typical 12-16 Hz range found in adults, slow oscillation showed a shift in occurrence patterns from posterior to anterior sites. Likewise, the coupling of fast spindles within slow oscillation up-states manifested with age, which is almost non-existing in 5- to 6-year-old children. However, and most intriguingly, a coupling analysis based on the adult-like 12-16 Hz range revealed an already existing SO-spindle phase-relation across all age ranges. Altogether, this data nicely demonstrates the trajectory of sleep spindles and SOs in children and highlights the almost inherent coupling between SOs and "adult" sleep spindles. In my view, these results not only provide a good overview of a healthy development but also interesting food for thought regarding the function of SO-spindle coupling in healthy or clinical development.

Overall, this work is well-written, and the performed analyses are well conceptualized. Hence, there are one general and a few minor aspects that could be addressed to hopefully strengthen this manuscript a bit further.

The biggest aspect that was striking is the shear amount of data reported, e.g., a supplement with 28 tables is too extensive. The authors should consider reducing a few aspects.<br /> For example, the authors employ a linear mixed effects model and report coefficient etc. in the supplement. However, in the main text, the authors mainly report ANOVA-based results. Obviously, a LMM and an ANOVA are equivalent, however, focusing on one approach could streamline everything.<br /> Another example is the assessment of spindle frequency via the discrete events: First spindle peak frequency is derived via power spectra. Using the then individually identified peaks, discrete events are detected. Shouldn't it be obvious that these events show the same behavior with regard to their frequency?<br /> As a final example, the authors first report changes in fast spindle properties across age and, e.g., find an increase in frequency towards 12-16 Hz adult range. They then repeat the whole analysis in the 12-16 Hz range and examine the "distance" to the individualized results. It should again be obvious that this approach comes to the same conclusion, a smaller distance in older children. Even more obvious is the conclusion "Hence, it appears as if fast centro-parietal SPs become more dominant and adult-like in their frequency and amplitude characteristics in older children" because it describes a normal development of a healthy child. Altogether, the authors could streamline a few aspects by removing hidden redundancies and focus on the - in my view - central aspect of an inherent 12-16 Hz coupling across all ages.

Reviewer #3 (Public Review):

Mesenchymal stem cells have been shown to have potent immunomodulatory and regenerative properties and have been tested and tried in kidney transplantation. In a previous paper, the authors of this paper reviewed the beneficial actions of nitric oxide (NO) on the beneficial action of MSC. In this manuscript, they describe a method to generate NO in the therapeutic MSC. While NO donors like the short-acting nitrates have been used for angina pectoris patients few therapeutic approaches have been published aiming at the local delivery of NO to specific tissues or organs like the kidney. Gene therapy with adenoviral vectors, overexpressing the eNOS gene itself failed due to the fact that the eNOS enzyme, when overexpressed quickly runs out of sufficient co-factors like BH4. As a consequence, the enzyme uncouples and becomes cytotoxic due to the generation of peroxynitrate. Hence, the current strategy to generate NO in the MSC itself is novel and interesting.

The authors first describe the cryoprotective effects and antioxidant effects of NO generation in MSC in vitro and subsequently in vivo in a mouse model of ischemia-reperfusion injury that may reflect acute kidney injury (or ischemia associated with kidney transplantation) in patients. While the MSC are transplanted intracortical on a local position in the kidneys, the manuscript describes surprising effectivity on serum creatinine, ureum, casts, and protection of brush border. Also, upon immunohistochemical analyses, fibrosis, and kidney injury markers decrease. Most likely there is a strong paracrine effect. It is unfortunate that the control "PBS + MGP" is lacking to exclude some low-grade background conversion of the compound with subsequent release of NO. MGP only is tested however, studies in kidney sections with state-of-the-art EPR, give the authors the wanted control.

The paper provides an interesting proof of concept for a novel therapeutic approach. However, in the clinical arena, some questions remain involving the survival of the MSC after transplantation and the introduction of novel antigens associated with the engineered cells