Reviewer #2 (Public Review):

Summary:<br /> CD8+ QFL T cells recognize a peptide, FYAEATPML (FL9), presented on Erap1-deficient cells. QFL T cells are present at a high frequency in the spleen of naïve mice. They express an antigen-experienced phenotype, and about 80% express an invariant TCRα chain Vα3.2Jα21.

Here, Guan and colleagues report that QFL T cells are present not only in the spleen but also in the intestinal epithelium, where they display several phenotypic and functional peculiarities. The establishment of spleen and gut Vα3.2+ QFL T cells is TAP-dependent, and their phenotype is regulated by the presence/absence of Qa-1b and Erap1. Maintenance of gut Vα3.2+ QFL T cells depends on the gut microbiota and is associated with colonization by Pediococcus pentosaceus.

Strengths:<br /> This article contains in-depth studies of a peculiar and interesting subset of unconventional CD8 T cells, based partly on generating two novel TCR-transgenic models.

The authors discovered a clear relation between the gut microbiome and the maintenance of gut QFL T cells. One notable observation is that monocolonization of the gut with Pediococcus pentosaceus is sufficient to sustain gut QFL T cells.

Weaknesses:<br /> In the absence of immunopeptidomic analyses, the presence or absence of the FL9 peptide on various cell types is inferred based on indirect evidence.

Analyses of the homology between the FL9 and bacterial peptides were limited to two amino acid residues (P4 and P6).

The potential function of QFL T cells remains elusive.

Reviewer #2 (Public Review):

The manuscript "IQCH regulates spermatogenesis by interacting with CaM to promote RNA-binding proteins' expression" by Ruan et al. identified a homozygous variant affecting the splicing of IQCH in two infertile men from a Chinese family. The authors also generated an IQCH knockout mouse model to confirm the abnormal sperm phenotypes associated with IQCH deficiency. Further molecular biological assays supported the important role and mechanism of IQCH in spermatogenesis. This manuscript is informative for clinical and basic research on male infertility.

Reviewer #2 (Public Review):

The manuscript by Chambert et al. describes a thorough and careful characterization of inositol pyrophosphate isomers and the PHO pathways in different genetic backgrounds in S. cerevisiae. The paper ultimately arrives at a proposed model in which the inositol pyrophosphate 1,5-IP8 signals phosphate abundance to SPX-domain containing proteins. To arrive at their conclusion, the authors rely heavily on CE-MS analysis of inositol pyrophosphates in different yeast strains, and monitoring inositol pyrophosphate depletion over time in response to phosphate starvation. This analysis is complemented by different reporter systems of PHO pathway activation, such as Pho4 translocation and Pho81 expression.

The experiments are well-designed and the results interpreted with care. With their findings, the authors demonstrate convincingly, that a previous study by O'Shea and co-workers (reference 15 and 16) had been misleading. Lee et al. claimed that the PHO pathway in S. cerevisiae is triggered by an increase in 1-IP7. This claim has been debated heavily in the community, and several groups were not able to reproduce this putative increase of inositol pyrophosphates (references 6, 11, 18). The confusion regarding these discrepancies has been resolved by the current study and is of significant importance to the community.

Reviewer #2 (Public Review):

Summary:

Planctomycetes encompass a group of bacteria with unique biological traits, the compartmentalized cells make them appear to be organisms in between prokaryotes and eukaryotes. However, only a few of the Planctomycetes bacteria are cultured thus far, and this hampers insight into the biological traits of these evolutionarily important organisms.

This work reports the methodology details of how to isolate the deep-sea bacteria that could be recalcitrant to laboratory cultivation, and further reveals the distinct characteristics of the new species of a deep-sea Planctomycetes bacterium, such as the chronic phage release without breaking the host and promote the host and related bacteria in nitrogen utilization. Therefore, the finding of this work is of importance in extending our knowledge of bacteria.

Strengths:

Through the combination of microscopic, physiological, genomics, and molecular biological approaches, this reports the isolation and comprehensive investigation of the first anaerobic representative of the deep-sea Planctomycetes bacterium, in particular in that of the budding division, and release phage without lysis of the cells. Most of the results and conclusions are supported by the experimental evidence.

Weaknesses:

1. While EMP glycolysis is predicted to be involved in energy conservation, no experimental evidence indicated any sugar utilization by the bacterium.<br /> 2. "anaerobic representative" is indicated in the Title, the contrary, TCA in energy metabolism is predicted by the bacterium.<br /> 3. The possible mechanisms of the chronic phage release without breaking the host are not discussed.

Reviewer #2 (Public Review):

Summary:

The authors develop a computational approach-avoidance-conflict (AAC) task, designed to overcome the limitations of existing offer based AAC tasks. The task incorporated likelihoods of receiving rewards/ punishments that would be learned by the participants to ensure computational validity and estimated model parameters related to reward/punishment and task induced anxiety. Two independent samples of online participants were tested. In both samples participants who experienced greater task induced anxiety avoided choices associated with greater probability of punishment. Computational modelling revealed that this effect was explained by greater individual sensitivities to punishment relative to rewards.

Strengths:

Large internet-based samples, with discovery sample (n = 369), pre-registered replication sample (n = 629) and test-retest sub group (n = 57). Extensive compliance measures (e.g. audio checks) seek to improve adherence.

There is a great need for RL tasks that model threatening outcomes rather than simply loss of reward. The main model parameters show strong effects and the additional indices with task based anxiety are a useful extension. Associations were broadly replicated across samples. Fair to excellent reliability of model parameters is encouraging and badly needed for behavioral tasks of threat sensitivity.

The task seems to have lower approach bias than some other AAC tasks in the literature.

Weaknesses:

The negative reliability of punishment learning rate is concerning as this is an important outcome.

The Kendall's tau values underlying task induced anxiety and safety reference/ various indices are very weak (all < 0.1), as are the mediation effects (all beta < 0.01). The interaction with P(punishment|conflict) does explain some of this.

The inclusion of only one level of reward (and punishment) limits the ecological validity of the sensitivity indices.

Appraisal and impact:

Overall this is a very strong paper, describing a novel task that could help move the field of RL forward to take account of threat processing more fully. The large sample size with discovery, replication and test-retest gives confidence in the findings. The task has good ecological validity and associations with task-based anxiety and clinical self-report demonstrate clinical relevance. Test-retest of the punishment learning parameter is the only real concern. Overall this task provides an exciting new probe of reward/threat that could be used in mechanistic disease models.

Additional context:

The sex differences between the samples are interesting as effects of sex are commonly found in AAC tasks. It would be interesting to look at the main model comparison with sex included as a covariate.

Reviewer #2 (Public Review):

The study is performed with old tool Spamo (12 year ago), source data from Encode (2010-2012), even peak caller tool version MACS is old ~ 2013. De novo motif search tool is old too (new one STREME is not mentioned). Any composite element search tool published for the recent 12 years are not cited, there are some issues in data analysis in presentation. Almost all references are from about 8-10 year ago (the most recent date is 2019)

The title is misleading<br /> Instead of<br /> A new pipeline SPICE identifies novel JUN-IKZF1 composite elements<br /> It should be written as<br /> Application of SpaMo tool identifies novel JUN-IKZF1 composite elements<br /> It reflects the pipeline better but honestly shows that the novelty is missed.

The study was performed on too old data from ENCODE, authors mentioned 343 Encode ChIP-Seq libraries, but authors even did not care even about to set for each library the name of target TF (Figure 1E, Figure S2, Table 2).

Reviewer #2 (Public Review):

Most farming is done by subtracting or adding what people want based in nature. However, in nature, crops interact with various objects, and mostly we are unaware of their effects. In order to increase agricultural productivity, finding useful objects is very important. However, in an uncontrolled environment, it coexists with so many biological objects that it is very inefficient to verify them all experimentally. It is therefore necessary to develop an effective screening method to identify external environmental factors that can increase crop productivity. This study identified factors presumed to be important to crop growth based on metabarcoding analysis, field sampling, and non-linear analysis/information theory, and conducted a mesocosm experiment to verify them experimentally. In conclusion, the object proposed by the author did not increase rice yield, but rather rice growth rate.

The authors responded to my general concerns and all of my specific comments. The manuscript has significantly improved. The flow of aims and approaches is more understandable. Extra supplementary material -especially the visual ones, is useful.

I agree with the other reviewers that the study needs more data and evidence. However, this study aims to introduce ecological concepts and advanced statistical methods to the field. Also, most time series analyses require absolute abundance data, but the manuscript provides solutions for the sequencing data.

Reviewer #2 (Public Review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The conclusions from the paper are mostly well supported by the simulations, however, they remain very descriptive and the key findings should be better described and validated. In particular:

It has been shown that the lipid composition of HIV membrane is rich in cholesterol [1], which accounts for almost 50% molar ratio. The authors use a very different composition and should therefore provide a reference. It has been shown for 4E10 that the change in lipid composition affects dynamics of the binding. The robustness of the results to changes of the lipid composition should also be reported.

The real advantage of the multiscale approach (coarse grained (CG) simulation followed by a back-mapped all atom simulation) remains unclear. In most cases, the binding mode in the CG simulations seem to be an artifact.

The results reported in this study should be better compared to available experimental data. For example how does the approach angle compare to cryo-EM structure of the bnAbs engaging with the MPER region, e.g. [2-3]? How do these results from this study compare to previous molecular dynamics studies, e.g.[4-5]?

References<br /> 1. Brügger, Britta, et al. "The HIV lipidome: a raft with an unusual composition." Proceedings of the National Academy of Sciences 103.8 (2006): 2641-2646.<br /> 2. Rantalainen, Kimmo, et al. "HIV-1 envelope and MPER antibody structures in lipid assemblies." Cell Reports 31.4 (2020).<br /> 3. Yang, Shuang, et al. "Dynamic HIV-1 spike motion creates vulnerability for its membrane-bound tripod to antibody attack." Nature Communications 13.1 (2022): 6393.<br /> 4. Carravilla, Pablo, et al. "The bilayer collective properties govern the interaction of an HIV-1 antibody with the viral membrane." Biophysical Journal 118.1 (2020): 44-56.<br /> 5. Pinto, Dora, et al. "Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01." Cell host & microbe 26.5 (2019): 623-637.

Reviewer #2 (Public Review):

The exact dynamics of responses to volatiles from herbivore-attacked neighbouring plants have been little studied so far. Also, we still lack evidence of whether herbivore-induced plant volatiles (HIPVs) induce or prime plant defences of neighbours. The authors investigated the volatile emission patterns of receiver plants that respond to the volatile emission of neighbouring sender plants which are fed upon by herbivorous caterpillars. They applied a very elegant approach (more rigorous than the current state-of-the-art) to monitor temporal response patterns of neighbouring plants to HIPVs by measuring volatile emissions of senders and receivers, senders only and receivers only. Different terpenoids were produced within 2 h of such exposure in receiver plants, but not during the dark phase. Once the light turned on again, large amounts of terpenoids were released from the receiver plants. This may indicate a delayed terpene burst, but terpenoids may also be induced by the sudden change in light. A potential caveat exists with respect to the exact timing and the day-night cycle. The timing may be critical, i.e. at which time-point after onset of light herbivores were placed on the plants and how long the terpene emission lasted before the light was turned off. If the rhythm or a potential internal clock matters, then this information should also be highly relevant. Moreover, light on/off is a rather arbitrary treatment that is practical for experiments in the laboratory but which is not a very realistic setting. Particularly with regard to terpene emission, the sudden turning on of light instead of a smooth and continuous change to lighter conditions may trigger emission responses that are not found in nature. As one contrasting control, the authors also studied the time-delay in volatile emission when plants were just kept under continuous light (just for the experiment or continuously?). Here they also found a delayed terpenoid production, but this seemed to be lower compared to the plants exposed to the day-night-cycle. Another helpful control would be to start the herbivory treatment in the evening hours and leave the light on. If then again plants only release volatiles after a 17 h delay, the response is indeed independent of the diurnal clock of the plant.

Interestingly, internal terpene pools of one of the leaves tested here remained more comparable between night and day, indicating that their pools stay higher in plants exposed to HIPVs. In contrast, terpene synthases were only induced during the light-phase, not in the dark-phase. Moreover, jasmonates were only significantly induced 22 h after the onset of the volatile exposure and thus parallel with the burst of terpene release.

An additional experiment exposing plants to the green leaf volatile (glv) (Z)-3-hexenyl acetate revealed that plants can be primed by this glv, leading to a stronger terpene burst. The results are discussed with nice logic and considering potential ecological consequences. Some data are not discussed, e.g. the jasmonate and gene induction pattern.

Overall, this study provides intriguing insights into the potential interplay between priming and induction, which may co-occur, enhancing (indirect and direct) plant defence. Follow-up studies are suggested that may provide additional evidence.

Reviewer #2 (Public Review):

The manuscript titled "Calcineurin Inhibition Enhances Caenorhabditis elegans Lifespan by Defecation Defects-Mediated Calorie Restriction and Nuclear Hormone Signaling" by Priyanka Das, Alejandro Aballay, and Jogender Singh reveals that inhibiting calcineurin, a conserved protein phosphatase, in C. elegans affects the defecation motor program (DMP), leading to intestinal bloating and increased susceptibility to bacterial infection. This intestinal bloating mimics calorie restriction, ultimately resulting in an enhanced lifespan. The research identifies the involvement of HLH-30 and NHR-8 proteins in this lifespan enhancement, providing new insights into the role of calcineurin in C. elegans DMP and mechanisms for longevity.

The authors present novel findings on the role of calcineurin in regulating the defecation motor program in C. elegans and how its inhibition can lead to lifespan enhancement. The evidence provided is solid with multiple experiments supporting the main claims.

Strengths:<br /> The manuscript's strength lies in the authors' use of genetic and biochemical techniques to investigate the role of calcineurin in regulating the DMP, innate immunity, and lifespan in C. elegans. Moreover, the authors' findings provide a new mechanism for calcineurin inhibition-mediated longevity extension, which could have significant implications for understanding the molecular basis of aging and developing interventions to promote healthy aging.

1. The study uncovers a new role for calcineurin in the regulation of C. elegans DMP and a potential novel pathway for enhancing lifespan via calorie restriction involving calcineurin, HLH-30, and NHR-8 in C. elegans.<br /> 2. Multiple signaling pathways involved in lifespan enhancement were investigated with fairly strong experimental evidence supporting their claims.

Weaknesses:<br /> The manuscript's weaknesses include the lack of mechanistic details regarding how calcineurin inhibition leads to defects in the DMP and induces calorie restriction-like effects on lifespan.

The exact site of calcineurin action, i.e., whether in the intestine or enteric muscles (Lee et al., 2005), and the possible molecular mechanisms linking calcineurin inhibition, DMP defects, and lifespan were not adequately explored. Although characterization of the full mechanism is probably beyond the scope of this paper, given the relative simplicity and advantages of using C. elegans as a model organism for this study, some degree of rigor is expected with additional straightforward control experiments as listed below:

The authors state that tax-6 knockdown animals had drastically reduced expulsion events (Figure 2G), leading to irregular DMP (Lines 144-145), but did not describe the nature of DMP irregularity. For example, did the reduced expulsion events still occur with regular intervals but longer cycle lengths? Or was the rhythmicity completely abolished? The former would suggest the intestine clock is still intact, and the latter would indicate that calcineurin is required for the clock to function. Therefore, ethograms of DMP in both wild-type and tax-6 mutant animals are warranted to be included in the manuscript. Along the same line, besides the cycle length, the three separable motor steps (aBoc, pBoc, EMC) are easily measurable, with each step indicating where the program goes wrong, hence the site of action, which is precisely the beauty of studying C. elegans DMP. Unfortunately, the authors did not use this opportunity to characterize the exact behavior phenotypes of the tax-6 mutant to guide future investigations. Furthermore, it is interesting that about 64% of tax-6 (p675) animals had normal DMP. The authors attributed this to p675 being a weak allele. It would be informative to further examine tax-6 RNAi as in other experiments or to make a tax-6 null mutant with CRISPR. In addition, in one of the cited papers (Lee et al., 2005), the exact calcineurin loss-of-function strain tax-6(p675) was shown to have normal defecation, including normal EMC, while the gain-of-function mutant of calcineurin tax-6(jh107) had abnormal EMC steps. It wasn't clear from Lee et al., 2005, if the reported "normal defecation" was only referring to the expulsion step or also included the cycle length. Nevertheless, this potential contradiction and calcineurin gain-of-function mutant is highly relevant to the current study and should be further explored as a follow-up to previously reported results. For some of the key experiments, such as tax-6's effects on susceptibility to PA14, DMP, intestinal bloating, and lifespan, additional controls, as the norm of C. elegans studies, including second allele and rescue experiments, would strengthen the authors' claims and conclusions.

The second weakness of this manuscript is the data presentation for all survival rate curves. The authors stated that three independent experiments or biological replicates were performed for each group but only showed one "representative" curve for each plot. Without seeing all individual datasets or the averaged data with error bars, there is no way to evaluate the variability and consistency of the survival rate reported in this study.

Overall, the authors' claims and conclusions are justified by their data, but further experiments are needed to confirm their findings and establish the detailed mechanisms underlying the observed effects of calcineurin inhibition on the DMP, calorie restriction, and lifespan in C. elegans.

Reviewer #2 (Public Review):

Summary:<br /> Laham et al. investigate how the projection from adult-born granule cells into CA2 affects the retrieval of social memories at various developmental points. They use chemogenetic manipulations and electrophysiological recordings to test how this projection affects hippocampal network properties during behavior. I find the study to be very interesting, the results are important for our understanding of how social memories of different natures (remote or immediate) are encoded and supported by the hippocampal circuitry. I have some points that I added below that I think could help clarify the conclusions:<br /> - My major concern with the manuscript was that making the transitions between the different experiments for each result section is not very smooth. Maybe they can discuss a bit in a summary conclusion sentence at the end of each result section why the next set of experiments is the most logical step.<br /> - In line 113, the authors say that "the DG is known to influence hippocampal theta-gamma coupling and SWRs". Another recent study Fernandez-Ruiz et al. 2021, examined how various gamma frequencies in the dentate gyrus modulate hippocampal dynamics.<br /> - Having no single cells in the electrophysiological recordings makes it difficult to interpret the ephys part. Perhaps having a discussion on this would help interpret the results. If more SWRs are produced from the CA2 region (perhaps aided by projections from abGC), more CA2 cells that respond to social stimuli (Oliva et al. 2020) would reactivate the memories, therefore making them consolidate faster/stronger. On the other hand, the projections from abGC that the authors see, also target a great deal of PV+ interneurons, which have been shown to pace the SWRs frequency (Stark et al 2014, Gan et al 2017), which further suggests that this projection could be involved in SWRs modulation.<br /> - The authors should cite and discuss Shuo et al., 2022 (A hypothalamic novelty signal modulates hippocampal memory).<br /> - I think the authors forgot to refer to Fig 3a-f, maybe around lines 163-168.<br /> - Are the SWRs counted only during interaction time or throughout the whole behavior session for each condition?<br /> - Figure 3t shows a shift in the preferred gamma phase within theta cycles as a result of abGC projections to CA2 ablation with CNO, especially during Mother CNO condition. I think this result is worth mentioning in the text.<br /> - Figure 3u in the legend mention "scale bars = 200um", what does this refer to?<br /> - What exactly is calculated as SWR average integral? Is it a cumulative rate? Please clarify.<br /> - Alexander et al 2017, "CA2 neuronal activity controls hippocampal oscillations and social behavior", examined some of the CA2 effects in the hippocampal network after CNO silencing, and the authors should cite it.

Strengths:<br /> Behavioral experiments after abGC projections to CA2 are compelling as they show clearly distinct behavioral readout.

Weaknesses:<br /> Electrophysiological experiments are difficult to interpret without additional quantifications (single-cell responses during interactions etc.)

Reviewer #2 (Public Review):

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

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

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

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

Reviewer #2 (Public Review):

The authors employed the Mendelian Randomization method to analyze the association between type 2 diabetes (T2D) and fracture using the UK Biobank data. They found that "genetically predicted T2D was associated with higher BMD and lower risk of fracture". Additionally, they identified 10 loci that were associated with both T2D and fracture risk, with the SNP rs4580892 showing the highest signal. While the negative relationship between T2D and fracture has been previously observed, the discovery of these 10 loci adds an intriguing dimension to the findings, although the clinical implications remain uncertain.

Reviewer #2 (Public Review):

In this manuscript, the authors use newly-generated, large-scale transcriptomic data along with histological data to attempt to dissect the mechanisms by which individuals with Tibetan ancestry are able to mitigate the negative effects of high elevation on birth weight. They present detailed analyses of the transcriptomic data and find significant sex differences in the placenta transcriptome.

I have significant concerns about the conclusions that are presented. The analyses also lack the information necessary to evaluate their reliability.

The experimental design does not include a low elevation comparison and thus cannot be used to answer questions about how ancestry influences hypoxia responses and thus birthweight at high elevations. Importantly, because the placenta tissues (and trophoblasts specifically) are quickly evolving, there are a priori good reasons to expect to find population differences irrespective of adaptive evolution that might contribute to fetal growth protection. There are also significant details missing in the analyses that are necessary to substantiate and replicate the analyses presented.

Although the datasets are ultimately valuable as reference sets, the absence of low elevation comparisons for Tibetans and Han Chinese individuals undermines the ability of the authors to assess whether differences observed between populations are linked to hypoxia responses or variation in the outcomes of interest (i.e., hypoxia-dependent fetal growth restriction).

The authors attempt to tackle this phenotypic association by looking for correlations between gene networks (WGCNA) and individual genes with birthweight and other measurements collected at birth. I have some reservations about this approach with only two groups (i.e., missing the lowland comparison), but it is further problematic that the authors do not present data demonstrating that there are differences in birthweight or any other traits between the populations in the samples they collected.

Throughout, I thus find conclusions about the adaptive value and hypoxia-responses made by the authors to be unsubstantiated and/or the data to be inadequate. There are also a gratuitous number of speculative statements about mechanisms by which differential gene expression leads to the protection of birthweight that are not evaluated and thus cannot be substantiated by the data presented.

As currently presented and discussed, these results thus can only be used to evaluate population differences and tissue-specific variation therein.

There is also some important methodological information missing that makes it difficult or impossible to assess the quality of the underlying data and/or reproduce the analyses, further limiting the potential impact of these data:<br /> 1. Transcriptome data processing and analyses: RNA quality information is not mentioned (i.e., RIN). What # of reads are mapped to annotated regions? How many genes were expressed in each tissue (important for contextualizing the # of DE genes reported - are these a significant proportion of expressed genes or just a small subset?).<br /> 2. The methods suggest that DE analyses were run using data that were normalized prior to reading them into DESeq2. DESeq2 has an internal normalization process and should not be used on data that was already normalized. Please clarify how and when normalization was performed.<br /> 3. For enrichment analyses, the background gene set (all expressed genes? all genes in the genome? or only genes expressed in the tissue of interest?) has deterministic effects on the outcomes. The background sets are not specified for any analyses.<br /> 4. In the WGCNA analysis, P-values for correlations of modules with phenotype data (birthweight etc.) should be corrected for multiple testing (i.e., running the module correlation for each outcome variables) and p.adjust used to evaluate associations to limit false positives given the large number of correlations being run.<br /> 6. The plots for umbilical histological data (Fig 5 C) contain more than 5 points, but the use of replicate sections is not specified. If replicate sections were used, the authors should control for non-independence of replicate sections in their analyses (i.e., random effects model).

On more minor notes:<br /> There is significant and relevant published data on sex differences and hypoxia in rodents (see Cuffe et al 2014, "Mid- to late-term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways, and nutrient transporters in a sex-specific manner" and review by Siragher and Sferuzzi-Perro 2021, "Placental hypoxia: What have we learnt from small animal models?"), and historical work reporting sex differences in placental traits associated with high elevation adaptation in Andeans (series of publications by Moira Jackson in the late 1980s, reviewed in Wilsterman and Cheviron 2021, "Fetal growth, high altitude, and evolutionary adaptation: A new perspective").

Reviewer #2 (Public Review):

In this manuscript, Xie and colleagues investigate the contribution of osteocytes to bone metastasis of non-small cell lung carcinoma (NSCLC) using a combination of clinical samples and in vitro and in vivo data. They find that metastatic NSCLC cells exhibit lower levels of the proliferation marker Ki-67 when located in areas adjacent to the bone surface in both NSCLC patients and an intraosseous animal model of NSCLC. Using in vitro approaches, they show that osteocyte-like cells inhibit the proliferation of NSCLC cells through the secretion of small extracellular vesicles (sEVs). They identify miR-99b-3p as a component of sEVs and demonstrate that miR-99b3p inhibits the proliferation of NSCLC cells by targeting the transcription factor MDM2. Interestingly, the data also shows that mechanical stimulation of osteocytes enhances the inhibitory effect of osteocytes on NSCLC cell proliferation via increasing sEVs release. By performing different in vivo studies, the authors show that tibial loading and moderate exercise (treadmill running), before and after tumor cell inoculation, suppress tumor progression in bone and protect bone mass. Intriguingly, the moderate exercise regime shows additive/synergistic effects with the co-administration of anti-resorptive therapy. These data add to the growing evidence pointing towards osteocytes as important cells of the tumor microenvironment capable of influencing the progression of tumors in bone.

The conclusions of the paper, however, are not well supported by the data, and some critical aspects of image analysis and data analysis need to be clarified and extended.

1) The histological images are analyzed in a qualitative manner, with no description of the methodology used. In bone metastases, cancer cells are frequently mixed with bone marrow cells. The lack of cell markers to identify NSCLC cells versus bone marrow cells makes the interpretation of the imaging data difficult. The authors rely on KI-67 as a marker of proliferation. Yet, it is intriguing that some osteocytes, non-proliferating cells by definition, are often positive for this marker, which questions the specificity of the staining. To make these results more solid, the authors should have provided the proper immunostaining controls to check for specificity and use additional markers of proliferation.

2) Adding control groups to fully assess the impact of the in vivo interventions (tibial loading, moderate exercise, anti-resorptive therapy) on bone mass would be needed. The authors should have used naive mice or analyzed the bones from the non-injected contralateral legs. Further, validating the in vivo work with other osteocyte-like cells or primary osteocytes would have strengthened the results.

3) The data on miRNA99b-3p on NSCLC in Supplementary Figure 3 is not convincing. The positive cells are difficult to see and most of the osteocyte lack nuclei. Better data, in humans and the mouse model, would have helped to confirm that osteocytes produce miRNA99b-3p.

4) The conclusions of the paper are not fully supported by the data provided. Osteocytes, as well as other bone cells, can respond to mechanical stimulation and thus could virtually be responsible for the protective effects of mechanical loading or moderate exercise. In vivo experiments demonstrating a direct role of osteocytes-produced miRNA99b-3p are needed to support the notion that osteocytes maintain tumor dormancy in NSCLC bone metastasis. Further, the authors solely rely on Ki-67 as a marker of dormancy. Completing this analysis with an assessment of a dormant gene expression signature or in vivo studies assessing tumor dormancy directly would be needed to confirm this notion.

Reviewer #2 (Public Review):

Summary:

This work presents a previously undescribed neuroanatomical and neurophysiological analog between mammals and songbirds. Juvenile zebra finches learn to sing by memorizing an adult song and then, through practice, converging to a close copy of the stored template. Previous work identified pathways emanating from the avian auditory cortical regions (AIV) and basal ganglia that, through ventral pallium (VP), and the subthalamic nucleus, innervate the finches' ventral tegmental area (VTA). As in mammals, the dopaminergic projections of the VTA onto the avian striatopallidal nucleus, area X, deliver a prediction error signal. This signal encodes a surprisingly better or worse performance of the ongoing song and therefore allows the birds to improve.

In mammals, lateral Habenula (LHb) neurons contribute to learning by signaling disappointing trial outcomes or aversive stimuli. Using viral tract tracing Roesner et al. identify projections from the zebra finch VP and AIV to the LHb as well as from the LHb to the VTA. The authors use functional mapping to show that the VP activates the LHb and that the LHb suppresses the Area X-projecting VTA neurons. Then, the authors show that lesioning the LHb in juvenile finches does not prevent them from copying their tutor's song but still leads to worse performance than controls due to the production of highly abnormal vocalizations, peppered in both lone and female-directed songs. In contrast, lesioning the LHb in adult finches has no effect on the song. Together, these findings suggest that the LHb may be part of a song evaluation system and may participate in learning by signaling vocalizations that deviate from the desired tutor template.

The LHb is an evolutionarily conserved structure that connects the forebrain and midbrain with the epithalamus in vertebrates. By identifying the LHb as a component in song learning, the authors lay the grounds for a trove of new research into the various emotional, biophysical, memory, and sensory processes that contribute to learning within and through the LHb. Most conclusions of this paper are well supported by data, but some conceptual and analytic aspects require framing with respect to methodological limitations.

Strengths:<br /> The use of both anatomical tracing and functional circuit mapping is a uniquely-powerful approach to addressing the main line of inquiry in this work. Specifically, collision testing and antidromic identification allow identifying LHb-->VTA and VTA-->X projecting neurons and therefore testing the response of these specific learning-related projections to stimulation in VP and LHb (respectively).

The evaluation of abnormal vocalizations using a variational autoencoder (VAE) is a particularly strong approach that is immune to observer biases. By training this artificial neural network model with sham or pre-lesion animals, the authors clearly distinguish abnormal syllables because of their significantly poorer reconstruction through the VAE. This approach allowed the authors to provide strong quantitative support to the effect of LHb lesion in juvenile finches on their adult song.

Weaknesses:<br /> The lesions in juveniles, as the authors discuss, were histologically examined at the end of the song development, months after their creation. The authors mention not being able to rule out damage to the medial part of the Hb. But the effect of the lesions could perhaps be mediated by damage to other brain regions, such as DLM, or passing fibers (when using electrolytic lesions).

Additionally, the effect on learning could also be mediated indirectly. In mammals, the outputs of the LHb target dopaminergic regions, serotonergic regions, and a cholinergic region. In birds, the LHb may also have a diverse impact on neuromodulators and therefore an impact on behavior states and on sleep. Disrupted behavior states may lead to poorer or less frequent practice and indirectly to abnormal results that do not stem from erroneous performance evaluation.

Reviewer #2 (Public Review):

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

Reviewer #2 (Public Review):

Summary:<br /> Sotiropoulou et al. present an interesting study of an incredibly rare premature aging disease (De Barsy syndrome), examining both the underlying mechanisms at play behind the condition as well as how that biology may have a larger role in understanding features of normal aging, and in particular, human skin aging. The authors link one of the underlying genetic defects in De Barsy syndrome (PYCR1 mutations) to its phenotypic manifestations and then extrapolate those findings to present more preliminary data to suggest that a loss of PYCR1 may be a biomarker of normal human skin aging.

Strengths:<br /> - The study is important as De Barsy syndrome is challenging to study given its rarity, thus making it an understudied condition. Here the authors combine both human patient samples and murine models to offer a nice contribution to further understanding the pathophysiology of this disease.

- The authors are able to link some of the observed features in De Barsy syndrome preliminarily to more common aging models and processes (senescence, human skin dermal aging). They nicely show that the loss of Pycr1 in mice can provoke thinning of the dermis of mice while not affecting the epidermis. Furthermore, they present compelling data to suggest that Pycr2 may be compensating in mice (while not in humans) and this may contribute to the differences in lifespan observed between the mice and humans.

- Should these results be further verified, this could suggest that further study of Pycr1 and Pycr2 biology may offer new insights into aging and senescence in other tissues.

Weaknesses:<br /> - Some of the data appears preliminary and seems like it needs further analysis as described further below in my suggestions for the authors:

1) While the authors report that there is no difference in the lifespan of the Pycr1-KO mice, can they report whether there was overall weight loss or any size differences between the mice? This is helpful particularly when comparing the dermal thickness as well as considering how the global loss of Pycr1 may affect overall systemic health.

2) In Figure S2E, the comparison "pairs" seems somewhat arbitrarily chosen and it seems from the quantifications of these pairs that depending upon which young sample you compare to which old sample, you may end up with differing results. I think the more appropriate way to make this quantitative comparison would be to average the young samples and average the old samples and then compare them and perform statistics. This seems critical to really assess whether PYCR1 loss would be a consistent marker of human skin dermal aging. Additionally, it would be helpful to also look at Pycr2 expression in the normal young versus old dermis to see if the reported difference in Pycr1 is really something specific for Pycr1 and not something more general.

3) Are the labels mixed up in Figures 1J and 1K or am I reading it incorrectly? From what I can see the graph is showing that the dermal thickness and collagen intensity is higher in the Pycr1-/- mice. Similarly, the authors state that there is "significantly less collagen fiber staining", although in Figure S1G neither the quantification of collagen I or collagen III are shown to be significant. These discrepancies need to be discussed or corrected.

4) Can the authors speculate further on why Pycr2 is also diminished in human patients (while it clearly remains present in the mice).

5) Can the authors comment on whether other canonical senescence features are seen in De Barsy syndrome (p16 positivity, senescence associated secretory phenotype, etc.)? Along these lines, there is an abundance of publicly available RNA-seq datasets from various forms of senescent cells. It would be interesting to examine these and see whether there is any loss of expression of PYCR1 or PYCR2 in these data, or is the loss of PYCR1 only seen at the protein level?

Reviewer #2 (Public Review):

Milovic, Duong, and Barbour investigate the inflammatory response of three species of small mammals (P. leucopus, M. musculus, and R. norvegicus) to endotoxin lipopolysaccharide (LPS) injection via genome-wide transcriptomics from blood samples. Understanding the inflammation response of P. leucopus is of importance as they are a reservoir for several pathogens. The study is a thorough, controlled, well researched analysis that will be valuable for designing and interpreting future studies. The authors discuss the limitations of the data and the potential directions. Clearly P. leucopus respond differently to the LPS exposure which is very interesting and opens the door for numerous other comparative studies.

The conclusions of the manuscript are thoughtful and mostly supported by the data, but there are a couple of points for clarification.

1) How were the number of animals for each experiment selected? Was a power analysis conducted?

2) The authors conducted a cursory evaluation of sex differences of P. leucopus and reported no difference in response except for Il6 and Il10 expression being higher in the males than the females in the exposed group. The data was not presented in the manuscript. Nor was sex considered for the other two species. A further discussion of the role that sex could play and future studies would be appreciated.

3) The ratio of Nos2 and Arg1 copies for LPS treated and control P. leucopus and M.musculus in Table 3 show that in P. leucopus there is not a significant difference but in M.musculus there is an increase in Nos2 copies with LPS treatment. The authors then used a targeted RNA-seq analysis to show that in P. leucopus the number of Arg1 reads after LPS treatment is significantly higher than the controls. These results are over oversimplified in the text as an inverse relationship for Nos2/Arg1 in the two species.

Reviewer #2 (Public Review):

Summary:

This work addresses the question of how human macrophages restrict intracellular replication of Salmonella.

Strengths:

Through a series of genetic knockouts and using specific inhibitors, Egan et al. demonstrated that the inflammasome components caspase-1, caspase-4, gasdermin D (GSDMD), and the final lytic death effector ninjurin-1 (NINJ1) are required for control of Salmonella replication in human macrophages. Interestingly, caspase-1 proved crucial in restricting Salmonella early during infection, whereas caspase-4 was essential in the later stages of infection. Furthermore, using a chloroquine resistance assay and state-of-the-art microscopy, the authors found that NAIP receptor and caspase-1 mostly regulate replication of cytosolic bacteria, with smaller, yet significant, impact on the vacuolar bacteria.

The finding that inflammasomes are critical in the restriction of replication of intracellular Salmonella in human macrophages contrasts with the published minimal role of inflammasomes in restriction of replication of intracellular Salmonella in murine macrophages. These findings demonstrate yet another example of interspecies and intercellular differences in regulation of bacterial infections by the immune system.

Weaknesses: none.

Reviewer #2 (Public Review):

Summary: The authors have previously demonstrated that the E3 ligase PDLIM2 inhibits NF-kB and STAT3 and is epigenetically repressed in human lung cancers (Sun et al. Nat. Comm. 2019 10: 5324); therefore, PDLIM2 is a tumor suppressor in lung cancer. In this manuscript, they follow up on their previous findings and show that expression of PDLIM2 is downregulated in human lung cancers by both genetic deletion and promoter methylation. They further describe a novel approach to restore the expression of PDLIM2 in mouse lung tumors by systemically administering PDLIM2 plasmids encapsulated in nanoparticles (termed "nanoPDLIM2"). The nanoPDLIM2 approach was shown to exhibit efficacy with low toxicity in a urethane-induced mouse lung cancer model. The authors further demonstrated the synergy of nanoPDLIM2 with chemotherapy and PD-1 blockade immunotherapy. The combination therapy of nanoPDLIM2, chemotherapy, and immunotherapy proved most effective with complete tumor remission in 60% of mice. Mechanistically, nanoPDLIM2 upregulated MHC-I expression, enhanced CD4/CD8 T cell activation and tumor infiltration, and suppressed MDR1 induction and nuclear expression of STAT3, RelA and prosurvival genes in tumors. Overall, this study is important because it reinforces the critical roles of PDLIM2 in suppressing lung cancer, and also identifies a potential approach to restoring PDLIM2 expression in lung tumors. The experiments were well executed; the data are convincing and support the conclusions made by the authors.

Reviewer #2 (Public Review):

In a study by Shen et al., the authors investigated YAP/TAZ target genes that play a role in the formation of processing bodies (P-bodies). P-bodies are membraneless cytoplasmic granules that contain translationally repressed mRNAs and components of mRNA turnover. GO enrichment analysis of the RNA-Seq data of colorectal cancer cells (HCT116) after YAP/TAZ knockdown showed that the downregulated genes were enriched in P-body resident proteins. Overexpression, knockdown, and ChIP-qPCR analyses showed that SAMD4A, PNRC1, AJUBA, and WTIP are YAP-TEAD target genes that also play a role in P-body biogenesis. Using P-body markers such as DDX6 and DCP1A, the authors showed that the knockdown of YAP in the HCT116 cell line causes a reduction in the number of P-bodies. Similarly, overexpression of constitutively active YAP (YAP 5SA) increased the P-body number. The YAP-TEAD target genes SAMD4A and AJUBA positively regulate P-body formation, because lowering their expression levels using siRNA reduces the number of P-bodies. The other YAP target gene, PNRC1, is a negative regulator of P-body biogenesis and consistently YAP suppresses its expression through the recruitment of the NuRD complex. YAP target genes that modulate P-body formation play prominent roles in oncogenesis. PNRC1 suppression is key to YAP-mediated proliferation, colony formation, and tumorigenesis in HCT116 xenografts. Similarly, SAMD4 and AJUBA knockdown abrogated cell viability. In summary, this study demonstrated that SAMD4, AJUBA, WTIP, and PNRC1 are bona fide YAP-TEAD target genes that play a role in P-body formation, which is also linked to the oncogenesis of colon cancer cells.

Major Strengths:

The majority of the experiments were appropriately planned so that the generated data could support the conclusions drawn by the authors. The phenotype observed with YAP/TAZ knockdown correlated inversely with YAP5SA overexpression, which is complementary. Where possible, the authors also used point mutations that selectively disrupt protein-protein interactions, such as YAP S94A and PNRC1 W300A. The CRC cell line HCT116 was used throughout the study; additionally, data from other cancer cell lines were used to support the generality of the findings.

Weaknesses:

The authors did not elucidate the mechanistic link between P-body formation and oncogenesis; therefore, it is unclear why an increase in the number of P-bodies is pro-tumorigenic. AJUBA and SAMD4 may have housekeeping functions and reduce the proliferation of YAP-independent cell lines. Figure 6 - Figure Supplement 4 shows a reduction in cell viability and migration in control HCT116 cell lines upon AJUBA/SAMD4 knockdown. Therefore, it is unclear whether their tumor suppressive role is YAP-dependent. The authors extrapolated and suggested that their findings could be exploited therapeutically, without providing much detail. How do they plan to stimulate the expression of PNRC1? It is not necessary for every scientific finding to lead to a therapeutic benefit; therefore, they can tone down such statements if therapeutic exploitation is not realistic. The authors elucidated a mechanism for PNRC1 repression and one wonders why no attempts were made to understand the mechanism of activation of SAMD4, AJUBA, and WTIP expression.

Reviewer #2 (Public Review):

Summary:<br /> ​In the present study, Gardeux et al provide a web-based tool for curated association mapping results from DRP studies. The tool lets users view association results for phenotypes and compare mean phenotype ~ phenotype correlations between studies. In the manuscript, the authors provide several example utilities associated with this new resource, including pan-study summary statistics for sex, traits, and loci. They highlight cross-trait correlations by comparing studies focused on longevity with phenotypes such as oxphos and activity.

Strengths:<br /> -Considerable efforts were dedicated toward curating the many DRG studies provided.<br /> -Available tools to query large DRP studies are sparse and so new tools present appeal

Weaknesses:<br /> The creation of a tool to query these studies for a more detailed understanding of physiologic outcomes seems underdeveloped. These could be improved by enabling usages such as more comprehensive queries of meta-analyses, molecular information to investigate given genes or pathways, and links to other information such as in mouse rat or human associations.

Reviewer #2 (Public Review):

Summary: In this study of the mouse homolog of acidic mammalian chitinase, the overall goal is to provide a mechanistic explanation for the unusual observation of two pH optima for the enzyme. The study includes biochemical assays to establish kinetic parameters at different solution pH, structural studies of enzyme/substrate complexes, and theoretical analysis of amino acid side chain pKas and molecular dynamics.

Strengths: The biochemical assays are rigorous and nicely complemented by the structural and computational analysis. The mechanistic proposal that results from the study is well rationalized by the observations in the study.

Weaknesses: The overall significance of the work could be made more clear. Additional details could be provided about the limitations of prior biochemical studies of mAMC that warranted the kinetic analysis. The mouse enzyme seems unique in terms of its behavior at high and low pH, so it remains unclear how the work will enhance broader understanding of this enzyme class. It was also not clear can the findings be used for therapeutic purposes, as detailed in the abstract, if the human enzyme works differently.

Reviewer #2 (Public Review):

This submission is about the role of Dbp5/Gle1 in tRNA export. The manuscript provides data showing that Dbp5/Gle1 are involved in tRNA export from the nucleus which is an essential process critical to translation. The authors provide data that largely supports conclusions, however, there are some pieces of data that are misinterpreted. (Figure 1A and B look the same; in Fig 1E, the DAPI staining is abnormal; in Fig 4 the bands can't be seen.)

Additionally, the methods used are fairly standard so the article does not contain any new technical achievements.

Reviewer #2 (Public Review):

The authors presented a well-written manuscript describing the comparison of active-learning methods with state-of-art methods for several datasets of pharmaceutical interest. This is a very important topic since active learning is similar to a cyclic drug design campaign such as testing compounds followed by designing new ones which could be used to further tests and a new design cycle and so on. The experimental design is comprehensive and adequate for proposed comparisons. However, I would expect to see a comparison regarding other regression metrics and considering the applicability domain of models which are two essential topics for the drug design modelers community.

Reviewer #2 (Public Review):

A limitation in using SNPs to understand recent histories of genomes is their low mutation frequency. Tellier et al. explore the possibility of adding hypermutable markers to SNP based methods for better resolution over short time frames. In particular, they hypothesize that epimutations (CG methylation and demethylation) could provide a useful marker for this purpose. Individual CGs in Arabidopsis tends to be either close to 100% methylated or close to 0%, and are inherited stably enough across generations that they can be treated as genetic markers. Small regions containing multiple CGs can also be treated as genetic markers based on their cumulative methylation level. In this manuscript, Tellier et al develop computational methods to use CG methylation as a hypermutable genetic marker and test them on theoretical and real data sets. They do this both for individual CGs and small regions. My review is limited to the simple question of whether using CG methylation for this purpose makes sense at a conceptual level, not at the level of evaluating specific details of the methods. I have a small concern in that it is not clear that CG methylation measurements are nearly as binary in other plants and other eukaryotes as they are in Arabidopsis. However, I see no reason why the concept of this work is not conceptually sound. Especially in the future as new sequencing technologies provide both base calling and methylating calling capabilities, using CG methylation in addition to SNPs could become a useful and feasible tool for population genetics in situations where SNPs are insufficient.

Reviewer #2 (Public Review):

In this study, Lewis et al seek to further define the role of ROM1. ROM1 is a tetraspanin protein that oligomerizes with another tetraspanin, PRPH2, to shape the rims of the membrane discs that comprise the light-sensitive outer segment of vertebrate photoreceptors. ROM1 knockout mice and several PRPH2 mutant mice are reexamined. The conclusion reached is that ROM1 is redundant to PRPH2 in regulating the size of newly forming discs, although excess PRPH2 is required to compensate for the loss of ROM1.

This replicates earlier findings while adding rigor using a mass spectrometry-based approach to quantitate the ratio of ROM1 and PRPH2 to rhodopsin (the protein packed in the body of the disc membranes) and careful analysis of tannic acid labeled newly forming discs using transmission electron microscopy.

In ROM1 knockout mice PRPH2 expression was found to be increased so that the level of PRPH2 in those mice matches the combined amount of PRPH2 and ROM1 in wildtype mice. Despite this, there are defects in disc formation that are resolved when the ROM1 knockout is crossed to a PRPH2 overexpressing line. A weakness of the study is that the molar ratios between ROM1, PRPH2 and rhodopsin were not measured in the PRPH2 overexpressing mice. This would have allowed the authors to be more precise in their conclusion that a 'sufficient' excess of PRPH2 can compensate for defects in ROM1.

Reviewer #2 (Public Review):

In this study, the authors examined how the maintenance of mitochondrial-associated endoplasmic reticulum membranes (MAM) is critical for the prevention of muscle atrophy under microgravity conditions. They observed, a reduction in MAM in myotubes placed in a microgravity condition; in addition, MFN2-deficient human iPS cells showed a decrease in the number of MAM, similar to in myotubes differentiated under microgravity conditions, in addition to the activation of the Notch signaling pathway. The authors, moreover, observed that treatment with the gamma-secretase inhibitor with DAPT preserved the atrophic phenotype of differentiated myotubes in microgravity and improve the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice.

The entire study was well conducted, bringing an interesting analysis in vitro and in vivo of aging conditions. In my opinion, it is necessary to improve the analysis of both genes and proteins to better support the conclusions

The study can contribute to a better understanding of one of the major problems of aging, such as muscle atrophy and inhibition of muscle regeneration, emphasizing the importance of the NOTCH pathway in these pathological situations. The work will be of interest to all scientists working on aging.

Reviewer #2 (Public Review):

Summary:

Cell-free DNA (cfDNA) are short DNA fragments released into the circulation when cells die. Plasma cfDNA level is thought to reflect the degree of cell-death or tissue injury. Indeed, plasma cfDNA is a reliable diagnostic biomarker for multiple diseases, providing insights into disease severity and outcomes. In this manuscript, Dr. Sender and colleagues address a fundamental question: What fraction of DNA released from cell death is detectable as plasma cfDNA? The authors use public data to estimate the amount of DNA produced from dying cells. They also utilize public data to estimate plasma cfDNA levels. Their calculations showed that <10% of DNA released is detectable as plasma cfDNA, the fraction of detectable cfDNA varying by tissue sources. The study demonstrates new and fundamental principles that could improve disease diagnosis and treatment via cfDNA.

Strengths:

1. The experimental approach is resource-mindful taking advantage of publicly available data to estimate the fraction of detectable cfDNA in physiological states. The authors did not assess if the fraction of detectable cfDNA changes in disease conditions. Nonetheless, their pioneering study lays the foundation and provides the methods needed for a similar assessment in disease states.<br /> 2. The findings of this study potentially explain discrepancies in measured versus expected tissue-specific cfDNA from some tissues. For example, the gastrointestinal tract is subject to high cell turnover and release of DNA. Yet, only a small fraction of that DNA ends up in plasma as gastrointestinal cfDNA.<br /> 3. The study proposes potential mechanisms that could account for the low fraction of detectable cfDNA in plasma relative to DNA released. This includes intracellular or tissue machinery that could "chew up" DNA released from dying cells, allowing only a small fraction to escape into plasma as cfDNA. Could this explain why the gastrointestinal track with an elaborate phagosome machinery contributes a small fraction of plasma cfDNA? Given the role of cfDNA as damage-associated molecular pattern in some diseases, targeting such a machinery may provide novel therapeutic opportunities.

Weaknesses: In vitro and in vivo studies are needed to validate these findings and define tissue machinery that contribute to cfDNA production. The validation studies should address the following limitations of the study design: -

1. Align the cohorts to estimate DNA production and plasma cfDNA levels. Cellular turnover rate and plasma cfDNA levels vary with age, sex, circadian clock, and other factors (Madsen AT et al, EBioMedicine, 2019). This study estimated DNA production using data abstracted from a homogenous group of healthy control males (Sender & Milo, Nat Med 2021). On the other hand, plasma cfDNA levels were obtained from datasets of more diverse cohort of healthy males and females with a wide range of ages (Loyfer et al. Nature, 2023 and Moss et al., Nat Commun, 2018).<br /> 2. "cfDNA fragments are not created equal". Recent studies demonstrate that cfDNA composition vary with disease state. For example, cfDNA GC content, fraction of short fragments, and composition of some genomic elements increase in heart transplant rejection compared to no-rejection state (Agbor-Enoh, Circulation, 2021). The genomic location and disease state may therefore be important factors to consider in these analyses.<br /> 3. Alternative sources of DNA production should be considered. Aside from cell death, DNA can be released from cells via active secretion. This and other additional sources of DNA should be considered in future studies. The distinct characteristics of mitochondrial DNA to genomic DNA should also be considered.

Reviewer #2 (Public Review):

Summary:<br /> This study seeks to advance our knowledge of how vitamin D may be protective in allergic airway disease in both adult and neonatal mouse models. The rationale and starting point are important human clinical, genetic/bioinformatic data, with a proposed role for vitamin D regulation of 2 human chromosomal loci (Chr17q12-21.1 and Chr17q21.2) linked to the risk of immune-mediated/inflammatory disease. The authors have made significant contributions to this work specifically in airway disease/asthma. They link these data to propose a role for vitamin D in regulating IL-2 in Th2 cells implicating genes associated with these loci in this process.

Strengths:<br /> Here the authors draw together evidence from multiple lines of investigation to propose that amongst murine CD4+ T cell populations, Th2 cells express high levels of VDR, and that vitamin D regulates many of the genes on the chromosomal loci identified to be of interest, in these cells. The bottom line is the proposal that vitamin D, via Ikfz3/Aiolos, suppresses IL-2 signalling and reduces IL-2 signalling in Th2 cells. This is a novel concept and whilst the availability of IL-2 and the control of IL-2 signalling is generally thought to play a role in the capacity of vitamin D to modulate both effector and especially regulatory T cell populations, this study provides new data.

Weaknesses:<br /> Overall, this is a highly complicated paper with numerous strands of investigation, methodologies etc. It is not "easy" reading to follow the logic between each series of experiments and also frequently fine detail of many of the experimental systems used (too numerous to list), which will likely frustrate immunologists interested in this. There is already extensive scientific literature on many aspects of the work presented, much of which is not acknowledged and largely ignored. For example, reports on the effects of vitamin D on Th2 cells are highly contradictory, especially in vitro, even though most studies agree that in vivo effects are largely protective. Similarly, other reports on adult and neonatal models of vitamin D and modulation of allergic airway disease are not referenced. In summary, the data presentation is unwieldy, with numerous supplementary additions, which makes the data difficult to evaluate and the central message lost. Whilst there are novel data of interest to the vitamin D and wider community, this manuscript would benefit from editing to make it much more readily accessible to the reader.

Wider impact: Strategies to target the IL-2 pathway have long been considered and there is a wealth of knowledge here in autoimmune disease, transplantation, GvHD etc - with some great messages pertinent to the current study. This includes the use of IL-2, including low dose IL-2 to boost Treg but not effector T cell populations, to engineered molecules to target IL-2/IL-2R.

Reviewer #2 (Public Review):

Summary:<br /> The present study explores the molecular function of LRRC23 in male fertility, specifically in the context of the regulation of spermiogenesis. The author initiates the investigation by identifying LRRC23 mutations as a potential cause of male sterility based on observations made in closely related individuals affected by asthenozoospermia (ASZ). To further investigate the function of LRRC23 in spermatogenesis, mutant mice expressing truncated LRRC23 proteins are created, aligning with the identified mutation site. Consequently, the findings confirm the deleterious effects of LRRC23 mutations on sperm motility in these mice while concurrently observing no significant abnormalities in the overall flagella structure. Furthermore, the study reveals LRRC23's interaction with the RS head protein RSPH9 and its active involvement in the assembly of the axonemal RS. Notably, LRRC23 mutations result in the loss of the RS3 head structure and disruption of the RS2-RS3 junction structure. Therefore, the author claimed that LRRC23 is an indispensable component of the RS3 head structure and suggests that mutations in LRRC23 underlie sterility in mice.

Strengths:<br /> The key contribution of this article lies in confirming LRRC23's involvement in assembling the RS3 head structure in sperm flagella. This finding represents a significant advancement in understanding the complex architecture of the RS3 structural complex, building upon previous studies. Moreover, the article's topic is interesting and originates from clinical research, which holds significant implications for potential clinical applications.

Weaknesses:<br /> 1. While the author generated mutant mice expressing truncated LRRC23 proteins, the expression of these truncated proteins was not detected in sperm. This implies that, in terms of sperm structure, the mutant LRRC23 protein behaves similarly to the complete knockout of the LRRC23 protein, which has been previously reported and characterized (Zhang et al., 2021).

2. This reviewer questions the proposal that LRRC23 is an integral component of RS3, as the results indicate not only the loss of the RS3 head structure but also an incomplete RS2-RS3 junction structure. In addition, the interaction of LRRC23 with RSPH9 alone does not fully explain its involvement solely in RS3 assembly. Additional evidence is required to examine the influence of LRRC23 on the RS2-RS3 junction.

3. The article does not explore how these mutations affect the flagella structure in human sperm, which needs further study. Expanding the study to include human sperm structure would undoubtedly enhance the quality of the article.

Reviewer #2 (Public Review):

This study investigates the excitability of neurons in the peri-infarct cortex during recovery from ischemic stroke. The excitability of neurons in the peri-infarct cortex during stroke recovery has produced contradictory findings: some studies suggest hyper-excitability to direct-brain stimulation, while others indicate diminished responsiveness to physical stimuli. However, most studies have used anesthetized animals, which can disrupt cortical activity and functional connectivity. The present study used two-photon Ca2+ imaging after focal photothrombotic stroke to examine neural activity patterns in awake mice. The authors found reduced neuronal spiking in the peri-infarct cortex that was strongly correlated with motor performance deficits. Additionally, the authors found disruptions in neural activation, functional connectivity, and assembly architecture in the immediate peri-infarct region but not in the distal cortex regions.

The findings of this study are very important as they show that there is no measurable change in terms of neuronal activation and reorganization in distal regions of remapped cortical response areas after stroke.

However, cortical response areas are calculated using a threshold of 95% peak activity within a trial. The threshold is presumably used to discriminate between the sensory-evoked response and collateral activation / less "relevant" response (noise). Since the peak intensity is lower after stroke, the "response" area is larger - lower main signal results in less noise exclusion. Predictably, areas that show a higher response before stroke than after are excluded from the response area before the stroke and included after.

We suggest a reinterpretation of the findings: much of the non-remapped areas are included when using a within-trial threshold as a criterion, and the absence of increased neuronal activation and reorganization is evidence for this claim. The take-home message of this study should be that we need a much better criterion for what we consider remapping.

Reviewer #2 (Public Review):

Summary:<br /> This is an interesting and impressive study that provides a rare opportunity to learn about brain-behaviour links of learning systems at a relatively early stage of development.

Strengths:<br /> The main strengths are that the authors followed a relatively large group of children over 2 years and used a reinforcement learning task aimed at assessing learning that depends on both the striatum and the hippocampus. The authors also included a thorough overview of the computational models and the choices they made. I think this paper would be of considerable interest and contributes to knowledge about how learning and memory systems change with development.

Weaknesses:<br /> There were a few things that I thought would be helpful to clarify. First, what exactly are the anatomical regions included in the striatum here? Second, it was mentioned that for the reduced dataset, object recognition memory focused on "sure" ratings. This seems like the appropriate way to do it, but it was not clear whether this was also the case for the full analyses in the main text. Third, the children's fitted parameters were far from optimal; is it known whether adults would be closer to optimal on the task?

The main thing I would find helpful is to better integrate the differences between the main results reported and the many additional results reported in the supplement, for example from the reduced dataset when excluding non-learners. I found it a bit challenging to keep track of all the differences with all the analyses and parameters. It might be helpful to report some results in tables side-by-side in the two different samples. And if relevant, discuss the differences or their implication in the Discussion. For example, if the patterns change when excluding the poor learners, in particular for the associations between delayed feedback and hippocampal volume, and those participants were also those less well fit by the value-based model, is that something to be concerned about and does that affect any interpretations? What was not clear to me is whether excluding the poor learners at one extreme simply weakens the general pattern, or whether there is a more qualitative difference between learners and non-learners. The discussion points to the relevance of deficits in hippocampal-dependent learning for psychopathology and understanding such a distinction may be relevant.

Reviewer #2 (Public Review):

This study examined the role of CRF neurons in the BNST in both phasic and sustained fear in males and females. The authors first established a differential fear paradigm whereby shocks were consistently paired with tones (Full) or only paired with tones 50% of the time (Part), or controls who were exposed to only tones with no shocks. Recall tests established that both Full and Part conditioned male and female mice froze to the tones, with no difference between the paradigms. Additional studies using the NSF and startle test, established that neither fear paradigm produced behavioral changes in the NSF test, suggesting that these fear paradigms do not result in an increase in anxiety-like behavior. Part fear conditioning, but not Full, did enhance startle responses in males but not females, suggesting that this fear paradigm did produce sustained increases in hypervigilance in males exclusively. Photometry studies found that while undifferentiated BNST neurons all responded to shock itself, only Full conditioning in males lead to a progressive enhancement of the magnitude of this response. BNST neurons in males, but not females, were also responsive to tone onset in both fear paradigms, but only in Full fear did the magnitude of this response increase across training. Knockdown of CRF from the BNST had no effect on fear learning in males or females, nor any effect in males on fear recall in either paradigm, but in females enhanced both baseline and tone-induced freezing only in Part fear group. When looking at anxiety following fear training, it was found in males that CRF knockdown modulated anxiety in Part fear trained animals and amplified startle in Fully trained males but had no effect in either test in females. Using 1P imaging, it was found that CRF neurons in the BNST generally decline in activity across both conditioning and recall trials, with some subtle sex differences emerging in the Part fear trained animals in that in females BNST CRF neurons were inhibited after both shock and omission trials but in males this only occurred after shock and not omission trials. In recall trials, CRF BNST neuron activity remained higher in Part conditioned mice relative to Full conditioned mice.

Overall, this is a very detailed and complex study that incorporates both differing fear training paradigms and males and females, as well as a suite of both state of the art imaging techniques and gene knockdown approaches to isolate the role and contributions of CRF neurons in the BNST to these behavioral phenomena. The strengths of this study come from the thorough approach that the authors have taken, which in turn helped to elucidate nuanced and sex specific roles of these neurons in the BNST to differing aspects of phasic and sustained fear. More so, the methods employed provide a strong degree of cellular resolution for CRF neurons in the BNST. In general, the conclusions appropriately follow the data, although the authors do tend to minimize some of the inconsistencies across studies (discussed in more depth below), which could be better addressed through discussion of these in greater depth. As such, the primary weakness of this manuscript comes largely from the discussion and interpretation of mixed findings without a level of detail and nuance that reflects the complexity, and somewhat inconsistency, across the studies. These points are detailed below:

-Given the focus on CRF neurons in the BNST, it is unclear why the photometry studies were performed in undifferentiated BNST neurons as opposed to CRF neurons specifically (although this is addressed, to some degree, subsequently with the 1P studies in CRF neurons directly). This does limit the continuity of the data from the photometry studies to the subsequent knockdown and 1P imaging studies. The authors should address the rationale for this approach so it is clear why they have moved from broader to more refined approaches.

-The CRF KD studies are interesting, but it remains speculative as to whether these effects are mediated locally in the BNST or due to CRF signaling at downstream targets. As the literature on local pharmacological manipulation of CRF signaling within the BNST seems to be largely performed in males, the addition of pharmacological studies here would benefit this to help to resolve if these changes are indeed mediated by local impairments in CRF release within the BNST or not. While it is not essential to add these experiments, the manuscript would benefit from a more clear description of what pharmacological studies could be performed to resolve this issue.

-While I can appreciate the authors perspective, I think it is more appropriate to state that startle correlates with anxiety as opposed to outright stating that startle IS anxiety. Anxiety by definition is a behavioral cluster involving many outputs, of which avoidance behavior is key. Startle, like autonomic activation, correlates with anxiety but is not the same thing as a behavioral state of anxiety (particularly when the startle response dissociates from behavior in the NSF test, which more directly tests avoidance and apprehension). Throughout the manuscript the use of anxiety or vigilance to describe startle becomes interchangeable, but then the authors also dissociate these two, such as in the first paragraph of the discussion when stating that the Part fear paradigm produces hypervigilance in males without influencing fear or anxiety-like behaviors. The manuscript would benefit from harmonization of the language used to operationally define these behaviors and my recommendation would be to remain consistent with the description that startle represents hypervigilance and not anxiety, per se.

-The interpretation of the anxiety data following CRF KD is somewhat confusing. First, while the authors found no effect of fear training on behavior in the NSF test in the initial studies, now they do, however somewhat contradictory to what one would expect they found that Full fear trained males had reduced latency to feed (indicative of an anxiolytic response), which was unaltered by CRF KD, but in Part fear (which appeared to have no effect on its own in the NSF test), KD of CRF in these animals produced an anxiolytic effect. Given that the Part fear group was no different from control here it is difficult to interpret these data as now CRF KD does reduce latency to feed in this group, suggesting that removal of CRF now somehow conveys an anxiolytic response for Part fear animals. In the discussion the authors refer to this outcome as CRF KD "normalizing" the behavior in the NSF test of Part fear conditioned animals as now it parallels what is seen after Full fear, but given that the Part fear animals with GFP were no different then controls (and neither of these fear training paradigms produced any effect in the NSF test in the first arm of studies), it seems inappropriate to refer to this as "normalization" as it is unclear how this is now normalized. Given the complexity of these behavioral data, some greater depth in the discussion is required to put these data in context and describe the nuance of these outcomes, in particular a discussion of possible experimental factors between the initial behavioral studies and those in the CRF KD arm that could explain the discrepancy in the NSF test would be good (such as the inclusion of surgery, or other factors that may have differed between these experiments). These behavioral outcomes are even more complex given that the opposite effect was found in startle whereby CRF KD amplified startle in Full trained animals. As such, this portion of the discussion requires some reworking to more adequately address the complexity of these behavioral findings.

Reviewer #2 (Public Review):

In this manuscript, González-Segarra et al. investigated how ISNs regulate sugar and water ingestion in Drosophila. In their previous paper, authors have shown that inhibiting neurotransmission in ISNs has opposite effects on sugar and water ingestion. In this manuscript, the authors first identified the effector molecules released by ISNs. Their RNAi screen found that, surprisingly, ISNs use ilp3 as a neuromodulator. Next, using light and electron microscopy, they investigated the downstream neural circuits ISNs connect with to regulate water or sugar ingestion. These analyses identified a new group of neurons named Bilateral T-shaped neurons (BiT) as the main output of ISNs, and several other peptidergic neurons as downstream effectors of ISNs. While BiT activity regulated both sugar and water ingestion, BiT downstream neurons, such as CCHa2R, only impacted water ingestion. These results suggested that ISNs might interact with distinct neural circuits to control sugar or water ingestion. The authors also investigated other ISN downstream neurons, such as ilp2 and CCAP, and revealed that their activity also contributes to ingestive behaviors in flies.

Major strengths:<br /> 1. This manuscript presents a comprehensive investigation of the downstream neurons connected to ISNs.

2. The authors have identified and characterized a diverse set of peptidergic neurons that regulate ingestive behaviors in the fly brain.

Weaknesses:<br /> 1. Only one RNAi hairpin is used to knock down Ilp3 in ISNs? There is a concern about off-targeting effects without the presence of another hairpin or mutant data. Do ilp3 mutants also have similar defects in sugar/water ingestion compared to ISN ilp3 knockdown?

2. Throughout the paper, authors use either voltage or calcium sensors without explaining why they choose to use either method to determine the functional connectivity between neurons.

3. How these diverse sets of peptidergic neurons interact to regulate ingestive behaviors is unclear and requires further investigation.

Reviewer #2 (Public Review):

Summary:

This manuscript by Kroon, Grunewald, Marrink and coworkers present the development of Vermouth library for coarse grain assignment and parameterization and an updated version of python script, the Martinize2 program, to build Martini coarse grained (CG) models, primarily for protein systems.

Strengths:

In contrast to many mature and widely used tools to build all-atom (AA) models, there are few well-accepted programs for CG model constructions and parameterization. The research reported in this manuscript is among the ongoing efforts to build such tools for Martini CG modeling, with a clear goal of high-throughput simulations of complex biomolecular systems and, ultimately, whole-cell simulations. Thus, this manuscript targets a practical problem in computational biophysics. The authors see such an effort to unify operations like CG mapping, parameterization, etc. as a vital step from the software engineering perspective.

Weaknesses:

However, the manuscript in this shape is unclear in the scientific novelty and appears incremental upon existing methods and tools. The only "validation" (more like an example application) is to create Martini models with two protein structure sets (I-TASSER and AlphaFold). The success rate in building the models was only 73%, while the significant failure is due to incomplete AA coordinates. This suggests a dependence on the input AA models, which makes the results less attractive for high-throughput applications (for example, preparation/creation of the AA models can become the bottleneck). There seems to be an improvement in considering the protonation state and chemical modification, but convincing validation is still needed. Besides, limitations in the existing Martini models remain (like the restricted dynamics due to the elastic network, the electrostatic interactions or polarizability).

Reviewer #2 (Public Review):

In this manuscript, authors firstly investigated the role of a transcriptional factor BATF in hepatic lipid metabolism both in vivo and in vitro. By using a AAV transfection to overexpress BATF in liver, the mice with overexpression of BATF resisted the high fat diets induced obesity and attenuated the hepatic steatosis. Mechanically, the PD1 mediated its effect on lipid accumulation in hepatocyte and IL-27 mediated its effect on adiposity reduction in vivo.

Strengths:

1) This work found the transcription factor BATF was positive to reduce hepatic lipid accumulation and offered a potential target to treat NAFLD.<br /> 2) PD1 antibody is always used to treat cancer, authors here have developed its new function in metabolic disease. PD1 antibody could help mice to combat obesity and hepatic steatosis induced by high fat diets.<br /> 3) Overexpression of BATF in the liver not only decreased the lipid accumulation in the liver but also reduced the fat mass. IL-27 secretion in the liver was enhanced to affect the adipose tissue. The cross talk in liver and adipose tissue was also validated in this paper.

Reviewer #2 (Public Review):

The manuscript by G. Viejo et al. describes a new open-source toolbox called Pynapple, for data analysis of electrophysiological recordings, calcium imaging, and behavioral data. It is an object-oriented python package, consisting of 5 main object types: timestamps (Ts), timestamped data (Tsd), TsGroup, TsdFrame, and IntervalSet. Each object has a set of built-in core methods and import tools for common data acquisition systems and pipelines.

Pynapple is a low-level package that uses NWB as a file format, and further allows for other more advanced toolsets to build upon it. One of these is called Pynacollada which is a toolset for data analysis of electrophysiological, imaging, and behavioral data.

Pynapple and Pynacollada have the potential to become very valuable and foundational tools for the analysis of neurophysiological data. NWB still has a steep learning curve and Pynapple offers a user-friendly toolset that can also serve as a wrapper for NWB.

Reviewer #2 (Public Review):

In this study, Lamire et al. use a calcium imaging approach, behavioural tests, and pharmacological manipulations to identify the molecular mechanisms behind visual habituation. They show a valuable drug screen paradigm to assess the impact of pharmacological compounds on the behaviour of larval zebrafish.

The pharmacological screen identifies an expected suppression of habituation by GABA receptor antagonists. More interestingly, it identifies potentially new contributions of melatonin receptor agonists, and oestrogen receptor agonists to habituation, as they seem to increase the rate of habituation.

The volumetric calcium imaging of habituation to dark flashes is valuable, but the mix of responses to visual cues that are not relevant to the dark flash escape, such as the slow increase back to baseline luminosity, lowers the clarity of the results. The link between the calcium imaging results and free-swimming behaviour is not especially convincing, however, that is a common issue of head-restrained imaging with larval zebrafish. The identification of a cluster of neurons with potentiating responses, which could drive the habituation is intriguing, but more characterizations of these neurons would be needed to fully understand their function in habituation. The pharmacological manipulation of the habituation circuits mapped in the first part does not arrive at any satisfying conclusion, which is acknowledged by the authors.

Overall, the authors did identify interesting new molecular pathways that may be involved in habituation to dark flashes. Their screening approach, while not novel, will be a powerful way to interrogate other behavioural profiles. The authors identified circuit loci apparently involved in habituation to dark flashes, and the potentiation and no adaptation clusters have not been previously observed and are interesting targets for future work. This work suggests that the circuits and mechanisms underlying habituation are likely more complex than anticipated. The data will be useful to guide follow-up experiments by the community on the new pathway candidates that this screen has uncovered, including behaviours beyond dark flash habituation.

Reviewer #3 (Public Review):

Moutard, Laura, et al. investigated the gene expression and functional aspects of Leydig cells in a cryopreservation/long-term culture system. The authors found that critical genetic markers for Leydig cells were diminished when compared to the in-vivo testis. The testis also showed less androgen production and androgen responsiveness. Although they did not produce normal testosterone concentrations in basal media conditions, the cultured testis still remained highly responsive to gonadotrophin exposure, exhibiting a large increase in androgen production. Even after the hCG-dependent increase in testosterone, genetic markers of Leydig cells remained low, which means there is still a missing factor in the culture media that facilitates proper Leydig cell differentiation. Optimizing this testis culture protocol to help maintain proper Leydig cell differentiation could be useful for future human testis biopsy cultures, which will help preserve fertility and child cancer patients.

Overall, the authors addressed most comments and questions from the previous review. The additional data regarding the necrotic area is helpful for interpreting the quality of the cultures.

The authors did not conduct a multiple comparison tests although there are multiple comparisons conducted on for a single dependent variable (Fig 2J, Fig 3F, among many others), however, the addition of this multiple comparison is unlikely to change the conclusions of the paper or the figure and, thus is a minor technical detail in this case.

Reviewer #2 (Public Review):

Summary:<br /> Ma et al. employed a myeloid progenitor/microglia differentiation protocol to produce human-induced pluripotent stem cell (hiPSC)-derived microglia in order to examine the potential of microglial cell replacement as a treatment for retinal disorders. They characterized the iPSC-derived microglia by gene expression and in vitro assay analysis. By evaluating xenografted microglia in the partly microglia-depleted retina, the function of the microglia was further assessed.

Strengths:

Overall, the study and the data are convincing, and xenografted microglia were also tested in a RPE injury paradigm.

Weaknesses:

Gene expression analysis of mature microglia cells should be better interpreted and it would be beneficial to compare the iPSC-derived microglia gene set to a human microglial cell line (for example, HMC3) instead of myeloid progenitor cells.<br /> The way that the manuscript has been written, unfortunately, is not optimal. I recommend that the entire manuscript be edited and proofread in English. The text contains spelling and grammar mistakes, and the manuscript is inconsistent in several parts. The manuscript should also be revised for a scientific paper format.

Reviewer #2 (Public Review):

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

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

Although the CRISPR-mediated knockdown of the Rai1 gene (Fig. 4) appears to be highly effective, given the broad transduction of AAV serotype 9, it may be helpful to exclude the possibility of other brain regions adjacent to the PVH, such as the DMH or VMH, being affected by this viral procedure. If the PVH-specificity is established, the majority of Rai1 cKO effects in Bdnf+ cells are primarily attributed to PVH-Bdnf+ cells based on the similarity of phenotypes observed. With regards to the apparent rescue of the body weight phenotype in Rai1 heterozygous mutants using a selective TrkB activator, the specific biological processes, and neurons responsible for this effect remain unclear to this reviewer. Elucidating these aspects would be significant when considering potential applications to human SMS cases.

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

Reviewer #2 (Public Review):

The authors provide convincing evidence that optogenetic stimulation of ChR2-expressing motor neurons implanted in muscles effectively restores innervation of severely affected skeletal muscles in the aggressive SOD1 mouse model of ALS, and conclude that this method can be applied to selectively control the function of implicated muscles. This was supported by convincing data presented in the paper.

This is an interesting paper providing new/improved optogenetic methods to restore or improve muscle strength in ALS. In general, it is of high significance in both the techniques and concept, and the paper was well written. The evidence supporting the conclusions is convincing, with rigorous muscle tension physiological analysis, and nerve and muscle histology and image analysis. The work will be of broad interest to medical biologists on muscle disorders.

One weak point is that proper control experiments were not clearly presented - these could be shown in the paper. For example, one control experiment with only YFP but no ChR2 expression with optogenetic stimulation should be performed, following similar procedures and analysis applied to the ChR2-transduced animals.

Reviewer #2 (Public Review):

Summary: Two early Cambrian taxa of linguliform brachiopods are assigned to the family Eoobolidae. The taxa exhibit a columnar shell structure and the phylogenetic implications of this shell structure in relation to other early Cambrian families are discussed.

Strengths: Interesting idea regarding the evolution of shell structure.

Weaknesses: The early record of shell structures of linguliform brachiopods is incomplete and partly contradictory. The authors maintain silence regarding contradictory information throughout the article to the extent that information is cited wrongly.<br /> The structure and language of the article need reworking in my opinion, the systematic part can be in the appendix but the main results and the results relevant for the discussion should be in the main article. A critical revision of the family Eoobolidae and Lingulellotretidae including a revision of the type species of Eoobolus and Lingulellotreta is needed.

Reviewer #2 (Public Review):

I have read with great interest the manuscript entitled "The optimal clutch size revisited: separating individual quality from the costs of reproduction" by LA Winder and colleagues. The paper consists in a meta-analysis comparing survival rates from studies providing clutch sizes of species that are unmanipulated and from studies where the clutch sizes are manipulated, in order to better understand the effects of differences in individual quality and of the costs of reproduction. I find the idea of the manuscript very interesting. However, I am not sure the methodology used allows to reach the conclusions provided by the authors (mainly that there is no cost of reproduction, and that the entire variation in clutch size among individuals of a population is driven by "individual quality").

I write that I am not sure, because in its current form, the manuscript does not contain a single equation, making it impossible to assess. It would need at least a set of mathematical descriptions for the statistical analysis and for the mechanistic model that the authors infer from it.<br /> The texts mixes concepts of individual vs population statistics, of within individual vs among-individuals measures, of allocation trade-offs and fitness trade-offs, etc ....which means it would also require a glossary of the definitions the authors use for these various terms, in order to be evaluated.

This problem is emphasised by the following sentence to be found in the discussion "The effect of birds having naturally larger clutches was significantly opposite to the result of increasing clutch size through brood manipulation". The "effect" is defined as the survival rate (see Fig 1). While it is relatively easy to intuitively understand what the "effect" is for the unmanipulated studies: the sensitivity of survival to clutch size at the population level, this should be mentioned and detailed in a formula. Moreover, the concept of effect size is not at all obvious for the manipulated ones (effect of the manipulation? or survival rate whatever the manipulation (then how could it measure a trade-off ?)? at the population level? at the individual level ?) despite a whole appendix dedicated to it. This absolutely needs to be described properly in the manuscript.

Despite the lack of information about the underlying mechanistic model tested and the statistical model used, my impression is still that the interpretation in the introduction and discussion is not granted by the outputs of the figures and tables. Let's use a model similar to that of (van Noordwijk and de Jong, 1986): imagine that the mechanism at the population level is<br /> a.c_(i,q)+b.s_(i,q)=E_q<br /> Where c_(i,q) are s_(i,q) are respectively the clutch size for individual i which is of quality q, and E_q is the level of "energy" that an individual of quality q has available during the given time-step (and a and b are constants turning the clutch size and survival rate into energy cost of reproduction and energy cost of survival, and there are both quite "high" so that an extra egg (c_(i,q) is increased by 1) at the current time-step, decreases s_(i,q) markedly (E_q is independent of the number of eggs produced), that is, we have strong individual costs of reproduction). Imagine now that the variance of c_(i,q) (when the population is not manipulated) among individuals of the same quality group, is very small (and therefore the variance of s_(i,q) is very small also) and that the expectation of both are proportional to E_q. Then, in the unmanipulated population, the variance in clutch size is mainly due to the variance in quality. And therefore, the larger the clutch size c_(i,q) the higher E_q, and the higher the survival s_(i,q).<br /> In the manipulated populations however, because of the large a and b, an artificial increase in clutch size, for a given E_q, will lead to a lower survival s_(i,q). And the "effect size" at the population level may vary according to a,b and the variances mentioned above. In other words, the costs of reproduction may be strong, but be hidden by the data, when there is variance in quality; however there are actually strong costs of reproduction (so strong actually that they are deterministic and that the probability to survive is a direct function of the number of eggs produced)

Moreover, it seems to me that the costs of reproduction are a concept closely related to generation time. Looking beyond the individual allocative (and other individual components of the trade-off) cost of reproduction and towards a populational negative relationship between survival and reproduction, we have to consider the intra-population slow fast continuum (some types of individuals survive more and reproduce less (are slower) than other (which are faster)). This continuum is associated with a metric: the generation time. Some individuals will produce more eggs and survive less in a given time-period because this time-period corresponds to a higher ratio of their generation time (Gaillard and Yoccoz, 2003; Gaillard et al., 2005). It seems therefore important to me, to control for generation time and in general to account for the time-step used for each population studied when analysing costs of reproduction. The data used in this manuscript is not just clutch size and survival rates, but clutch size per year (or another time step) and annual (or other) survival rates.

Finally, it is important to relate any study of the costs of reproduction in a context of individual heterogeneity (in quality for instance), to the general problem of the detection of effects of individual differences on survival (see, e.g., Fay et al., 2021). Without an understanding of the very particular statistical behaviour of survival, associated to an event that by definition occurs only once per life history trajectory (by contrast to many other traits, even demographic, where the corresponding event (production of eggs for reproduction, for example) can be measured several times for a given individual during its life history trajectory).

References:<br /> Fay, R. et al. (2021) 'Quantifying fixed individual heterogeneity in demographic parameters: Performance of correlated random effects for Bernoulli variables', Methods in Ecology and Evolution, 2021(August), pp. 1-14. doi: 10.1111/2041-210x.13728.<br /> Gaillard, J.-M. et al. (2005) 'Generation time: a reliable metric to measure life-history variation among mammalian populations.', The American naturalist, 166(1), pp. 119-123; discussion 124-128. doi: 10.1086/430330.<br /> Gaillard, J.-M. and Yoccoz, N. G. (2003) 'Temporal Variation in Survival of Mammals: a Case of Environmental Canalization?', Ecology, 84(12), pp. 3294-3306. doi: 10.1890/02-0409.<br /> van Noordwijk, A. J. and de Jong, G. (1986) 'Acquisition and Allocation of Resources: Their Influence on Variation in Life History Tactics', American Naturalist, p. 137. doi: 10.1086/284547.

Reviewer #2 (Public Review):

To reveals cellular and molecular heterogeneity in enthesis, the authors established a single-cell temporal atlas during development. This study provides a transcriptional resource for further investigation of fibrocartilage development.

Reviewer #2 (Recommendations for the authors):

1. As known, Fei Fang et al. have established single-cell transcriptomes of mouse supraspinatus tendon enthesis cells (Cell Stem Cell, 2022). It is suggested that the authors introduced Fei Fang et al.'s work in Introduction and emphasize the significant novelty compared with Fei Fang et al.'s work.<br /> 2. In Fig1, the authors highlighted P7 was a critical stage for enthesis differentiation. But this section was less associated with the following content. The authors should link these results with the scRNASeq data. Is there any time-dependent change/signaling in scRNASeq data at this critical time point?<br /> 3. In the H&E staining of Fig1a, the tendon structure was separated and random. It is suggested that the authors provide high-quality staining figures.<br /> 4. Fig2 showed that the Scx+ or Sox9+ cells was decreased in enthesis over time. At least it should be co-staining to show the distribution and frequency of double positive and single positive cell populations. However, a previous study has demonstrated this finding (PLOS ONE, 2020). It is suggested to verify some new findings by IF or IHC staining.<br /> 5. There are some conflicts about trajectory analysis. In Fig3c, RNA velocity showed that the arrow flowed from BTJ to MTJ and CTFb. However, in Fig3d, PAGA plot indicated that BTJ cells is independent of other cells. Furthermore, in supplementary figure S3, RNA velocity showed that the trajectory flowed from TC to BTJ. These figures were inconsistent with the described results. Please provide detailed explanation to avoid misleading readers.<br /> 6. Fig5 showed that C1 was the original cluster, and whether C1 cluster expressed canonical progenic/stem cell markers.<br /> 7. The authors performed cell-cell interaction based on cellchat analysis. But the cell-cell interaction was not actively examined.

Reviewer #2 (Public Review):

Diffusion MRI is sensitive to the brain microstructure, and it has been used to assess the integrity of white matter for nearly 3 decades. Its main limitation is the limited specificity, which makes it difficult to link changes in diffusion parameters to a given pathological substrate. Recently methods based on diffusion MRI that enable the estimation of axonal diameter, non invasively, have become available. This paper aims at validating one of such methods using an experimental model of neurodegeneration. The authors found a significant correlation between axonal diameter estimated by MRI and an histological marker of neurodegeneration. Although this is of great interest, as it demonstrates that this method is sensitive to neurodegeneration, a direct validation would require a measurement of axonal diameter using electron or confocal microscopy, rather than a correlation with a measure of axonal degeneration not directly related to axonal diameter. So, although these data are compelling, they do not prove that the increase in axonal diameter suggested by diffusion MRI corresponds to actual axonal swelling. The Authors also apply the same method to compare the white matter of patients with multiple sclerosis (MS) and healthy controls, showing widespread increases in axonal diameter in the patients. These data are compelling, but again, not conclusive. Other factors such as gloss could bias the MRI measurement and lead to an apparent increase in axonal diameter.

Reviewer #2 (Public Review):

The paper "Polymerization cycle of actin homolog MreB from a Gram-positive bacterium" by Mao et al. provides the second biochemical study of a gram-positive MreB, but importantly, the first study examines how gram-positive MreB filaments bind to membranes. They also show the first crystal structure of a MreB from a Gram-positive bacterium - in two nucleotide-bound forms, finally solving structures that have been missing for too long. They also elucidate what residues in Geobacillus MreB are required for membrane associations. Also, the QCM-D approach to monitoring MreB membrane associations is a direct and elegant assay.

While the above findings are novel and important, this paper also makes a series of conclusions that run counter to multiple in vitro studies of MreBs from different organisms and other polymers with the actin fold. Overall, they propose that Geobacillus MreB contains biochemical properties that are quite different than not only the other MreBs examined so far but also eukaryotic actin and every actin homolog that has been characterized in vitro. As the conclusions proposed here would place the biochemical properties of Geobacillus MreB as the sole exception to all other actin fold polymers, further supporting experiments are needed to bolster these contrasting conclusions and their overall model.

1. (Difference 1) - The predominant concern about the in vitro studies that makes it difficult to evaluate many of their results (much less compare them to other MreB/s and actin homologs) is the use of a highly unconventional polymerization buffer containing 500(!) mM KCL. As has been demonstrated with actin and other polymers, the high KCl concentration used here (500mM) is certain to affect the polymerization equilibria, as increasing salt increases the hydrophobic effect and inhibits salt bridges, and therefore will affect the affinity between monomers and filaments. For example, past work has shown that high salt greatly changes actin polymerization, causing: a decreased critical concentration, increased bundling, and a greatly increased filament stiffness(Kang et al., 2013, 2012). Similarly, with AlfA, increased salt concentrations have been shown to increase the critical concentration, decrease the polymerization kinetics, and inhibit the bundling of AlfA filaments (Polka et al., 2009). A more closely related example comes from the previous observation that increasing salt concentrations increasingly slow the polymerization kinetics of B. subtilis MreB (Mayer and Amann, 2009). Lastly, These high salt concentrations might also change the interactions of MreB(Gs) with the membrane by screening charges and/or increasing the hydrophobic effect.

Given that 500mM KCl was used throughout this paper, many (if not all) of the key experiments should be repeated in more standard salt concentration (~100mM), similar to those used in most previous in vitro studies of polymers. This would test if the many divergent properties of MreB(Gs) reported here arise from some difference in MreB(Gs) relative to other MreBs (and actin homologs), or if they arise from the 400mM difference in salt concentration between the studies. Critically, it would also allow direct comparisons to be made relative to previous studies of MreB (and other actin homologs) that used much lower salt, thereby allowing them to definitively demonstrate whether MreB(Gs) is indeed an outlier relative to other MreB and actin homologs. I would suggest using 100mM KCL, as historically, all polymerization assays of actin and numerous actin homologs have used 50-100mM KCL: 50mM KCl (for actin in F buffer) or 100mM KCl for multiple prokaryotic actin homologs and MreB (Deng et al., 2016; Ent et al., 2014; Esue et al., 2006, 2005; Garner et al., 2004; Polka et al., 2009; Rivera et al., 2011; Salje et al., 2011) Likewise, similar salt concentrations are standard for tubulin (80 mM K-Pipes) and FtsZ (100 mM KCl or 100mM KAc in HMK100 buffer).

2. (Difference 2) - One of the most important differences claimed in this paper is that MreB(Gs) filaments are straight, a result that runs counter to the curved T. Maritima and C. crescentus filaments detailed by the Löwe group (Ent et al., 2014; Salje et al., 2011). Importantly, this difference could also arise from the difference in salt concentrations used in each study (500mM here vs. 100mM in the Löwe studies), and thus one cannot currently draw any direct comparisons between the two studies.

One example of how high salt could be causing differences in filament geometry: high salts are known to greatly increase the bending stiffness of actin filaments, making them more rigid (Kang et al., 2013). Likewise, increasing salt is known to change the rigidity of membranes. As the ability of filaments to A) bend the membrane or B) Deform to the membrane depends on the stiffness of filaments relative to the stiffness of the membrane, the observed difference in the "straight vs. curved" conformation of MreB filaments might simply arise from different salt concentrations.

Thus, in order to draw several direct comparisons between their findings and those of other MreB orthologs (as done here), the studies of MreB(GS) confirmations on lipids should be repeated at the same buffer conditions as used in the Löwe papers, then allowing them to be directly compared.

3. (Difference 3) - The next important difference between MreB(Gs) and other MreBs is the claim that MreB polymers do not form in the absence of membranes.

A) This is surprising relative to other MreBs, as MreBs from 1) T. maritime (multiple studies), E.coli (Nurse and Marians, 2013), and C. crescentus (Ent et al., 2014) have been shown to form polymers in solution (without lipids) with electron microscopy, light scattering, and time-resolved multi-angle light scattering. Notably, the Esue work was able to observe the first phase of polymer formation and a subsequent phase of polymer bundling (Esue et al., 2006) of MreB in solution. 2) Similarly, (Mayer and Amann, 2009) demonstrated B. subtilis MreB forms polymers in the absence of membranes using light scattering.

B) The results shown in figure 5A also go against this conclusion, as there is only a 2-fold increase in the phosphate release from MreB(Gs) in the presence of membranes relative to the absence of membranes. Thus, if their model is correct, and MreB(Gs) polymers form only on membranes, this would require the unpolymerized MreB monomers to hydrolyze ATP at 1/2 the rate of MreB in filaments. This high relative rate of hydrolysis of monomers compared to filaments is unprecedented. For all polymers examined so far, the rate of monomer hydrolysis is several orders of magnitude less than that of the filament. For example, actin monomers are known to hydrolyze ATP 430,000X slower than the monomers inside filaments (Blanchoin and Pollard, 2002; Rould et al., 2006).

C) Thus, there is a strong possibility that MreB(Gs) polymers are indeed forming in solution in addition to those on the membrane, and these "solution polymers" may not be captured by their electron microscopy assay. For example, high salt could be interfering with the absorption of filaments to glow discharged lacking lipids.<br /> In order to definitively prove that MreB(Gs) does not have polymers in solution, the authors should:

i) conduct orthogonal experiments to test for polymers in solution. The simplest test of polymerization might be conducting pelleting assays of MreB(Gs) with and without lipids, sweeping through the concentration range as done in 2B and 5a.

ii) They also could examine if they see MreB filaments in the absence of lipids at 100mM salt (as was seen in both Löwe studies), as the high salt used here might block the charges on glow discharged grids, making it difficult for the polymer to adhere.

iii) Likewise, the claim that MreB lacking the amino-terminus and the α2β7 hydrophobic loop "is required for polymerization" is questionable as if deleting these resides blocks membrane binding, the lack of polymers on the membrane on the grid is not unexpected, as these filaments that cannot bind the membrane would not be observable. Given these mutants cannot bind the membrane, mutant polymers could still indeed exist in solution, and thus pelleting assays should be used to test if non-membrane associated filaments composed of these mutants do or do not exist.

A final note, the results shown in "Figure 1 - figure supplement 2, panel C" appear to directly refute the claim that MreB(Gs) requires lipids to polymerize. As currently written, it appears they can observe MreB(Gs) filaments on EM grids without lipids. If these experiments were done in the presence of lipids, the figure legend should be updated to indicate that. If these experiments were done in the absence of lipids, the claim that membrane association is required for MreB polymerizations should be revised.

4. (Difference 4) - The next difference between this study and previous studies of MreB and actin homologs is the conclusion that MreB(Gs) must hydrolyze ATP in order to polymerize. This conclusion is surprising, given the fact that both T. Maritima (Salje · 2011, Bean 2008) and B. subtilis MreB (Mayer 2009) have been shown to polymerize in the presence of ATP as well as AMP-PNP. Likewise, MreB polymerization has been shown to lag ATP hydrolysis in not only T. maritima MreB (Esue 2005), eukaryotic actin, and all other prokaryotic actin homologs whose polymerization and phosphate release have been directly compared: MamK (Deng et al., 2016), AlfA (Polka et al., 2009), and two divergent ParM homologs (Garner et al., 2004; Rivera et al., 2011).

Currently, the only piece of evidence supporting the idea that MreB(Gs) must hydrolyze ATP in order to polymerize comes from 2 observations: 1) using electron microscopy, they cannot see filaments of MreB(Gs) on membranes in the presence of AMP-PNP or ApCpp, and 2) no appreciable signal increase appears testing AMPPNP- MreB(Gs) using QCM-D. This evidence is by no means conclusive enough to support this bold claim: While their competition experiment does indicate AMPPNP binds to MreB(Gs), it is possible that MreB(Gs) cannot polymerize when bound to AMPPNP. For example, it has been shown that different actin homologs respond differently to different non-hydrolysable analogs: Some, like actin, can hydrolyze one ATP analog but not the other, while others are able to bind to many different ATP analogs but only polymerize with some of one of them. Thus, to further verify their "hydrolysis is needed for polymerization" conclusion, they should:<br /> A. Test if a hydrolysis deficient MreB(Gs) mutant (such as D158A) is also unable to polymerize by EM.<br /> B. They also should conduct an orthogonal assay of MreB polymerization aside from EM (pelleting assays might be the easiest). They should test if polymers of ATP, AMP-PNP, and MreB(Gs)(D158A) form in solution (without membranes) by conducting pelleting assays. These could also be conducted with and without lipids, thereby also addressing the points noted above in point 3.<br /> C. Polymers may indeed form with ATP-gamma-S, and this non-hydrolysable ATP analog should be tested.<br /> D. They could also test how the ADP-Phosphate bound MreB(Gs) polymerizes in bulk and on membranes, using beryllium phosphate to trap MreB in the ADP-Pi state. This might allow them to further refine their model.<br /> E. Importantly, the Mayer study of B. subtilis MreB found the same results in regard to nucleotides, "In polymerization buffer, MreB produced phosphate in the presence of ATP and GTP, but not in ADP, AMP, GDP or AMP-PNP, or without the readdition of any nucleotide". Thus this paper should be referenced and discussed

5. (Difference 5) - The introduction states (lines 128-130) "However, the need for nucleotide binding and hydrolysis in polymerization remains unclear due to conflicting results, in vivo and in vitro, including the ability of MreB to polymerize or not in the presence of ADP or the non-hydrolyzable ATP analog AMP-PNP."

A) While this is a great way to introduce the problem, the statement is a bit vague and should be clarified, detaining the conflicting results and appropriate references. For example, what conflicting in vivo results are they referring to? Regarding "MreB polymerization in AMP-PNP", multiple groups have shown the polymerization of MreB(Tm) in the presence of AMP-PNP, but it is not clear what papers found opposing results.

B) The statement "However, the need for nucleotide binding and hydrolysis in polymerization remains unclear due to conflicting results, in vivo and in vitro, including the ability of MreB to polymerize or not in the presence of ADP or the non-hydrolyzable ATP analog AMP-PNP" is technically incorrect and should be rephrased or further tested.

i. For all actin (or tubulin) family proteins, it is not that a given filament "cannot polymerize" in the presence of ADP but rather that the ADP-bound form has a higher critical concentration for polymer formation relative to the ATP-bound form. This means that the ADP polymers can indeed polymerize, but only when the total protein exceeds the ADP critical concentration. For example, many actin-family proteins do indeed polymerize in ADP: ADP actin has a 10-fold higher critical concentration than ATP actin, (Pollard, 1984) and the ADP critical concentrations of AlfA and ParM are 5X and 50X fold higher (respectively) than their ATP-bound forms(Garner et al., 2004; Polka et al., 2009)

ii. Likewise, (Mayer and Amann, 2009) have already demonstrated that B. subtilis MreB can polymerize in the presence of ADP, with a slightly higher critical concentration relative to the ATP-bound form.

Thus, to prove that MreB(Gs) polymers do not form in the presence of ADP would require one to test a large concentration range of ADP-bound MreB(Gs). They should test if ADP- MreB(Gs) polymerizes at the highest MreB(Gs) concentrations that can be assayed. Even if this fails, it may be the MreB(Gs) ADP polymerizes at higher concentrations than is possible with their protein preps (13uM). An even more simple fix would be to simply state MreB(Gs)-ADP filaments do not form beneath a given MreB(Gs) concentration.

Other Points to address:

1. There are several points in this paper where the work by Mayer and Amann is ignored, not cited, or readily dismissed as "hampered by aggregation" without any explanation or supporting evidence of that fact.

A) Lines 100-101 - While the irregular 3-D formations seen formed by MreB in the Dersch 2020 paper could be interpreted as aggregates, stating that the results from specifically the Gaballah and Meyer papers (and not others) were "hampered by aggregation" is currently an arbitrary statement, with no evidence or backing provided. Overall, these lines (and others in the paper) dismiss these two works without giving any evidence to that point. Thus, they should provide evidence for why they believe all these papers are aggregation, or remove these (and other) dismissive statements.

One important note - There are 2 points indicating that dismissing the Meyer and Amann work as aggregation is incorrect: 1) the Meyer work on B. subtilis MreB shows both an ATP and a slightly higher ADP critical concentration. As the emergence of a critical concentration is a steady-state phenomenon arising from the association/dissociation of monomers (and a kinetically limiting nucleation barrier), an emergent critical concentration cannot arise from protein aggregation, critical concentrations only arise from a dynamic equilibrium between monomer and polymer. 2) Furthermore, Meyer observed that increased salt slowed and reduced B. subtilis MreB light scattering, the opposite of what one would expect if their "polymerization signal" was only protein aggregation, as higher salts should increase the rate of aggregation by increasing the hydrophobic effect.

B) Lines 113-137 -The authors reference many different studies of MreB, including both MreB on membranes and MreB polymerized in solution (which formed bundles). However, they again neglect to mention or reference the findings of Meyer and Amann (Mayer and Amann, 2009), as it was dismissed as "aggregation". As B. subtilis is also a gram-positive organism, the Meyer results should be discussed.

2. Lines 387-391 state the rates of phosphate release relative to past MreB findings: "These rates of Pi release upon ATP hydrolysis (~ 1 Pi/MreB in 6 min at 53{degree sign}C) are comparable to those observed for MreBTm and MreB(Ec) in vitro". While the measurements of Pi release AND ATP hydrolysis have indeed been measured for actin, this statement does not apply to MreB and should be corrected: All MreB papers thus far have only measured Pi release alone, not ATP hydrolysis at the same time. Thus, it is inaccurate to state "rates of Pi release upon ATP hydrolysis" for any MreB study, as to accurately determine the rate of Pi release, one must measure: 1. The rate of polymer over time, 2) the rate of ATP hydrolysis, and 3) the rate of phosphate release. For MreB, no one has, so far, even measured the rates of ATP hydrolysis and phosphate release with the same sample.

3. The interpretation of the interactions between monomers in the MreB crystal should be more carefully stated to avoid confusion. While likely not their intention, the discussions of the crystal packing contacts of MreB can appear to assume that the monomer-monomer contacts they see in crystals represent the contacts within actual protofilaments. One cannot automatically assume the observations of monomer-monomer contacts within a crystal reflect those that arise in the actual filament (or protofilament).

A) They state, "the apo form of MreBGs forms less stable protofilaments than its G- homologs ." Given filaments of the Apo form of MreB(GS) or b. subtilis have never been observed in solution, this statement is not accurate: while the contacts in the crystal may change with and without nucleotide, if the protein does not form polymers in solution in the apo state, then there are no "real" apo protofilaments, and any statements about their stability become moot. Thus this statement should be rephrased or appropriately qualified.

B) Another example: while they may see that in the apo MreB crystal, the loop of domain IB makes a *single* salt bridge with IIA and none with IIB. This contrasts with every actin, MreB, and actin homolog studied so far, where domain IB interacts with IIB. This might reflect the real contacts of MreB(Gs) in the solution, or it may be simply a crystal-packing artifact. Thus, the authors should be careful in their claims, making it clear to the reader that the contacts in the crystal may not necessarily be present in polymerized filaments.

4. lines 201-202 - "Polymers were only observed at a concentration of MreB above 0.55 μM (0.02 mg/mL)". Given this concentration dependence of filament formation, which appears the same throughout the paper, the authors could state that 0.55 μM is the critical concentration of MreB on membranes under their buffer conditions. Given the lack of critical concentration measurement in most of the MreB literature, this could be an important point to make in the field.

5. Both mg/ml and uM are used in the text and figures to refer to protein concentration. They should stick to one convention, preferably uM, as is standard in the polymer field.

6. Lines 77-78 - (Teeffelen et al., 2011) should be referenced as well in regard to cell wall synthesis driving MreB motion.

7. Line 90 - "Do they exhibit turnover (treadmill) like actin filaments?". This phrase should be modified, as turnover and treadmilling are two very different things. Turnover is the lifetime of monomers in filaments, while treadmilling entails monomer addition at one end and loss at the other. While treadmilling filaments cause turnover, there are also numerous examples of non-treadmilling filaments undergoing turnover: microtubules, intermediate filaments, and ParM. Likewise, an antiparallel filament cannot directionally treadmill, as there is no difference between the two filament ends to confer directional polarity.

8. Throughout the paper, the term aggregation is used occasionally to describe the polymerization shown in many previous MreB studies, almost all of which very clearly showed "bundled" filaments, very distinct entities from aggregates, as a bundle of polymers cannot form without the filaments first polymerizing on their own. Evidence to this point, polymerization has been shown to precede the bundling of MreB(Tm) by (Esue et al., 2005).

9. lines 106-108 mention that "The N-terminal amphipathic helix of E. coli MreB (MreBEc) was found to be necessary for membrane binding. " This is not accurate, as Salje observed that one single helix could not cause MreB to mind to the membrane, but rather, multiple amphipathic helices were required for membrane association (Salje et al., 2011). The Salje results imply that dimers (or further assemblies) of MreB drive membrane association, a point that should be discussed in regard to the question "What prompts the assembly of MreB on the inner leaflet of the cytoplasmic membrane?" posed on lines 86-87.

10. On lines 414-415, it is stated, "The requirement of the membrane for polymerization is consistent with the observation that MreB polymeric assemblies in vivo are membrane-associated only." While I agree with this hypothesis, it must be noted that the presence or absence of MreB polymers in the cytoplasm has not been directly tested, as short filaments in the cytoplasm would diffuse very quickly, requiring very short exposures (<5ms) to resolve them relative to their rate of diffusion. Thus, cytoplasmic polymers might still exist but have not been tested.

11. lines 429-431 state, "but polymerization in the presence of ADP was in most cases concluded from light scattering experiments alone, so the possibility that aggregation rather than ordered polymerization occurred in the process cannot be excluded."

A) If an increased light scattering signal is initiated by the addition of ADP (or any nucleotide), that signal must come from polymerization or multimerization. What the authors imply is that there must be some ADP-dependent "aggregation" of MreB, which has not been seen thus far for any polymer. Furthermore, why would the addition of ADP initiate aggregation?

B) Likewise, the statement "Differences in the purity of the nucleotide stocks used in these studies could also explain some of the discrepancies" is unexplained and confusing. How could an impurity in a nucleotide stock affect the past MreB results, and what is the precedent for this claim?

12. lines 467-469 state, "Thus, for both MreB and actin, despite hydrolyzing ATP before and after polymerization, respectively, the ADP-Pi-MreB intermediate would be the long-lived intermediate state within the filaments."

A) For MreB, this statement is extremely speculative and unbiased, as no one has measured 1) polymerization, 2) ATP hydrolysis, and 3) phosphate release. For example, it could be that ATP hydrolysis is slow, while phosphate release is fast, as is seen in the actin from Saccharomyces cerevisiae.

B) For actin, the statement of hydrolysis of ATP of monomer occurring "before polymerization" is functionally irrelevant, as the rate of ATP hydrolysis of actin monomers is 430,000 times slower than that of actin monomers inside filaments(Blanchoin and Pollard, 2002; Rould et al., 2006).

13. Lines 442-444. "On the basis of our data and the existing literature, we propose that the requirement for ATP (or GTP) hydrolysis for polymerization may be conserved for most MreBs." Again, this statement both here (and in the prior text) is an extremely bold claim, one that runs contrary to a large amount of past work on not just MreB, but also eukaryotic actin and every actin homolog studied so far. They come to this model based on 1) one piece of suggestive data (the behavior of MreB(GS) bound to 2 non-hydrolysable ATP analogs in 500mM KCL), and 2) the dismissal (throughout the paper) of many peer-reviewed MreB papers that run counter to their model as "aggregation" or "contaminated ATP stocks ." If they want to make this bold claim that their finding invalidates the work of many labs, they must back it up with further validating experiments.

References cited.

Blanchoin L, Pollard TD. 2002. Hydrolysis of ATP by Polymerized Actin Depends on the Bound Divalent Cation but Not Profilin †. Biochemistry-us 41:597-602. doi:10.1021/bi011214b

Deng A, Lin W, Shi N, Wu J, Sun Z, Sun Q, Bai H, Pan Y, Wen T. 2016. In vitro assembly of the bacterial actin protein MamK from 'Candidatus Magnetobacterium casensis' in the phylum Nitrospirae. Protein Cell 7:267-280. doi:10.1007/s13238-016-0253-x

Dersch S, Reimold C, Stoll J, Breddermann H, Heimerl T, Soufo HJD, Graumann PL. 2020. Polymerization of Bacillus subtilis MreB on a lipid membrane reveals lateral co-polymerization of MreB paralogs and strong effects of cations on filament formation. Bmc Mol Cell Biology 21:76. doi:10.1186/s12860-020-00319-5

Ent F van den, Izoré T, Bharat TA, Johnson CM, Lowe J. 2014. Bacterial actin MreB forms antiparallel double filaments. eLife 3:e02634. doi:10.7554/elife.02634

Esue O, Cordero M, Wirtz D, Tseng Y. 2005. The Assembly of MreB, a Prokaryotic Homolog of Actin. J Biol Chem 280:2628-2635. doi:10.1074/jbc.m410298200

Esue O, Wirtz D, Tseng Y. 2006. GTPase Activity, Structure, and Mechanical Properties of Filaments Assembled from Bacterial Cytoskeleton Protein MreB. J Bacteriol 188:968-976. doi:10.1128/jb.188.3.968-976.2006

Garner EC, Campbell CS, Mullins RD. 2004. Dynamic instability in a DNA-segregating prokaryotic actin homolog. Science (New York, NY) 306:1021-1025. doi:10.1126/science.1101313

Kang H, Bradley MJ, Elam WA, De La Cruz EM. 2013. Regulation of Actin by Ion-Linked Equilibria. Biophys J 105:2621-2628. doi:10.1016/j.bpj.2013.10.032

Kang H, Bradley MJ, McCullough BR, Pierre A, Grintsevich EE, Reisler E, Cruz EMDL. 2012. Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness. Proc National Acad Sci 109:16923-16927. doi:10.1073/pnas.1211078109

Mayer JA, Amann KJ. 2009. Assembly properties of the Bacillus subtilis actin, MreB. Cell Motil Cytoskel 66:109-118. doi:10.1002/cm.20332

Nurse P, Marians KJ. 2013. Purification and Characterization of Escherichia coli MreB Protein. J Biol Chem 288:3469-3475. doi:10.1074/jbc.m112.413708

Polka JK, Kollman JM, Agard DA, Mullins RD. 2009. The Structure and Assembly Dynamics of Plasmid Actin AlfA Imply a Novel Mechanism of DNA Segregation. J Bacteriol 191:6219-6230. doi:10.1128/jb.00676-09

Pollard TD. 1984. Polymerization of ADP-actin. J Cell Biology 99:769-777. doi:10.1083/jcb.99.3.769

Rivera CR, Kollman JM, Polka JK, Agard DA, Mullins RD. 2011. Architecture and assembly of a divergent member of the ParM family of bacterial actin-like proteins. The Journal of biological chemistry 286:14282-14290. doi:10.1074/jbc.m110.203828

Rould MA, Wan Q, Joel PB, Lowey S, Trybus KM. 2006. Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States*. J Biol Chem 281:31909-31919. doi:10.1016/s0021-9258(19)84105-4

Salje J, van den Ent F, de Boer P, Löwe J. 2011. Direct Membrane Binding by Bacterial Actin MreB. Mol Cell 43:478-487. doi:10.1016/j.molcel.2011.07.008

Teeffelen S van, Wang S, Furchtgott L, Huang KC, Wingreen NS, Shaevitz JW, Gitai Z. 2011. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proceedings of the National Academy of Sciences of the United States of America 108:15822-15827. doi:10.1073/pnas.1108999108

Reviewer #2 (Public Review):

The current study by Taylor and colleagues investigated the role of microRNA-218 in hippocampal development and discover that disturbances in miR-218 during a key developmental window can lead to persistent changes in network excitability which could have implications for neurodevelopmental and neurological diseases. They found that miR-218 is developmentally regulated in the mouse hippocampus and resides in both excitatory pyramidal neurons and interneurons. Using antagomirs (inhibitors) specifically targeted to miR-218 they find that persistent inhibition of miR-218 elevates network activity and renders mice more susceptible to seizures when challenged with a chemoconvulsant. Additionally antagomir treated mice displayed altered cognitive processing when compared to control-treated mice. Taylor and colleagues then identified potential pathways and targets through which miR-218 may exert control over network formation and stabilisation and identified cell-type-specific targets through which it may function. Overall they find that the activity of miR-218 and its effects on network development may be mediated through its activity in interneurons.

The conclusions of this paper are mostly excellently supported by extensive and advanced experimentation.

The data on miR-218 is the least convincing element of the paper but there are inherent difficulties in assessing miR-mediated targeting which the authors may have encountered. Firstly the justification for performing gene ontology on genes with an FC of greater than 0 must be included. Similarly, the use of p values of less than 0.2 lacks stringency and authors should specify why these parameters were chosen. Otherwise, the gene ontology data is difficult to interpret. Protein data may add to this section also.

The authors state they do not analyse known developmental miRs such as miR-124. But the reasoning behind this is not explained. As known developmental miRNAs, analysing their expression would add confidence to the data. Furthermore, the statistical significance of Fig 1B is unclear.

Reviewer #2 (Public Review):

The authors present findings on a designed peptide, PITCR, and its role in inhibiting TCR activation through an extensive series of experiments. These include the measurement of phosphorylation in the TCR zeta chain and a number of associated signaling proteins such as Zap70, LAT, PLCg1, and SLP76. In addition, the authors measure the impact of PITCR on the TCR intracellular calcium response and examine the peptide-induced inhibition of TCR activation by antigen-presenting cells. They also present data indicating that the fluorescently labeled PITCR co-localizes with TCR in Jurkat cells and with ligand-bound TCR in primary murine cells.

Overall the experiments provide useful insights into the mechanism of T cell activation and generally support an allosteric model of activation, while not necessarily excluding alternative models.

However, some aspects of the study do need clarification.

1) The authors do not provide a clear structural basis for their peptide design, which makes it difficult to understand the rationale for choosing this particular peptide. The use of a structural model based on the TCR zeta domain, for example, and how it becomes modified to generate PITCR would provide some clarity on what types of putative interactions are being engineered.

2) The inhibitory effects of PITCR are not large. Measurement of dose dependence might improve confidence in the results.

3) Use of control peptides is not uniform. Control peptides similar to PITCR in Figure 1 and Figure 2 studies, for example, could strengthen the authors' arguments.

Reviewer #2 (Public Review):

Mattes et al. used a CRISPR screen to determine tumor-intrinsic factors modulating the efficacy of T cell-mediated cell lysis. For this purpose, colon carcinoma cell lines were modified with gain-of-function (CRISPRa) and loss-of-function (CRISPRi) sgRNA libraries. Modified colon cancer cells were subsequently exposed to antigen-specific tumor cell lysis by CD8+ cytotoxic T cells and surviving colon cancer cells were analyzed for over- and underrepresented genes. The screen replicated findings from previous studies showing the importance of IFNy, TNFa, and autophagy pathways for T cell-mediated lysis of cancer cells. In addition, the authors identified two genes involved in cell adhesion that modulate T cell-mediate cell lysis: ILKAP and ICAM1. Subsequently, the authors validate their findings in in-vitro experiments and show that a soluble form of ICAM1 is negatively affecting tumor cell lysis. Finally, they analyze publicly available gene expression data from cancer patient cohorts and show that high ICAM1 expression, in combination with high expression of genes associated with soluble ICAM1 generation, has a negative impact on patient survival. Beyond these findings, the CRISPR screening dataset from this study serves as a comprehensive resource for other researchers in the onco-immunology space.

The conclusions of this paper are well supported by data, but some aspects of the role of soluble ICAM1 in T cell-mediated tumor cell lysis and the limitations of the employed experimental system should be clarified and extended.

1. For their screening, the authors use an in-vitro model of antigen-specific tumor cell lysis based on expanded CMV-specific CD8+ T cells and CMV peptide-pulsed colon cancer cells. While this model allows for the efficient induction of cellular cytotoxicity by expanded primary T cells, it has one critical caveat: pulsing colon cancer cells with CMV peptide adds the antigen artificially to the MHC complex on the cell surface. Cell-intrinsic factors of antigen processing and presentation are not required for tumor cell recognition in this system. However, antigen processing and presentation pathways represent important targets of tumor evasion in cancer patients. Factors affecting these processes won't be detected in this study. To consider antigen processing and presentation as well, the authors could, for instance, have used an additional model with T cells containing a transgenic TCR specific for an antigen typically expressed on colon cancer cells (or another cancer cell line).

2. The authors demonstrate the negative impact of soluble ICAM1 on T cell-mediated cytotoxicity in their co-culture assay. However, they lack to provide evidence on how this is facilitated. One option, as the authors speculate in their discussion and cartoon, could be that soluble ICAM1 occupies LFA1 on T cells thereby preventing the binding of T cells to ICAM1 on the surface of tumor cells. To demonstrate that this is indeed the case, the authors could, for instance, have used microscopy and measured T cell and tumor cell interaction duration and frequency under conditions with and without soluble ICAM1 present.

3. Regarding the analysis of clinical relevance, the authors show that patients with high levels of ICAM1 expression in combination with high levels of protease expression have poor survival. The rationale behind this is that the proteases cleave ICAM1 off the membrane leading to high levels of soluble ICAM1 that then negatively affects T cell-mediate tumor cell lysis. To demonstrate that indeed the combination of both factors, ICAM1 expression, and protease expression, is responsible for poor survival, the authors should also have analyzed the impact of each of these factors alone on patient survival. If their hypothesis is true, the combination of high ICAM1 and protease expression should have a worse impact on survival than each factor alone.

Reviewer #2 (Public Review):

Mikula et al. have a large experience studying the escape distances of birds as a proxy of behavioral adaptation to urban environments. They profited from the exceptional conditions of social distance and reduced mobility during the covid-19 pandemic to continue sampling urban populations of birds under exceptional circumstances of low human disturbance. Their aim was to compare these new data with data from previous "normal" years and check whether bird behavior shifted or not as a consequence of people's lockdown. Therefore, this study would add to the growing body of literature assessing the effect of the covid-19 shutdown on animals. In this sense, this is not a novel study. However, the authors provide an interesting conclusion: birds have not changed their behavior during the pandemic shutdown. This lack of effects disagrees with most of the previously published studies on the topic. I think that the authors cannot claim that urban birds were unaffected by the covid-19 shutdown. I think that the authors should claim that they did not find evidence of covid-19-shutdown effects. This point of view is based on some concerns about data collection and analyses, as well as on evolutionary and ecological rationale used by the authors both in their hypotheses and results interpretation. I will explain my criticisms point by point:

1) The authors used ambivalent, sometimes contradictory, reasoning in their predictions and results interpretation. Some examples:<br /> 1.1) The authors claimed that urban birds perceive humans as harmless (L224), but birds actually escape from us, when we approach them... Furthermore, they escape usually 5 to 20 m away. This is more distance that would be necessary just to be not trampled.<br /> 1.2) If we are harmless, why birds should spend time monitoring us as a potential threat (L102)? Indeed, I disagree with the second prediction of the authors. I could argue that reduced human activity should increase animal vigilance because real bird predators (e.g., raptors) may increase their occurrence or activity in empty cities. If birds should increase their vigilance because the invisible shield of human fear of their predators is no longer available, then I would expect longer escape distances.<br /> 1.3) To justify the same escape behavior shown by birds in pre- and pandemic conditions from an adaptive point of view, the authors argued a lack of plasticity and a strong genetic determination of such behavior. This contravenes the plasticity proposed in the previous point or the expected effect of the stringency index (L112). In my opinion, some degree of plasticity in the escape behavior would be really favorable for individuals from an adaptive perspective, as they may face quite different fear landscapes during their lives. Looking at the figures, one can see notable differences in the escape distance of the same species between sites in the same city. As I can hardly imagine great genetic differences between birds sampled in a park or a cemetery in Rovaniemi, for instance, I would expect a major role of plasticity to explain the observed variability. Furthermore, if escape behavior would not be plastic, I would not expect date or hour effects. By including them in their models, the authors are accepting implicitly some degree of plasticity.

2) Looking at the figures I do not see the immense stochasticity (L156, Fig. S3, S5) claimed by the authors. Instead, I can see that some species showed an obvious behavioral change during the shutdown. For instance, Motacilla alba, Larus ridibundus, or Passer domesticus clearly reduced their escape distances, while others like the Dendrocopos major, Passer montanus, or Turdus merula tended to increase it. On the other hand, birds in Poland tended to have larger escape distances during the shutdown for most species, while in Rovaniemi there was an apparent reduction of escape distances in most cases. The multispecies and multisite approach is a strength of this study, but it is an Achilles' heel at the same time. The huge heterogeneity in bird responses among species and sites counterbalanced and as a result, there was an apparent lack of shutdown effects overall. Furthermore, as most data comes from a few (European) species (i.e., Columba, Passer, Parus, Pica, Turdus, Motacilla) I would say that the overall results are heavily influenced (or biased) by them. The authors realize that results are often area- or species-specific (L203), therefore, does a whole approach make sense?

3) The previous point is worsened by the heterogeneity of cities and periods sampled. For instance:<br /> 3.1) I can hardly imagine any common feature between a small city in northern Finland (Rovaniemi) and a megacity in Australia (Melbourne). Thus, I would not be surprised to find different results between them.<br /> 3.2) Prague baseline data was for 2014 and 2018, while for the rest of the study sites were for 2018 and 2019. If study sites used a different starting point, you cannot compare differences at the final point.<br /> 3.3) Due to the obvious seasonal differences between the northern and southern hemispheres, data collection in Australia began five months later than in the rest of the sites (Aug vs Mar 2020). There, urban birds faced already too many months of reduced human disturbances, while European birds were sampled just at the beginning of the lockdown.<br /> 3.4) Some cities were sampled by a single observer, while others by many of them. Even if all of them are skilled birders, they represent different observers from a statistical point of view and consequently, observer identity was an extra source of noise in your data that you did not account for.

4) Although I liked the stringency index as a variable, I am not sure if it captured effectively the actual human activity every day. Even if restrictive measures were similar between countries, their actual accomplishment greatly depended on people's commitment and authorities' control and sanctions. I would suggest using a more realistic measure of human activity, such as google mobility reports.

5) The authors used escape trials from birds on the ground and perched birds. I think that they are not comparable, as birds on the ground probably perceive a greater risk than those placed some meters above the ground, i.e. I would expect shorter escape distances for perched birds. As this can be strongly dependent on the species preferences or sampling site (i.e, more or less available perches), I wonder how this mixture of observations from birds on the ground and perched birds could be affecting the results.

6) The authors did not sample the same location in the same breeding season to avoid repeated sampling of the same individuals (L331). This precaution may help, but it does not guarantee a lack of pseudoreplication. Birds are highly mobile organisms and the same individuals may be found in different places in the same city. This pseudoreplication seems particularly plausible for Rovaniemi, where sampling points must be necessarily close due to the modest size of this city.

7) An intriguing result was that the authors collected data for 135 species during the shutdown, while they collected data only for 68 species before the pandemic. Such a two-fold increase in bird richness would not be expected with a 36% increase in sampling effort during 2020-21. I wonder if this could be reflecting an actual increase in bird richness in urban areas as a positive result of the shutdown and reduced human presence.

8) The authors dismissed the multicollinearity problem of explanatory variables unjustifiably (L383). However, looking at fig. S1, I can see strong correlations between some of them. For instance, period and stringency index were virtually identical (r=0.95), while temperature and date were also strongly correlated.

9) The random structure of the models is a key element of the statistical analyses but those random factors are poorly explained and justified. I needed to look up the supplementary tables to fully understand the complex architecture of the random part of the models. To the best of my knowledge, random variables aim to account for undesirable correlations in the covariance matrix, which is expected in hierarchical designs, such as the present one. However, the theoretical violation of data independence may happen or not. As the random structure is usually of little interest, you should keep it as simple as necessary, otherwise random factors may be catching part of data variability that you would like to explain by fixed variables. I think that this is what is happening (at least, in part) here, as the authors included a too-complex random structure. For instance, if you include the year as a random factor, I think that you are leaving little room for the period effect. The authors simplified the random structure of the models (L387), but they did not explain how. Nevertheless, this model selection was not important at all, as the authors showed the results for several models. I assume, consequently, that the authors are considering all these models equally valid. This approach seems quite contradictory.

Reviewer #2 (Public Review):

Fuks et al. provide extensive paleobotanical data from several sites in the Negev desert to address hypotheses regarding the relative importance of the Roman Agricultural Diffusion (RAD) and the Islamic Green Revolution (IGR) in the dispersal of crops across Eurasia.

While the overall claims from the authors are convincing, I found the presentation of the data somewhat difficult to follow.<br /> Graphical visualization of the data with respect to the proposed hypotheses would go a long way towards making the argument clearer for a non-specialist audience.

The authors apply appropriate caveats in the discussion about their ability to assess IGR given their timeline only incorporates the first few hundred years and some IGR plants may not leave macrobotanical remains. Yet I think more could be done to explain how the data they do find provides positive evidence for RAD. Many of their findings are inferred to be RAD introductions not because of the timing in their sites, but because of previous evidence of introductions at other sites. It would thus be helpful to be more explicit about what additional evidence these findings provide beyond previously published data of introductions of many of these crops into the Levant.

Reviewer #2 (Public Review):

Antiretroviral therapy (ART) can control HIV replication and improve the quality of life for people living with HIV (PLWH); however, it does not cure infection, nor does it revert T cell exhaustion. Inhibitory receptor expression is a characteristic of CD8+ T cell exhaustion and a better understanding of the differences in receptor expression dynamics between healthy donors and PLWH on ART is of interest. In this comparative study, Blanch-Lombarte et al. use single-cell analysis of flow cytometric PBMC profiling to examine inhibitory receptor expression (IR) and functional markers in CD8+ T cells derived from PLWH on ART and healthy donors.

The authors first perform a mix of cross-sectional and longitudinal characterization of IR expression and memory differentiation markers in donors who are healthy controls, are in the early stages of HIV infection, PLWH on ART for ~ 2 years, and PLWH on ART for ~10 years. They conduct both supervised and unsupervised analyses of the phenotypic results. The authors use three experimental conditions (unstimulated, SEB stimulation, HIV Gag peptide pool stimulation) to perform cluster analyses. The longitudinal paired samples allow determination of the persistence of the alterations observed early after initiation of ART. The analyses show inverse correlations between frequencies of TIGIT+ and TIGIT+ TIM+ CD8 subsets and CD4 counts. However, findings for HIV-specific CD8 were different, with a selective reduction of TIGIT+ clusters whose functionality in terms of CD107 expression was recovered by anti-TIGIT blockade.

The authors conclude that TIGIT could be a therapeutic target to revive exhausted T cells (Tex) at all ART stages.

Strengths:<br /> - The study addressed relevant questions for the field.<br /> - The is a logical sequence of experiments and analyses.<br /> - The authors investigate interesting samples - longitudinal time points on ART several years apart are a significant asset.<br /> - Assessment of CD8 T cell populations as bulk unstimulated cells, broad stimulation with a superantigen (SEB), and HIV-specific responses (Gag peptide pool stimulation).<br /> - Complementary use of supervised and detailed unsupervised analyses of flow cytometry data.<br /> - The analyses are overall detailed and carefully presented, except for minor issues in color coding and font size.<br /> - Functional assays to assess the functional impact of TIGIT upregulation on CD8 T cell function.<br /> -<br /> Weaknesses:<br /> - While the paper reads overall well, the hypotheses and concepts should be clarified in several instances. For example, the authors speak of T cell exhaustion, which in principle is understood as antigen-specific T dysfunction associated with antigen persistence. However, a good part of the paper is focused on total CD8 T cells, and the links between findings in the different populations of CD8 examined (total, SEB-stimulated, Gag-stimulated) are hard to understand.<br /> - Upregulation of IRs can be associated with the state of T cell differentiation and also modulated by chronic inflammation independently of TCR signaling (eg, common gamma-chain cytokines upregulate PD-1), so defining these cells as univocally Tex is not correct.<br /> - The study mostly focuses on descriptive phenotypic analyses of CD8 T cells rather than dynamics studies which would imply more in-depth investigations of T cell evolution and fate.<br /> - HIV-specific CD8 T cells can be both quantitatively and quantitatively impaired, but the quantitative aspects are not considered, nor shifts in phenotype. For example, HIV-specific TIGIT+ CD8 responding to blockade proportionally over time - It is unclear if this is compensated by other subsets.<br /> - The functional assays with TIGIT blockade are limited and do not include other markers of cytotoxic cells (perforin, granzyme B expression...). It is not clear how do these subsets compare to the other clusters in terms of CD107 expression.<br /> - The statistical analyses do apparently not include correction for multiple comparisons.

Reviewer #2 (Public Review):

Summary:<br /> In the paper from Hartman, Vandenberg, and Hill entitled "assessing drug safety, by identifying the access of arrhythmia and cardio, myocytes, electro physiology", the authors, define a new metric, the axis of arrhythmia" that essentially describes the parameter space of ion channel conductance combinations, where early after depolarization can be observed.

Strengths:<br /> There is an elegance to the way the authors have communicated the scoring system. The method is potentially useful because of its simplicity, accessibility, and ease of use. I do think it adds to the field for this reason - a number of existing methods are overly complex and unwieldy and not necessarily better than the simple parameter regime scan presented here.

Weaknesses:<br /> The method described in the manuscript suffers from a number of weaknesses that plague current screening methods. Included in these are the data quality and selection used to inform the drug-blocking profile. It's well known that drug measurements vary widely, depending on the measurement conditions.

There doesn't seem to be any consideration of pacing frequency, which is an important consideration for arrhythmia triggers, resulting from repolarization abnormalities, but also depolarization abnormalities. Extremely high doses of drugs are used to assess the population risk. But does the method yield important information when realistic drug concentrations are used? In the discussion, the comparison to conventional approaches suggests that the presented method isn't necessarily better than conventional methods.

In conclusion, I have struggled to grasp the exceptional novelty of the new metric as presented, especially when considering that the badly needed future state must include a component of precision medicine.

Reviewer #2 (Public Review):

Anderson et al profiled chromatin features, including active chromatin marks, RNA polymerase II distribution, and histone modifications in the sex chromosomes of spermatogenic cells in Drosophila. The results are new and the experiments and analyses look well done, including with appropriate numbers of replicates. Results were parsed by comparing them among two arrest mutants and wildtype, as well as in FACS-sorted spermatocytes. The authors also profiled larval wing discs to serve as reference-somatic cells, which allowed them to focus only on features in their testis data that were associated with germ cells. Their results were further refined by categorizing the genes of interest based on available single nucleus RNA seq expression profiles. The authors document interesting phenomena, such as differences in the distribution of RNAPIIS2p on some genes in germ cells vs somatic cells, the presence of a uH2A body beginning in early spermatocytes, and high levels of uH2A on the Y chromosome and little or none on the X. The former is intriguing because this modification is usually associated with silencing, yet the Y chromosome is active in spermatogenic cells. The authors interpret some of their data as implying a lack of dosage compensation of the X chromosome in spermatocytes.

The data are believable and new, but it is not fully clear how to interpret them. The paper's interpretations rely on subtractive logic to parse results from mixtures of cells down to cell type, extracting spermatogonia, spermatocyte, etc. features by comparing bam mutants (only spermatogonia) to aly mutants (spermatogonia and early spermatocytes but no later stages) to wildtype (all spermatogenic stages), and extracting testis germline data by comparison to wing disc soma; their FACS sorted spermatocytes also have heterogeneity. I recognize that the present paper was a lot of work and am not suggesting that the authors redo their study using methods that give more purity and precision of stage (https://doi.org/10.1126/science.aal3096, https://doi.org/10.1101/gad.335331.119), but they should be aware of them and of their results.

The conclusions about dosage compensation are indirect, but are consistent with the current model documented in the studies cited by the authors, as well as earlier studies (doi: 10.1186/jbiol30).

Reviewer #2 (Public Review):

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

Reviewer #2 (Public Review):

The manuscript by Maio et al attempts to examine the bioenergetic mechanisms involved in the delayed migration of DC's during Mtb infection. The authors performed a series of in vitro infection experiments including bioenergetic experiments using the Agilent Seahorse XF, and glucose uptake and lactate production experiments. This is a well-written manuscript and addresses an important question in the TB field. A major weakness is the use of dead Mtb in virtually all the experiments. Unfortunately, the authors did not attempt to address this critical confounding factor. As a result, data was interpreted, and conclusions were made as if live Mtb was used. Also, previous studies (PMID: 30444490 and PMID: 31914380) have shown that live Mtb suppresses glycolysis, which contradicts findings in this study, perhaps because dead Mtb was used here. For these reasons, obtaining any pertinent conclusions from the study is not possible, which diminishes the significance of the work.

Reviewer #2 (Public Review):

In this work, the authors address the problem of missing data imputation in the life sciences domain and propose several new algorithms which improve on the current state-of-the-art. In particular (i) they modify two existing Random Forest-based imputation methods -- MissForest and miceRanger -- to use either determinantal sampling or deterministic determinantal sampling, and show slightly improved classification performance on two datasets (one synthetic, one real); in addition, (ii) the authors present a quantum circuit for performing the determinantal sampling which scales asymptotically better than the best-known classical methods, and perform small scale experiments using both a (noiseless) quantum simulator as well as a 10 qubit IBM quantum computer to validate that the approach works in principle.

The problem of data imputation is important in practice, and results that improve on existing methods should be of interest to those in the field. The use of determinantal sampling for applications beyond data imputation should also be of broader interest, and the connection to quantum computing warrants further investigation and analysis.

The use of classification accuracy (as measured by AUC) as a measure of success is well-motivated, and the authors use both real and synthetic datasets to evaluate their methods, which consistently (if only marginally) outperform the existing state-of-the-art. The results obtained here motivate the further study of this approach to a wider class of datasets, and to areas beyond life sciences.

As it stands, in my opinion, two points need addressing.

1. Additional clarity is required on what is novel:

While the application of determinantal and deterministic determinantal sampling to the specific case of data imputation appears to be novel, the authors should make it more clear that both of these methods themselves are not new, and have been directly lifted from the literature. As it stands, the current wording in the main body of the paper gives the impression that the deterministic determinantal algorithm is novel, e.g. "this motivated us to develop a deterministic version of determinantal sampling" (p.2), and it is only in the methods section that a reference is made to the paper of Schreurs et al. which proposed the algorithm.

Similarly, in the abstract and main body of the text, the wording gives the impression that the quantum circuits presented here are new (e.g., "We also develop quantum circuits for implementing determinantal point processes") whereas they have been previously proposed (although one of the authors of the current paper was also an author of the original paper proposing the quantum circuits for determinantal sampling).

2. Additional analysis is needed to support the claims of potential for quantum advantage:

The authors claim that the quantum algorithm for implementing determinantal point processes provides a computational advantage over classical ones, in that the quantum circuits scale linearly in the number of features compared with cubic scaling classically. While this may be true asymptotically, in my opinion, more discussion is required about the utility and feasibility of this method in practice, as well as the realistic prospects of this being a potential area of application for quantum computing.

For example, the authors mention that a quantum computer of 150 qubits capable of running circuits of depth 400 is needed to perform the determinantal sampling for the MIMIC-III dataset considered, and say "while [such hardware is] not available right now, it seems quite possible that they will be available in the not so far future". The authors also state "This suggests that with the advent of next-generation quantum computers... one could expect a computational speedup in performing determinantal sampling" and "it is expected that next-generation quantum computers will provide a speedup in practice". These are strong assertions (even if 'next generation' is not clearly defined), and in my opinion, are not sufficiently backed by evidence to date. Given that datasets of the size of MIMIC-III (and presumably much larger) can be handled efficiently classically, the authors should clarify whether one expects a quantum advantage by this approach in the "NISQ" (pre-error-corrected) era of quantum computing. This seems unlikely, and any argument that this is the case should include an analysis accounting for the absolute operation speeds and absolute times required to perform such computations, including any time required for inputting data, resetting quantum circuits etc. On the other hand, if by 'next generation' the authors mean quantum computers beyond the NISQ era (i.e., assuming fault-tolerant quantum computers and logical qubits), then the overhead costs of quantum error correction (both in terms of physical qubit numbers as well as computational time) should be analyzed, and the crossover regime (i.e., data size where a quantum computation takes less absolute time than classical) estimated in order to assess the prospects of a practical quantum advantage, especially in light of recent analyses e.g., [1,2] below.

[1] Hoefler, Haner, Troyer. Communicatios of the ACM, 66.5 (2023):82-87<br /> [2] Babbush et al., PRX Quantum 2.1 (2021):010103

Other comments and suggestions:<br /> The authors measure "running time [as] the depth of the necessary quantum circuits." While circuit depth may indeed correspond to wall-clock time, quantum circuit size (i.e. number of gates) is the fairer complexity metric for comparison with classical running time. If depth is used, then a fair comparison to classical methods should be to compare with classical parallel processing time using N processors. However, if circuit size is used, then the quantum complexity is Nd, which contrasts with the classical value of Nd^2 (pre-processing) + d^3 (per sample). This yields a subquadratic quantum speedup over classical, as opposed to a qubic speedup.

The results (e.g Table 1) show that the new algorithms consistently outperform the original miceRanger and MissForest methods, although the degree of improvement is small, typically of order 1% or less. Some discussion is therefore warranted on the practical benefits of this method, and any tradeoff in terms of efficiency. In particular, while Table 1 compares the classification accuracy (as measured by AUC) of the newly proposed methods vs the existing state-of-the-art, a discussion on the scalability and efficiency would be welcome. The determinantal sampling takes time Nd^2, how does this compare with the original methods? For what dataset and feature sizes are the determinantal methods feasible (which will determine the scale at which other approaches, e.g. those based on quantum computing may be required).

A discussion (or at least mention) of the algorithmic complexity of the classical deterministic determinantal sampling (which seems to also be Nd^2) in the main body of the text would be welcome.

The final paragraph of the Methods section discusses sampling many times from the quantum circuits to estimate the most probable outcome, and hence perform the deterministic determinantal sampling. A more careful analysis of the number of samples needed (for bounded variance/error) and the impact on the running time (and whether one still expects an advantage over classical (although one must define some bounded error version of the deterministic algorithm to do so) or performance of the algorithm would be welcome.

A discussion on the absolute running time required for the quantum experiments performed (and how they compare to classical) would be interesting.

A mention of which quantum simulator was used would be welcome.

In the introduction, three kinds of data missingness (MCAR, MAR, MNAR) are mentioned, although experiments are only performed for MCAR and MNAR. Can some explanation for excluding MAR be given?

Reference 24 (Shadbar et al., the study that demonstrated the effectiveness of miceRanger and MissForest) used 4 datasets: MIMIC-III, Simulated, Breast Cancer, and NHSX COVID-19. Of these, MIMIC-III is used in the current paper, and Simulated appears similar (although with 1000 instead of 2000 rows) to the synthetic dataset of the current paper. An analysis of the determinantal sampling methods applied to the Breast Cancer and NHSX COVID-19 datasets (which have naturally occurring missingness), and a comparison to the results of Shadbar et al. would be interesting.

Reviewer #2 (Public Review):

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

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

Weaknesses<br /> 1. Use of the word "predict" with the SLM in this advance is confusing, and to this reviewer seems to make a stronger claim than shown by the authors. For example, in their abstract, the authors state "We found that measures of biodiversity at a given scale can be consistently predicted using predictions (sic) derived from a minimal model of ecology." This appears to imply that a minimal model predicted the behavior of a system when in reality it accurately described the data it was trained on. This potential for confusion extends throughout the text and obscures what was actually achieved.<br /> 2. More generally, to my mind the presentation in the manuscript could benefit from a clearer delineation between the question of "what patterns are explainable by a noninteracting model vs require interactions" (which could be assessed with no reference to SLM, but by a simple randomization test), and whether specifically, SLM is a good null model / better than UNTB.

Overall Impact<br /> The authors achieve their aims, even though the text is at times dense. The use of SLM as a non-interacting null model for macroecological quantities and relationships is well supported by the text, and SLM should be used as a null model for these types of phenomena going forward.

Reviewer #2 (Public Review):

The study focuses on a mechanism of pest/pathogen resistance identified in Solanum commersonii, which appears to offer dominant resistance to Alternaria solani (potato early blight) through the activity of specific glycosyltransferases which facilitate the production of tetraose glycoalkaloids in leaf tissue. The authors demonstrated that these glycoalkaloids are suppressive to the growth of multiple pathogenic ascomycetes and furthermore, that transgenic plants expressing these glycosyltransferases in susceptible S. commersonii clones demonstrate improved resistance to specific strains of A. solani and a genotype of Colorado Potato Beetle. The study design is straightforward, yet thorough, and does a good job demonstrating the importance of these genes in resistance. This work is significant because it demonstrates the mechanism behind resistance to a necrotrophic pathogen. Resistance to this group of pathogens has historically relied on mechanisms that do not include the use of typical dominant resistance gene products (nucleotide-binding, leucine-rich repeat proteins). The identification of these glycosyltransferases and their role in resistance will give potato breeders options for the development of markers associated with resistance to this group of pathogens. However, this may demonstrate an important battle to balance between production traits (like disease resistance) and quality traits (like glycoalkaloid content), as the two may be mutually exclusive in the development of new varieties.

Reviewer #2 (Public Review):

This is an interesting follow-up study that uses long-read sequencing to examine previously constructed mutation accumulation lines between wild populations of S. cerevisiae and S. paradoxus. They also complement this work with reporter assays in hybrid backgrounds. The authors are attempting to test the hypothesis that hybridization leads to genome shock and unrestrained transposition. The paper largely confirms previous results (suggesting hybridization does not increase transposition) that are well cited and discussed in the paper, both from this group and from the Smukowski Heil/Dunham group but extends them to a new set of species/hybrids and with some additional resolution via the long read sequencing. The paper is well written and clear and I have no serious complaints.

In the abstract, the authors make three primary claims:

Structural variation plays a strong role in TE load.<br /> Transposition plays only a minor role in shaping the TE landscape in MA lines.<br /> Transposition rates are not increased by hybridization but are affected by genotype-specific factors.

I found all three claims supported, albeit with some minor questions below:

Structural variation plays a strong role in TE load.<br /> Convinced of this result. However:<br /> Line 185-187/Figure 3C: I'm curious given that the changes in Ty count are so often linked to changes in gross DNA sequence whether the count per total DNA sequence is actually changing on average in these genomes. Ie., does hybridization tend to increase TE count via CNV or does hybridization tend to increase DNA content in the MA lines and TEs come along for the ride?

One question about ploidy (lines 175-177):

Both aneuploidy and triploidy seem easy to call from this data. A 3:1 tetraploidy as well. However, in Figure 2B there are tetraploids that are around the 1:1 line. How are the authors calling ploidy for these strains? This was not clear to me from the text.

Reviewer #2 (Public Review):

This was an interesting study, and I enjoyed seeing different experimental approaches used to compare the properties of the different native proteins, the ancestral reconstructions, and the other mutants. I think it provides convincing mechanistic evidence as to how these small heat shock proteins have evolved. Thus, I think it represents a valuable contribution to the field. However, to a certain extent, I think the authors have at times over-interpeted their results, and over-simplified their explanations, as the differences between the ancestral proteins, and the changes induced by the two mutations, only partially explain the differences between IbpA proteins from the two different species. Furthermore, in some places, I found this difficult to follow and figures were not properly explained or labelled. If these issues were addressed, I think the paper would be considerably more accessible to readers.

Reviewer #2 (Public Review):

In this manuscript, the authors introduced an explicit ion model using the coarse-grained modelling approach to model the interactions between nucleosomes and evaluate their effects on chromatin organization. The strength of this method lies in the explicit representation of counterions, especially divalent ions, which are notoriously difficult to model. To achieve their aims and validate the accuracy of the model, the authors conducted coarse-grained molecular dynamics simulations and compared predicted values to the experimental values of the binding energies of protein-DNA complexes and the free energy profile of nucleosomal DNA unwinding and inter-nucleosome binding. Additionally, the authors employed umbrella sampling simulations to further validate their model, reproducing experimentally measured sedimentation coefficients of chromatin under varying salt concentrations of monovalent and divalent ions.

The significance of this study lies in the authors' coarse-grained model which can efficiently capture the conformational sampling of molecules while maintaining a low computational cost. The model reproduces the scale and, in some cases, the shape of the experimental free energy profile for specific molecule interactions, particularly inter-nucleosome interactions. Additionally, the authors' method resolves certain experimental discrepancies related to determining the strength of inter-nucleosomal interactions. Furthermore, the results from this study support the crucial role of intrinsic physicochemical interactions in governing chromatin organization within the nucleus.

The method is simple but can be useful, given the authors can provide more details on their ion parameterization. The paper says that parameters in their "potentials were tuned to reproduce the radial distribution functions and the potential of mean force between ion pairs determined from all-atom simulations." However, no details on their all-atom simulations were provided; at some point, the authors refer to Reference 67 which uses all-atom simulations but does not employ the divalent ions. Also, no explanation is given for their modelling of protein-DNA complexes.

Overall, the paper is well-written, concise and easy to follow but some statements are rather blunt. For example, the linker histone contribution (Figure 5D) is not clear and could be potentially removed. The result on inter-nucleosomal interactions and comparison to experimental values from Ref#44 is the most compelling. It would be nice to see if the detailed shape of the profile for restrained inter-nucleosomal interactions in Figure 4B corresponds to the experimental profile. Including the dependence of free energy on a vertex angle would also be beneficial.

Another limitation of this study is that the authors' model sacrifices certain atomic details and thermodynamic properties of the modelled systems. The potential parameters of the counter ions were derived solely by reproducing the radial distribution functions (RDFs) and potential of mean force (PMF) based on all-atom simulations (see Methods), without considering other biophysical and thermodynamic properties from experiments. Lastly, the authors did not provide any examples or tutorials for other researchers to utilize their model, thus limiting its application.

Reviewer #2 (Public Review):

Summary:<br /> The major purpose of this manuscript is to examine whether leucine treatment would be a potential strategy to treat cytokine storm syndrome (CSS). CSS is a common symptom in multiple infectious diseases in clinic, which gradually leads to multiple organ failure and high mortality. Strategies to treat CSS including pulse steroid therapy normally lead to severe side effects. Therefore, it is still required to develop a safe strategy with high efficacy to treat CSS. In clinic, sepsis is well characterized to exhibit CSS and therefore multiple studies utilized a LPS-induced sepsis model to evaluate CSS symptoms. In this study, the authors examined whether leucine, an essential amino acid that has been absorbed daily in our body, could ameliorate CSS symptoms in the LPS-induced sepsis mouse model. They found a potential protective effect of leucine in terms of the survival rate and inflammatory responses.

Strengths:<br /> The study is overall well designed and the results are well analyzed with only minor issues. The methods utilized are appropriate.

Weaknesses:<br /> The mechanistic insights are not sufficient and could not fully explain the phenotype they found. Considering the importance of this study is to identify the potential protective role of leucine in CSS, the authors could also consider investigator-initiated clinical trials to further expand the significance of this study.

Reviewer #2 (Public Review):

In this paper, Boi et al. thoroughly classified the electrophysiological and morphological characteristics of serotonergic and dopaminergic neurons in the DRN and examined the alterations of these neurons in the 6-OHDA-induced mouse PD model. Using whole-cell patch clamp recording, they found that 5-HT and dopamine (DA) neurons in the DRN are electrophysiologically well-distinguished from each other. In addition, they characterized distinct morphological features of 5-HT and DA neurons in the DRN. Notably, these specific features of 5-HT and DA neurons in the DRN exhibited different changes in the 6-OHDA-induced PD model. Then the authors utilized desipramine (DMI) to separate the effects of nigrostriatal DA depletion and noradrenalin (NA) depletion which are induced by 6-OHDA. Interestingly, protection from NA depletion by DMI pretreatment reversed the changes in 5-HT neurons, while having a minor impact on the changes in DA neurons in the DRN. These data indicate that the role of NA lesion in the altered properties of DRN 5-HT neurons by 6-OHDA is more critical than the one of DA lesion.

Overall, this study provides foundational data on the 5-HT and DA neurons in the DRN and their potential involvement in PD symptoms. Given the defects of the DRN in PD, this paper may offer insights into the cellular mechanisms that may underlie non-motor symptoms associated with PD. Despite the importance of the primary claim proposed by the authors, however, several weaknesses undermine the significance of the data.

Reviewer #2 (Public Review):

This manuscript describes experiments that further investigate the actions of the transcription factor Bcl11b in regulating mossy fiber (MF) synapses in the hippocampus. Prior work from the same group had demonstrated that loss of Bcl11b results in loss of MF synapses as well as a decrease in LTP. Here the authors focus on a target of Bcl11b a secreted synaptic organizer C1ql2 which is almost completely lost in Bcl11b KO. Viral reintroduction of C1ql2 rescues the synaptic phenotypes, whereas direct KD of C1ql2 recapitulates the Bcl1 phenotype. C1ql2 itself interacts directly with Nrxn3 and replacement with a binding deficient mutant C1q was not able to rescue the Bcl11b KO phenotype. Overall there are some interesting observations in the study, however there are also some concerns about the measures and interpretation of data.

The authors state that they used a differential transcriptomic analysis to screen for candidate targets of Bcl11b, yet they do not present any details of this screen. This should be included and at the very least a table of all DE genes included. It is likely that many other genes are also regulated by Bcl11b so it would be important to the reader to see the rationale for focusing attention on C1ql2 in this study.

All viral-mediated expression uses AAVs which are known to ablate neurogenesis in the DG (Johnston DOI: 10.7554/eLife.59291) through the ITR regions and leads to hyperexcitability of the dentate. While it is not clear how this would impact the measurements the authors make in MF-CA3 synapses, this should be acknowledged as a potential caveat in this study.

The authors claim that the viral re-introduction "restored C1ql2 protein expression to control levels. This is misleading given that the mean of the data is 2.5x the control (Figure 1d and also see Figure 6c). The low n and large variance are a problem for these data. Moreover, they are marked ns but the authors should report p values for these. At the least, this likely large overexpression and variability should be acknowledged. In addition, the use of clipped bands on Western blots should be avoided. Please show the complete protein gel in primary figures of supplemental information.

Measurement of EM micrographs: As prior work suggested that MF synapse structure is disrupted the authors should report active zone length as this may itself affect "synapse score" defined by the number of vesicles docked. More concerning is that the example KO micrographs seem to have lost all the densely clustered synaptic vesicles that are away from the AZ in normal MF synapses e.g. compare control and KO terminals in Fig 2a or 6f or 7f. These terminals look aberrant and suggest that the important measure is not what is docked but what is present in the terminal cytoplasm that normally makes up the reserve pool. This needs to be addressed with further analysis and modifications to the manuscript.

The study also presents correlated changes in MF LTP in Bcl11b KO which are rescued by C1ql2 expression. It is not clear whether the structural and functional deficits are causally linked and this should be made clearer in the manuscript. It is also not apparent why this functional measure was chosen as it is unlikely that C1ql2 plays a direct role in presynaptic plasticity mechanisms that are through a cAMP/ PKA pathway and likely disrupted LTP is due to dysfunctional synapses rather than a specific LTP effect. The authors should consider measures that might support the role of Bcl11b targets in SV recruitment during the depletion of synapses or measurements of the readily releasable pool size that would complement their findings in structural studies.<br /> Bcl11b KO reduces the number of synapses, yet the I-O curve reported in Supp Fig 2 is not changed. How is that possible? This should be explained.

Matsuda et al DOI: 10.1016/j.neuron.2016.04.001 previously reported that C1ql2 organizes MF synapses by aligning postsynaptic kainate receptors with presynaptic elements. As this may have consequences for the functional properties of MF synapses including their plasticity, the authors should report whether they see deficient postsynaptic glutamate receptor signaling in the Bcl11b KO and rescue in the C1ql2 re-expression.

Reviewer #2 (Public Review):

Schommartz et al. present a manuscript characterizing neural signatures of reinstatement during cued retrieval of middle-aged children compared to adults. The authors utilize a paradigm where participants learn the spatial location of semantically related item-scene memoranda which they retrieve after short or long delays. The paradigm is especially strong as the authors include novel memoranda at each delayed time point to make comparisons across new and old learning. In brief, the authors find that children show more forgetting than adults, and adults show greater engagement of cortical networks after longer delays as well as stronger item-specific reinstatement. Interestingly, children show more category-based reinstatement, however, evidence supports that this marker may be maladaptive for retrieving episodic details. The question is extremely timely both given the boom in neurocognitive research on the neural development of memory, and the dearth of research on consolidation in this age group. Also, the results provide novel insights into why consolidation processes may be disrupted in children. Despite these strengths, there are quite a few important design and analytical choices that derail my enthusiasm for the paper. If the authors could address these concerns, this manuscript would provide a solid foundation to better understand memory consolidation in children.

Reviewer #2 (Public Review):

The paper 'Complexes of vertebrate TMC1/2 and CIB2/3 proteins 1 form hair-cell mechanotransduction cation channels' by Giese and coworkers is quite an intense reading. The manuscript is packed with data pertaining to very different aspects of MET apparatus function, scales, and events. I have to praise the team that combined molecular genetics, biochemistry, NMR, microscopy, functional physiology, in-vivo tests for vestibulo-ocular reflexes, and other tests for vestibular dysfunction with molecular modeling and simulations. The authors nicely show the way CIBs are associated with TMCs to form functional MET channels. The authors clarify the specificity of associations and elucidate the functional effects of the absence of specific CIBs and their partial redundancy.

Reviewer #2 (Public Review):

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

Overall, the authors have significantly improved the manuscript, but there is still an unclarified point. In response to the inquiry in the initial review on how extent E80A KI mice function as a pathological model of dominant CoRD, the authors add data (Figures S7) and described the sixth section in the discussion. However, the authors mentioned that it is technically too challenging because of a small number of cones. The point is not clear to me, but it is possible to analyze cone differentiation and degeneration by immunostaining at multiple stages even though cone number is small. Cone arrestin and S- and M-opsins become positive at early postnatal stages in the mouse retina. Cone arrestin seems earlier than cone opsins. Cones seem born by detecting RXRg at P0, but are cone arrestin and/or cone opsins expressed in early postnatal E80A/+ retina? If positive, how about an apoptosis marker? If negative, it seems to be a cone development phenotype rather than cone degeneration phenotype. If so, authors should modify the expression to say that the E80A retina underlies CoRD-like phenotype. It seems an overstatement.

Reviewer #2 (Public Review):

Zheng et al., investigated the molecular and functional mechanisms of two homeodomain missense mutations causing human retinal photoreceptor degeneration diseases in photoreceptor development regulated by the CRX transcription factor. They analyzed the E80A mutation associated with dominant cone-rod dystrophy (CRD) and the K88N mutation associated with dominant Leber Congenital Amaurosis (LCA). The authors found that E80A CRX binds to the same target DNA sites as WT CRX, but the binding specificity of K88N CRX is altered from that of WT in an in vitro assay. They generated Crx(E80A) and Crx(K88N) KI mice and performed ChIP assay and observed that K88N CRX binds to novel genomic regions from the WT-binding sites, while E80A binds to the WT sites. In addition, using the KI mice, they found that E80A and K88N differently affect the expression of Crx target genes. The authors may want to provide explicit clarification on whether CRX E80A mice exhibit cone development and/or degeneration defects.

This study is well executed with proper and solid methodologies, and the manuscript is clearly written. This study gives us the insights into how single missense CRX mutations lead to different types of human retinal photoreceptor degeneration diseases.

Reviewer #2 (Public Review):

Kraus, Aurora et al. investigated the potential immune response of the olfactory bulb after exposure of the infectious hematopoietic necrosis virus (IHNV), via the olfactory epithelia. Specifically, they show that a) viral-specific neuronal activation of "OSNs" (Crypt cells), b) changes in behaviour of both adult and larval zebrafish after viral exposure, c) Pituitary adenylate-cyclase-activating polypeptide (PACAP), was enriched when assayed by single cell transcriptomic profiling of cells in the OB after OSNs are exposed to IHNV

Although the paper does have strengths in principle, the weaknesses of the manuscript are that these strengths are not directly demonstrated and the referencing of the manuscript omits many references important for the understanding of the questions and the results of the study. Furthermore, the data presented are not sufficient to fully support the key claims in the manuscript. In particular:

a) Viral-specific neuronal activation of OSNs:<br /> What type of neurons? The authors are a bit elusive and do not clearly state that the neurons are crypt cells (Sepahi et al.: rainbow trout) which have a very specific axonal projection to the brain and whose response characteristics are not well characterized (see work of Korsching lab). Crypt cells are not present in the olfactory epithelia of mammals. Furthermore, in their previous work the crypt cells die; so how do they think the (inflammatory) virus response is transmitted to the olfactory bulbs in order to protect the brain?<br /> The authors state from previous work that they never detected virus in the brain, but why would they? Does INHV move trans-synaptically?<br /> The neuronal activity was monitored using a pan-neuronal marker thus these data are of limited use when trying to understand the role of neuronal activity (crypt cells) in the IHNV-triggered activity: the authors may be looking at a generalized inflammation response, and the image presented is not particularly informative it is difficult to decipher the results. The authors assume IHNV is an odorant without carefully ruling out the possibility of a generalized inflammation response.<br /> b) Changes in behaviour of both adult and larval zebrafish after viral exposure:<br /> What is the motivating question for looking at behaviour of the virus infected animals? Do we know the effects of crypt cell loss on the behaviour in any fish species? Authors need to build a better conceptual framework for the behaviour experiments.

c) Pituitary adenylate-cyclase-activating polypeptide (PACAP) was enriched when assayed by single cell transcriptomic profiling of cells in the OB after OSNs are exposed to IHNV. Authors draw many generous conclusions from limited data. Authors seem to have forgotten to cite papers previously published showing that PACAP-38 has anti-viral activities in fish (VHSV: trout) such as: Velasquez et al 2020, First in vivo evidence of pituitary adenylate cyclase-activating polypeptide antiviral activity in teleost.<br /> The histology for PACAP presented in the manuscript is not convincing. The antibody is against the human form of PACAP thus any labelling should be treated with caution (and called PACAP-38-like).

Summary: The authors need to better develop their model (perhaps a diagram would be helpful) explaining exactly which neurons are transmitting the information. Because of the elusive nature of some referencing and the skirting of important issues such as clearly stating which neurons are affected (crypt cells), what the point of the behaviour is (relate to neuronal type infected by virus), and, the lack of an antibody specific to the zebrafish protein, the model appears to be built on an unstable base.

DOI: 10.1101/2023.08.09.552694

Resource: (ZFIN Cat# ZDB-ALT-060919-2,RRID:ZFIN_ZDB-ALT-060919-2)

Curator: @olekpark

SciCrunch record: RRID:ZFIN_ZDB-ALT-060919-2

What is this?

Reviewer #2 (Public Review):

Summary:<br /> In their preprint, Fang et al present data on extending a spatial transcriptomics method, MERFISH, to 3D using a spinning disc confocal. MERFISH is a well-established method, first published by Zhaung's lab in 2015 with multiple follow-up papers. In the last few years, MERFISH has been used by multiple groups working on spatial transcriptomics, including approximately 12 million cell maps measured in the mouse brain atlas project. Variants of MERFISH were used to map epigenetic information complementary to gene expression and RNA abundance. However, MERFISH was always limited to thin ~10um sections to this date. The key contribution of this work by Fang et al. was to perform the optimization required to get MERFISH working in thick (100-200um) tissue sections.

Major strengths and weaknesses:<br /> Overall the paper presents a technical milestone, the ability to perform highly multiplexed RNA measurements in 3D using MERFISH protocol. This is not the first spatial transcriptomics done in thick sections. Wang et al. 2018 - StarMAP used thick sections (150 um), and recently, Wang 2021 (EASI-FISH, not cited) performed serial HCR FISH on 300um sections. Data so far suggest that MERFISH has better sensitivity than in situ sequencing approaches (StarMAP) and has built-in multiplexing that EASI-FISH lacks. Therefore, while there is an innovation in the current work, i.e., it is a technically challenging task, the novelty, and overall contribution are modest compared to recently published work.

The authors could improve the writing and the manuscript text that places their work in the right context of other spatial transcriptomics work. Out of the 25 citations, 12 are for previous MERFISH work by Zhaung's lab, and only one manuscript used a spatial transcriptomics approach that is not MERFISH. Furthermore, even this paper (Wang et al, 2018) is only discussed in the context of neuroanatomy findings. The fact that Wang et al. were the first to measure thick sections is not mentioned in the manuscript. The work by Wang et al. 2021 (EASI-FISH) is not cited at all, as well as the many other multiplexed FISH papers published in recent years that are very relevant. For example, a key difference between seqFISH+ and MERFISH was the fact that only seqFISH+ used a confocal microscope, and MERFISH has always been relying on epi. As this is the first MERFISH publication to use confocal, I expect citations to previous work in seqFISH and better discussions about differences.

To get MERFISH working in 3D, the authors solved a few technical problems. To address reduced signal-to-noise due to thick samples, Fang et al. used non-linear filtering (i.e., deep learning) to enhance the spots before detection. To improve registrations, the authors identified an issue specific to their Z-Piezo that could be improved and replaced with a better model. Finally, the author used water immersion objectives to mitigate optical aberrations. All these optimization steps are reasonable and make sense. In some cases, I can see the general appeal (another demonstration of deep learning to reduce exposure time). Still, in other cases, the issue is not necessarily general enough (i.e., a different model of Piezo Z stage) to be of interest to a broad readership. There were a few additional optimization steps, i.e., testing four concentrations of readout and encoder probes. So while the preprint describes a technical milestone, achieving this milestone was done with overall modest innovation.

Data and code sharing - the only link in the preprint related to data sharing sends readers to a deleted Dropbox folder. Similarly, the GitHub link is a 404 error. Both are unacceptable. The author should do a better job sharing their raw and processed data. Furthermore, the software shared should not be just the MERlin package used to analyze but the specific code used in that package.

Reviewer #2 (Public Review):

In this study, the authors measured extracellular electrical features of colliding APs travelling in different directions down an isolated earthworm axon. They then used these features to build a model of the potential ephaptic effects of AP annihilation, i.e. the electrical signals produced by colliding/annihilating APs that may influence neighbouring tissue. The model was then applied to some different hypothetical scenarios involving synaptic connections. The conclusion was that an annihilating AP at a presynaptic terminal can ephaptically influence the voltage of a postsynaptic cell (this is, presumably, the 'electrical coupling between neurons' of the title), and that the nature of this influence depends on the physical configuration of the synapse.

As an experimental neuroscientist who has never used computational approaches, I am unable to comment on the rigour of the analytical approaches that form the bulk of this paper. The experimental approaches appear very well carried out, and here I just have one query - an important assumption made is that the conduction velocity of anti- and orthodromically propagating APs is identical in every preparation, but this is never empirically/statistically demonstrated.

My major concern is with the conclusions drawn from the synaptic modelling, which, disappointingly, is never benchmarked against any synaptic data. The authors state in their Introduction that a 'quantitative physical description' of ephaptic coupling is 'missing', however, they do not provide such a description in this manuscript. Instead, modelled predictions are presented of possible ephaptic interactions at different types of synapses, and these are then partially and qualitatively compared to previous published results in the Discussion. To support the authors' assertion that AP annihilation induces electrical coupling between neurons, I think they need to show that their model of ephaptic effects can quantitatively explain key features of experimental data pertaining to synaptic function. Without this, the paper contains some useful high-precision quantitative measurements of axonal AP collisions, some (I assume) high-quality modelling of these collisions, and some interesting theoretical predictions pertaining to synaptic interactions, but it does not support the highly significant implications suggested for synaptic function.

Reviewer #2 (Public Review):

The authors have provided evidence for a rostral-caudal organisation of locus coeruleus connectivity, which they show i) differs across the lifespan, ii) is associated with relevant cognitive and mood measures. They have taken a data-driven, gradient-based approach, which was applied in the CamCan dataset and then replicated in the HCP dataset. This is a useful contribution to the field as it comprehensively shows a rostral-caudal pattern of connectivity in vivo, which has mostly been supported by tracer studies to date.

The strengths of the study are the large sample sizes and replication across two cohorts. The connectomic mapping approach they have applied is very well suited to the question at hand, as it allows a continuous gradient of organisation to be identified.

Reviewer #2:

The authors have significantly improved the manuscript, where assumptions and analytical and numerical results are now presented more clearly.

I still have some comments, more of less specific, that I list below, starting with the conceptual ones.

1. Citation of previous work on dynamical quorum sensing (lines 51 & 52) I think misses two important points: first these works (and others following them) deal with the appearance of collective oscillations at high density (therefore, the same general problem addressed here); second, Taylor et al. studied also a transition where the oscillators involved did not oscillate at low density, whereas above a density threshold, they display coherent collective oscillations whose period decreases with density - similar to what observed here. I do not think this takes anything away from the originality of this work, which refers to a different system, and models it with different equations, but the parallelism between integrate-and-fire dynamics with quenched noise and excitable dynamics in the presence of noise should in my opinion not be overlooked.<br /> 2. As the authors stress in lines 105 and 132, the analytical model shows that all that really matters in this phenomenon is the fastest frequency of the system. This could be used as an argument to say that the actual frequency distribution of individual fireflies is not all that important, as long as their fastest frequency is comparable. The assumption that they are identical would then sound less radical. Ideally, one could use the numerical simulations to check this, as well as the fact that the phenomenon does not break down when the shortest individual interburst interval Tb_min is narrowly distributed (which could also explain why having a few individuals who can flash at a higher frequency does not affect the outcome).<br /> 3. I still feel that the agreement between the model and observations is a bit overstated (line 120). At least, I think the authors may stress that whereas the model predicts that the frequency of the 7-14 minutes oscillations should increase a lot with N, this is not observed in the data. Maybe this mismatch would be reduced if inter-individual variability was added.<br /> 4. In paragraph 4.2, I found it unclear why the authors find it unsurprising that different experiments would correspond to different betas. I think that this point should be discussed, as beta and N appear in combination in determining the interaction strength. Otherwise, they could try to fit all distributions with the same beta, which would be more natural for me. I guess that the fits would be anyway good to the eye, though quantitatively suboptimal (which could be quantified with the distance introduced).

Minor stylistic comments:<br /> 1. Lines 98-100. Are all three 'Thus' needed?<br /> 2. 114: 'sufficiently identical' sounds like an oxymoron: what about just 'identical', or 'sufficiently close, so that they can be approximately considered identical'?<br /> 3. It would be more in line with the text (line 122) if panel F was the first panel of Figure 3. Also, the two orange lines are very hardly visible in print. They could be thickened. The inset, which I guess represents a zoom into the low Tbs, should be explained in the figure caption.<br /> 4. The caption of figure 5 relative to panels A-E does not say what is depicted. On line 3, row -> rows<br /> 5. Line 195 provide -> provides

Reviewer #2 (Public Review):

In this paper, Wang and colleagues build on previous technical and analytical achievements in establishing tetraploid human-chimpanzee hybrid iPSCs to investigate the cell type-specificity of allele-specific expression and allele-specific chromatin accessibility across six differentiated cell types (here, "allele-specific" indicates species differences with a cis-regulatory basis). The combined body of work is remarkable in its creativity and ambition and has real potential for overcoming major challenges in understanding the evolutionary genetics of between-species differences. The present paper contributes to these efforts by showing how differentiated cells can be used to test a long-standing hypothesis in evolutionary genetics: that cis-regulatory changes may be particularly important in divergence because of their potential for modularity.

In my view, the paper succeeds in making this case: allele (species)-specific expression (ASE) and allele-specific chromatin accessibility (ASCA) are enriched in genes asymmetrically expressed in one cell type, and many cases of ASE/ASCA are cell type-specific. The authors do an excellent job showing that these results are robust across a set of possible analysis decisions. It is somewhat less clear whether these enrichments are primarily a product of relaxed constraint on cell type-specific genes or primarily result from positive selection in the human or chimp lineage. While the authors attempt to control for constraint using several variables (variance in ASE in humans and the sequence-based probability of haploinsufficiency score, pHI), these are imperfect proxies for constraint. For the pHI scores, enrichments for ASE also appear to be strongest in the least constrained genes. Overall, the relative role of relaxation of constraint versus positive selection is unresolved, although the manuscript's language leans in favor of an important role for selection.

The remainder of the manuscript draws on the cell type-specific ASE/ASCA data to nominate candidate genes and pathways that may have been important in differentiating humans and chimpanzees. Several approaches are used here, including comparing human-chimp ASE to the distribution of ASE observed in humans and investigating biases in the direction of ASE for genes in the same pathway. The authors also identify interesting candidate genes based on their role in development or their proximity to human accelerated regions (where many changes have arisen on the human lineage in otherwise deeply conserved sequence) and use a deep neural network to identify sequence changes that might be causally responsible for ASE/ASCA. These analyses have value and highlight potential strategies for using ASE/ASCA and hybrid cell line data as a hypothesis-generating tool. Of course, the functional follow-up that experimentally tested these hypotheses or linked sequence/expression changes in the candidate pathways to organismal phenotype would have strengthened the paper further- but this is a lot to ask in an already technically and analytically challenging piece of work.

As a minor critique, the present paper is very closely integrated with other manuscripts that have used the hybrid human-chimp cell lines for biological insight or methods development. Although its contributions make it a strong stand-alone contribution, some aspects of the methods are not described in sufficient detail for readers to understand (even on a general conceptual level) without referencing that work, which may somewhat limit reader understanding.

Reviewer #2 (Public Review):

In this paper, the authors presented a compelling rationale for investigating the role of UBCs in prolonging and diversifying signals. Based on the two types of UBCs known as ON and OFF UBC subtypes, they have highlighted the existing gaps in understanding UBCs connectivity and the need to investigate whether UBCs target UBCs of the same subtype, different subtypes, or both. The importance of this knowledge is for understanding how sensory signals are extended and diversified in the granule cell layer.

The authors designed very interesting approaches to study UBCs connectivity by utilizing transgenic mice expressing GFP and RFP in UBCs, Brainbow approach, immunohistochemical and electrophysiological analysis, and computational models to understand how the feed-forward circuits of interconnected UBCs transform their inputs.

This study provided evidence for the existence of distinct ON and OFF UBC subtypes based on their electrophysiological properties, anatomical characteristics, and expression patterns of mGluR1 and calretinin in the cerebellum. The findings support the classification of GRP UBCs as ON UBCs and P079 UBCs as OFF UBCs and suggest the presence of synaptic connections between the ON and OFF UBC subtypes. In addition, they found that GRP and P079 UBCs form parallel and convergent pathways and have different membrane capacitance and excitability. Furthermore, they showed that UBCs of the same subtype provide input to one another and modify the input to granule cells, which could provide a circuit mechanism to diversify and extend the pattern of spiking produced by mossy fiber input. Accordingly, they suggested that these transformations could provide a circuit mechanism for maintaining a sensory representation of movement for seconds.

Overall, the article is well written in a sound detailed format, very interesting with excellent discovery and suggested model, however, I have some comments/suggestions that may help to improve this manuscript:

• The discovery of UBCs innervating each other and their own subtypes, suggesting the presence of feed-forward networks in the cerebellum, is an incredibly fascinating and exciting finding followed by an intriguing model by authors. However, it is worth considering an alternative model as well. I acknowledge that visualizing such interactions using current tools and methods can be challenging ("The approaches used here were not able to determine the existence of networks of more than 2 UBCs connected one after the other. If present, 3 or more UBCs in series could extend and transform the input in even more dramatic ways. The temporal diversity that UBC circuits generate may underlie the flexibility of the cerebellum to coordinate movements over a broad range of behaviors."). Therefore, if this is the case in which more than 2 UBCs connected one after the other, then an alternative model PERHAPS resembles the basal nuclei, with its direct and indirect circuits, can be considered (maybe a type of circular model). The basal nuclei circuits are also regulated by modulators such as D1 dopamine receptors in the direct pathway, causing depolarization, and D2 dopamine receptors in the indirect pathway, resulting in hyperpolarization upon dopamine activation. This approach could involve using computational models to gain insight into potential alternatives within this pathway (may be a future direction).

• GRP UBCs are more densely distributed in lobes VI-IX, while P079 UBCs are more densely distributed in the dorsal leaflet of lobe X in sagittal sections. While the cerebellum is well known for its characteristic stripy pattern, are UBC distributions the same in coronal/transverse section?

• The extension of the axons from both subtypes of UBCs show they are long enough to pass several UBCs and even projections are directed toward the white matter (e.g. Fig 9A), suggesting targeting the UBCs or granule cells in other lobules. Is it suggesting UBCs connectivity between different lobules (perhaps longitudinal connectivity)? Is there any observation or information in coronal/transverse section to visualize mediolateral connectivity?

• The limitation in identifying networks involving more than two sequentially connected UBCs was briefly noted. I suggest including a paragraph describing limitations and discussing the implications of the findings would enhance the overall impact of the research and broaden our understanding of cerebellar function.

• It is a pity that there is no clear conclusion to the discussion of this very interesting study. I suggest providing the key points as a conclusion.

• Please make the correction in Figure 2A by relabeling it as IXa, IXb, and IXc to correct the typographical error.

• I recommend rotating Figure 7A to align its orientation with the other figures for consistency.

Reviewer #2 (Public Review):

The authors developed an algorithm that allows for deconvoluting of plasmid sequences from a mixture of plasmids that have been sequenced by nanopore long read technology. As library preparations and barcoding of individual samples increase sequencing costs, the algorithm bypasses this need and thus decreases time on sample prep and sequencing costs. In the first step, the tool assesses which of the plasmid constructions can be mixed in a single library preparation by calculating a distance matrix between the reference plasmid and the constructions producing sequence clusters. The user is given groups of plasmids, from different clusters, to be pooled together for sequencing. After sequencing, the algorithm deconvolutes the reads by classifying them based on alignments to the reference sequence. A Bayesian analysis approach is used to obtain a consensus sequence and quality scores.

Strengths<br /> The authors exploit one of the main advantages of long-read sequencing which is to accurately resolve regions of high complexity, as regularly found in plasmids, and developed a tool that can validate plasmid constructions by reducing sequencing costs. Multiple plasmids (up to six) can be analyzed simultaneously in a single library without the need for sample barcoding, also reducing sample preparation time. Although inserts must be different, just 2 bases difference would be enough for a correct assignation. It maximizes cost-efficiency for projects that require large amounts of plasmid constructions and high-throughput validation.

Weaknesses<br /> The method proposed by the authors requires prior knowledge of plasmid sequences (i.e., blueprints or plasmid reference) and is not suitable for small experiments. The plasmid inserts or backbones must be different e.g., multiple colonies from the same plasmid construction effort cannot be submitted together.

Reviewer #2 (Public Review):

Using standard and widely used tools, the authors revealed the factors (cultural, phenotypic, phylogenetic, etc.) shaping societal and scientific interest in natural species around the globe. The strength of this manuscript (and the authors') lies in its command of the available literature, database and variable management and analysis, and its solid discussion. The authors thus achieved a manuscript that was pleasant to read.

While I agree that doing a global study requires losing details of local patterns, maybe this is exactly the biggest shortcoming of the manuscript, oblivious to how different cultures (compare USA to PNG, for example) are reflected in these global patterns.

Related to this previous point, my only other comment is about using English as a reference of societal interest (i.e., the presence of a common name in English). While English may be widespread in Academia, it is still not that common in other societal circles, especially those not using Wikipedia for lack of internet access.

Reviewer #2 (Public Review):

In this study, the authors tried to gauge the effect of human activity on three species, (1) the Hooded grow, an urban exploiter, (2) the Rose ring parakeet, an invasive, alien species that has adapted to exploit human resources, and (3) the Graceful Prinia, an urban adapter, which is relatively shy of humans. A goal of the study was to increase awareness of the importance of urban parks.

Strengths:<br /> Strengths of the study include the fact that it was conducted at 17 different sites, including parks, roads and residential areas, and included three species with different habitat preferences. Each species produced relatively loud and repeatable vocalizations. To avoid the effect of seasonal changes, sounds were sampled within a 10 day period of the lockdown as well as post-lockdown. The analysis included a comparison of the number of sound files, binary values indicating emission of a common syllable, and also the total number of syllables emitted as a measurement of bird activity. Ambient temperatures and sound levels of human activity were also recorded. All of these factors speak to the comprehensive approach and analysis adopted in this study. The results are based on a rigorous statistical analysis, ruling out effects of various extraneous parameters.

Weaknesses:<br /> The explanation of methods can be improved. For example, it is not clear if data were low-pass filtered before resampling to avoid aliasing.<br /> It is quite possible that birds move into the trees and further from the recorders with human activity. Since sound level decreases by the square of the distance of the source from the recorders, this could significantly affect the data. As indicated in the Discussion, this is a significant parameter that could not be controlled.<br /> In interpreting the data, the authors mention the effect of human activity on bird vocalizations in the context of inter-species predator-prey interactions; however, the presence of humans could also modify intraspecies interactions by acting as triggers for communication of warning and alarm, and/or food calls (as may sometimes be the case) to conspecifics. Along the same lines, it is important to have a better understanding of the behavioral significance of the syllables used to monitor animal activity in the present study.<br /> Another potential effect that may influence the results but is difficult to study, relates to the examination of vocalizations near to the ambient noise level. This is the bandwidth of sound levels where most significant changes may occur, for example, due to the Lombard effect demonstrated in bird and bat species. However, as indicated, these are also more difficult to track and quantify. Moreover, human generated noise, other than speech, may be a more relevant factor in influencing acoustic activity of different bird species. Speech, per se, similar to the vocalizations of many other species, may simply enrich the acoustic environment so that the effects observed in the present study may be transient without significant long-term consequences.

In general, the authors achieved their aim of illustrating the complexity of the effect of human activity on animal behavior. At the same time, their study also made it clear that estimating such effects is not simple given the dynamics of animal behavior. For example, seasonality, temperature changes, animal migration and movement, as well as interspecies interactions, such as related to predator-prey behavior, and inter/intra-species competition in other respects can all play into site-specific changes in the vocal activity of a particular species.

Reviewer #2 (Public Review):

Iske et al. provide experimental data that NAD+ lessens disease severity in bacterial sepsis without impacting on the host pathogen load. They show that in macrophages, NAD+ prevents Il1b secretion potentially mediated by Caspase11.

While the in vivo and in vitro data is interesting and hints towards a crucial role of NAD+ to promote metabolic adaptation in sepsis, the manuscript has shortcomings and would profit from several changes and additional experiments that support the claims.

Conceptually, the definition of sepsis is outdated. Sepsis is not SIRS, as in sepsis-2. Sepsis-3 defines sepsis as infection-associated organ dysfunction. This concept needs to be taken into account for the introduction and when describing the potential effects of NAD+ in sepsis. Also, LPS application cannot be considered a sepsis model, since it only recapitulates the consequence of TLR-4 activation. It is a model of endotoxemia. Also, the LPS data does not allow to draw conclusions about bacterial clearance (L135).

The authors state that protective effects by NAD were independent of the host pathogen load. This clearly indicates that NAD confers protection via enhancing a disease tolerance mechanism, potentially via reducing immunopathology. This aspect is not considered by the authors. The authors should incorporate the concept of disease tolerance in their work, cite the relevant literature on the topic and discuss it their findings in light of the published evidence for metabolic alteration sand adaptations in sepsis.

For the in vitro data, the manuscript would benefit from additional experiments using in vitro infection models.

In the merge manuscript, the authors provide two different versions of the figures. In one, bar plots are shown without individual data and in the other with scatter blots. All bar plots need to be provided as scatter plots showing individual values.

The authors should show further serology data for kidney and liver failure etc. as well as further cytokine data such as IL-6 and TNF to better characterize their models.

Careful revision of the entire manuscript, the figure legends and figures is required. The figure legend should not repeat the methods and materials section. The nomenclature for mouse protein and genes needs to be thoroughly revised.

L350. The authors write that they dissect the capacity of NAD+ to dampen auto- and alloimmunity. In this work, no data that supports this statement is shown and experiments with autoantigens or alloantigens are not performed.

L163 The authors describe pyroptosis but in the figure legend call it apoptosis. Specific markers for each cell death should be measured and determined which cell death mechanisms is involved.

Animal data comes from an infection model and LPS application. The RNAseq data is obtained from cells primed with Pam3CSK4 and subsequently subjected to LPS. It is unclear how the cell culture model reflects the animal model. As such the link between IFN signaling and the bacterial infection/LPS model are not convincing and need to be further elaborated.

Figure 5: It is unclear how many independent survival experiments were done, how many mice per group were used and whether the difference between groups was statistical significant. This information should be added.

Further experiments with primary cells from Il10 k.o. and Caspase11 k.o. animals should be provided that support the findings in macrophages.

Reviewer #2 (Public Review):

This manuscript describes the functional and structural characterization of an anaerobic (Class III) ribonucleotide reductase (RNR) with an ATP cone domain from Prevotella copri (PcNrdD). Most significantly, the cryo-EM structural characterization revealed the presence of a flap domain that connects the ATP cone domain and the active site and provides structural insights about how nucleotides and deoxynucleotides bind to this enzyme. The authors also demonstrated the catalytic functions and the oligomeric states. However, many of the biochemical characterizations are incomplete, and it is difficult to make mechanistic conclusions from the reported structures. The reported nucleotide-binding constants may not be accurate because of the design of the assays, which complicates the interpretation of the effects of ATP and dATP on PcNrdD oligomeric states. Importantly, statistical information was missing in most of the biochemical data. Also, while the authors concluded that the dATP binding makes the GRD flexible based on the absence of cryo-EM density for GRD in the dATP-bound PcNrdD, no other supports were provided. There was also a concern about the relevance of the proposed GRD flexibility and the stability of Gly radical. Overall, the manuscript provides structural insights about Class III RNR with ATP cone domain and how it binds ATP and dATP allosteric effectors. However, ambiguity remains about the molecular mechanism by which the dATP binding to the ATP cone domain inhibits the Class III RNR activity.

Strengths:<br /> 1. The manuscript reports the first near-atomic resolution of the structures of Class III RNR with ATP domain in complex with ATP and dATP. These structures revealed the NxN flap domain proposed to form an interaction network between the substrate, the linker to the ATP cone domain, the GRD, and loop 2 important for substrate specificity. The structures also provided insights into how ATP and dATP bind to the ATP cone domain of Class III RNR. Also, the structures suggested that the ATP cone domain is directly involved in the tetramer formation by forming an interaction with the core domain in the presence of dATP. These observations serve as an important basis for future study on the mechanism of Allosteric regulation of Class III RNR.

2. The authors used a wide range of methodologies including activity assays, nucleotide binding assays, oligomeric state determination, and cryo-EM structural characterization, which were impressive and necessary to understand the complex allosteric regulation of RNR.

3. The activity assays demonstrated the catalytic function of PcNrdD and its ability to be activated by ATP and low-concentration dATP and inhibited by high-concentration dATP.

4. ITC and MST were used to show the ability of PcNrdD to bind NTP and dATP.

5. GEMMA was used successfully to determine the oligomeric state of PcNrdD, which suggested that PcNrdD exists in dimeric and tetrameric forms, whose ratio is affected by ATP and/or dATP.

Weaknesses:<br /> 1. Activity assays.<br /> The activity assays were performed under conditions that may not represent the nucleotide reduction activity. The authors initiated the Gly radical formation and nucleotide reduction simultaneously. The authors also showed that the amount of Gly radical formation was different in the presence of ATP vs dATP. Therefore, it is possible that the observed Vmax is affected by the amount of Gly radical. In fact, some of the data fit poorly into the kinetic model. Also, the number of biological and technical replicates was not described, and no statistical information was provided for the curve fitting.

2. Binding assays.<br /> The interpretation of the binding assays is complicated by the fact that dATP binds both a- and s-sites and ATP binds a- and active sites. dATP may also bind the active site as the product. It is unknown if ATP binds s-site in PcNrdD. Despite this complexity, the binding assays were performed under the condition that all the binding sites were available. Therefore, it is not clear which event these assays are reporting.

3. Oligomeric states.<br /> Due to the ambiguity in the kinetic parameters and the binding constants determined above, the effects of ATP and dATP on the oligomeric states are difficult to interpret. The concentrations of ATP used in these experiments (50 and 100 uM) were significantly lower than KL determined by the activity assays (780 uM), while it is close to the Kd values determined by ITC or MST (~25 uM). Since it is unclear what binding events ITC and MST are reporting, the data in Figure 3 does not provide support for the claimed effects of ATP binding. For the effects of dATP, the authors did not observe a significant difference in oligomeric states between 50 or 100 uM dATP alone vs 50 uM dATP and 100 uM CTP. The former condition has dATP ~ 2x higher than the Kd and KL (Figure 1b) and therefore could be considered as "inhibited". On the other hand, NrdD should be fully active under the latter condition. Therefore, these observations show no correlation between the oligomeric state and the catalytic activity.

4. Effects of dATP binding on GRD structure<br /> One of the key conclusions of this manuscript is that dATP binding induces the dissociation of GRD from the active site. However, the structures did not provide an explanation for how the dATP binding affects the conformation of GRD or whether the dissociation of GRD is a direct consequence of dATP binding or it is due to the absence of nucleotide substrate. Also, Gly radical is unlikely to be stable when it is not protected from the bulk solvent. Therefore, it is unlikely that the GRD dissociates from the active site unless the inhibition by dATP is irreversible. Further evidence is needed to support the proposed mechanism of inhibition by dATP.

5. Functional support for the observed structures.<br /> Evidence for connecting structural observations and mechanistic conclusions is largely missing. For example, the authors proposed that the interactions between the ATP cone domain and the core domain are responsible for tetramer formation. However, no biochemical evidence was provided to support this proposal. Similarly, the functional significance of the interaction through the NxN flap domain was not proved by mutagenesis experiments.

Reviewer #2 (Public Review):

In this work, Xin et al. describe cryo-EM structures of the native and carotenoid-depleted forms of RC-LH from R. castenholzii, attempting to reveal how differences in the carotenoid composition may result in the structural and functional differences in the RC-LH complex. Previously, the authors obtained the nRC-LH structure at 4.1 angstrom resolution. The current work extends the earlier moderate-resolution to a higher resolution (2.8 angstrom), which allowed them to identify 14 additional carotenoid molecules located at the external positions between adjacent LHs. These external carotenoids, together with bacteriochlorophylls, result in an impenetrable LH ring surrounding the RC, leaving only the LH opening shaped by subunit X and c-TM as the pathway for quinone exchange. They further solve the dRC-LH structure at 3.1 angstrom resolution, and find that while nRC-LH binds 15 internal and 14 external carotenoids, dRC-LH contains only five internal carotenoids, as well as a highly mobile c-TM, but no subunit X. Comparing the two types of complexes at both structural and biochemical levels, they show that these structural changes may result in the accelerated quinone exchange in dRC-LH than that in nRC-LH.<br /> The structural data in this work are solid. The cryo-EM structures are well discussed and presented by the authors to highlight the structural features that may arise from carotenoid depletion. The authors also measured the oxidation rate of the auracyanin to characterize the quinone exchange rate. The work carried out by the authors is useful in the understanding of the regulatory role of carotenoids in complex assembly and quinone exchange.

Reviewer #2 (Public Review):

This is a modeled analysis of the impact of disruptions in school-based HPV vaccination due to the COVID-19 pandemic. Different catch-up scenarios were considered, ranging from a rapid catch-up period to no catch-up vaccination, and the impact of these on future HPV-related malignancies was approximated. The approach in this study could shed light on strategies for catch-up vaccination due to disruptions caused by the COVID-19 pandemic.

Strengths:<br /> - Using the context of Australia, which has led the world in vaccination, allows us to consider a best-case scenario for the impact of disruptions in a well-running HPV vaccination program with good population coverage.<br /> - The model accounts for multiple factors, including HPV transmission dynamics, mitigation of disease development by screening

Suggested clarifications:<br /> - It could benefit from fleshing out concepts instead of using parentheses, particularly in the abstract.<br /> - There is space to expand on the results presented in Table 1, including an explanation of Affected cohorts 2008 vs Affected cohorts 2008-2009. It may also be useful to explain this analysis in the methods section.<br /> - Given that Australia is a best-case scenario and other countries have not had the same success in HPV vaccination coverage, in the discussion would it be possible to give a comparison of how these three scenarios would look different in a population with school-based vaccination but lower coverage volume, such that readers could understand how much of the success / failures of each of the three catch-up scenarios? It would be particularly helpful for readers who are not familiar with the modeling tool used in this analysis.

Reviewer #2 (Public Review):

The purpose of this study is unclear from the introduction. Additionally, the methods are incomplete and did not describe how data was collected and analyzed. The results do not describe the sample. Once these are described more clearly, further comments can be made about what the authors were trying to achieve and the impact of the work on the field.

Reviewer #2 (Public Review):

In studying the neural control of action generation there is a presumption that different nodes within a connected neural control circuit contribute differentially to the production of a given gesture. In many cases, these circuits also receive inputs that can bias ongoing motor commands to alter output and therefore the motor gesture itself. Showing the specific role that each of the different areas play in motor control and how inputs might bias motor output is challenging. Taking advantage of a precisely controlled error-correction learning task of adult birdsong, Tian et al. perform simultaneous neural recording in both the primary forebrain song motor output nucleus (RA) as well as in an input structure (LMAN) known to be necessary for biasing motor output during such learning tasks. By comparing the activity pattern and timing between recorded activity in both structures, they show that LMAN activity leads RA activity for each of the song syllables but that there is a preferential gain in activity level in LMAN after learning only during the precise time window (10 - 50 msec) associated with the specific syllable that is targeted during the error-correction paradigm. They then follow these recordings with short focal electrical stimulation in LMAN targeted to the precise time window that shows increased gain in the dual recording paradigm. This stimulation is intended to scramble the bias signal and they show that such manipulation, in a temporally specific manner, does indeed eliminate the acoustic bias imposed by LMAN.

The precise combination of dual recording and targeted stimulation, in my opinion, convincingly shows that LMAN provides a temporally precise command that can bias motor output in RA. It is assumed that LMAN inputs onto RA are mapped with some level of functional topography, especially given that RA is thought to have some degree of motor mapping. The more dorsal areas, for example, likely contribute more to respiratory control while the more ventral portion contributes to acoustic control with a possible acoustic motor map within that region. Unfortunately, the spatial precision of the recording electrodes in both RA as well as LMAN is rather coarse and a careful functional spatial mapping of spike timing correlation is not possible. Hopefully in future studies, more precise spatial mapping will provide correlations within these two structures that might be able to target subareas that encode the signal bias for subcomponents of the specific acoustic features that are being targeted in this error-correction learning paradigm.

Reviewer #2 (Public Review):

This study by Pentz et al. aims to understand how cellular attachments and/or development affect the fitness of the transition to undifferentiated multicellularity. This work has the potential to better understand why some types of multicellular development (e.g. clonal development) versus others (e.g. aggregative development) are more or less commonly observed in nature.

Presently, much of our understanding of these processes comes from observation and theoretical work. This work aims to bridge this gap by rewiring the evolutionary clock and testing if different selected undifferentiated multicellular developmental strategies are better or worse.

The authors compare the fitness of Snowflake and Floc yeast under settling-based selection. They find that Snowflake is fitter under these conditions than Floc. They augment these findings with a simplified mathematical model that supports these findings.

On their face, the findings seem interesting but have limitations in that the authors did not consider alternate selective conditions and may come to different conclusions, potentially supporting the null hypothesis. In addition, doing experiments in related multicellular model systems that the authors have previously worked in would substantially improve generalizability.

Reviewer #2 (Public Review):

This work attempts to connect the diet of a mother to the physiology and feeding behaviors of multiple generations of her offspring. Using genetic and molecular biology approaches in the fruit fly model, the authors argue that this Lamarckian inheritance is mediated by germline-inherited chromatin and is regulated by the general activity of a histone methylase. However, many of the measured effects are small and variable, the statistical tests to prove their significance are missing or poorly described, and some experiments are inadequately described and lack important controls.

1) The authors claim that the diet of a mother can influence the physiology of her progeny for several generations. However, the observed effects of maternal diet on later generations were small and variable for most assays (see Fig1C, S1.1A, B, D). Additionally, the effect size between F0 HSD to ND was often larger than the effect size between the progeny of F0 parents and ND. To put it another way, if the authors were to compare the F1, F2, etc. to the F0 HSD flies, they would conclude that the majority of the response to diet is not maternally transmitted, and is directly controlled by the diet of the individual being measured.<br /> 2) The authors chose to study PER, which had the largest average effect sizes between conditions. However, PER was highly variable in the averaged data, with some individuals showing large effects and others having no effects. A better characterization of transgenerational PER may increase the robustness of this assay and confidence in its results. For example, the authors could measure PER in lineages derived from individual flies to determine when transgenerational effects on PER decline or disappear. This form of data collection could help to explain the high variation in the averaged data presented in the paper.<br /> 3) What do the error bars represent on any figure? There are many examples where the data is highly variable and lies completely outside of the error bars. What is the statistical test for significance that is carried out in each figure? The brief comment about statistics in the methods section is inadequate. The authors should also supply the raw data used to generate the figures so that readers can perform their own statistical tests.<br /> 4) The model that global H3K27me3 is regulated by ancestral diet is unconvincing without further experimental validation and explanation. Points 4-10 address specific issues. The authors performed ChIP on cycle 11 embryos. This stage is extremely short (11 min) and contains roughly 10 times less chromatin than embryos only 30 minutes older. These features make it very difficult to collect large numbers of precisely staged embryos without significant contamination. It is also debatable whether early cell cycles (including and preceding cycle 11) are slow enough to deposit and propagate histone marks in the presence of new histone incorporation. See the opposing arguments in Zenk et al 2017 and Li et al 2014. The authors could perform ChIP on older embryos to avoid this controversy. Surely any maternally inherited information will also be present in cycle 14 or 15 embryos if it is to influence the development or physiology of the brain. The observed differences in global H3K27me3 levels in F1 vs ND flies could be explained by slightly different aged embryo collections or technical variations in the ChIP protocol. The authors could strengthen their conclusion by performing more ChIP replicates. Alternatively, the authors could use orthogonal approaches like antibody staining or western blots to measure global H3K27me3 levels in precisely staged embryos.<br /> 5) The authors measure PRC2 subunit mRNA levels in adult fly heads to attempt to explain the observed differences in inherited H3K27me3 levels in fly embryos. The authors should examine PRC2 components in germ cells and early embryos to understand how germ cells and early embryos generate H3K27me3 patterns.<br /> 6) The RNAi experiment targeting PRC2 components in embryos is uninterpretable without appropriate controls and an explanation of the genotypes used in the experimental paradigm. Are the authors crossing nosNGT mothers to UAS-RNAi fathers and assaying the progeny? What is the genotype of the F1 flies and how does it compare to the genotype of the ND flies? The authors should also note that the Gal4 drivers they use are not necessarily restricted to the ovary, and could directly affect other tissues controlling PER like neurons and muscle. Additionally, the authors should supply the appropriate controls to verify that their experimental paradigm has the intended effect. PRC2 proteins are presumably loaded into embryos and would be immune to zygotic-expressed RNAi. The authors could validate when PRC2 RNAi is effective by staining embryos for H3K27me3.<br /> 7) Although the authors do not note this, nosNGT>RNAi affects the PER of ND flies (compare Gal4>RNAi to just RNAi or just Gal4 in ND columns in Fig3A-D). This could be due to RNAi expression in neurons or muscles or some other indirect effect. Regardless of the mechanism, this result makes it difficult to interpret how RNAi treatments affect the transgenerational inheritance of PER if there is an equivalently strong non-transgenerational effect.<br /> 8) The matalpha gal4 experiment is inadequately explained in the text or methods. Are the authors expressing RNAi in the ovaries of the F0 flies that are fed an HSD? Does the ovary influence their PER somehow? Similar to point 8, there appears to be a non-transgenerational component to the RNAi phenotype that clouds the interpretation of the transgenerational effect (compare F0 in S3.1A-C).<br /> 9) For the EED inhibitor experiments (both PER and calcium imaging), it is unclear whether the authors fed the mothers or their adult progeny the EED inhibitor. If adult progeny were fed, what tissues were affected? The authors should stain various tissues with an H3K27me3 antibody to verify the effectiveness of their inhibitor. Finally, the effect of the EED inhibitor on calcium imaging was not convincing because the variation was so large.<br /> 10) In all of the PRC2 RNAi and inhibitor experiments, are there any other phenotypes that would suggest that the treatments are working? There are many published PRC2 loss-of-function phenotypes (molecular and developmental) in different tissues. The authors could assure the reader that their treatments are working as expected by doing these controls.<br /> 11) The authors propose that a transgenerationally inherited state of the caudal gene is responsible for the transgenerationally inherited PER. However, the experiments investigating the methylation state and expression level of caudal are unconvincing. Cad mRNA abundance varied immensely in the ND RNAseq samples. When the authors compared cad levels across generations, the effect size was small. A single outlier in the ND sample in both the RNAseq and the RTPCR experiments appears to drive up its mean and effect size. The H3K27me3 ChIP on cad is very similar in the F1 and ND samples and the acetylation peak on its promoter appears unchanged. The authors could vastly improve the caudal experiments in this paper by simply using cad antibodies to stain the relevant tissues that contribute to PER. For example, the authors could stain GR5a neurons for cad expression in different generations that inherit (or don't inherit) maternal PER to more accurately determine if cad levels are indeed transgenerationally regulated. The authors could also perform more ChIP experiments at a less variable stage to convincingly correlate epigenetic marks on cad with its expression level.

Reviewer #2 (Public Review):

This work describes the development of a new structure-based learning approach to predict transcription binding specificity and its application in the modeling of regulatory complexes in cis-regulatory modules. The development of accurate computer tools to model protein-DNA complexes and to predict DNA binding specificity is a very relevant research topic with significant impact in many areas.

This article highlights the importance of transcriptional regulatory elements in gene expression regulation and the challenges in understanding their mechanisms. Traditional definitions of activating regulatory elements, such as promoters and enhancers, are becoming unclear, suggesting an updated model based on DNA accessibility and enhancer/promoter potential. Experimental techniques can assess the sequence preferences of transcription factors (TFs) for binding sites. Recent models propose a cooperative model in which regulatory elements work together to increase the local concentrations of TFs, RNA polymerase II, and other co-factors. Co-operative binding can be mediated through protein-protein or DNA interactions. The authors developed a structure-based learning approach to predict TF binding features and model the regulatory complex(es) in cis-regulatory modules, integrating experimental knowledge of structures of TF-DNA complexes and high-throughput TF-DNA interactions. They developed a server to characterize and model the binding specificity of a TF sequence or its structure, which was applied to the examples of interferon-β enhanceosome and the complex of factors SOX11/SOX2 and OCT4 with the nucleosome. The models highlight the co-operativity of TFs and suggest a potential role for nucleosome opening.

The results presented by the authors have a large variability in performance upon the different TF families tested. Therefore, it would be ideal if the performance/accuracy of the method is tested in some simple predictions and validated with prospective experimental data before applying it to model difficult scenarios such as those described here: SOX11/SOX2/OCT4 and nucleosome or interferon beta and enhanceosome. This will give more support to the models generated and thus the validity of the conclusions and hypothesis derived from them.

Reviewer #2 (Public Review):

Toker et al. use a frequency-resolved analysis of cortico-thalamic and thalamo-cortical information transfer to determine at which combinations of frequencies a frequency-specific transfer of information exists, and how this transfer is modulated by anesthesia, spike-and-wave seizures, and psychedelic states. They find that anesthesia and seizures lower the transfer of information at a specific combination of frequencies (sending: 1.5-13Hz, receiving 50-100Hz), whereas psychedelic states induced by 5-MeO-DMT increase. The reductions were observed for both directions whereas significant increases were only observed from cortex to thalamus.

Neural mean-field modeling shows that these empirical observations may be linked to a deviation of neural dynamics from the critical point between ordered and chaotic dynamics.

The manuscript tackles an important question using innovative methods. Yet, the analysis of spectrally resolved information transfer at present suffers from an unfortunate choice of analysis parameters (especially a history length of 1, and a low number of surrogate data), that need to be changed to fully install trust in the presented results. The statistical analysis seems to suffer from so-called 'double-dipping', but there are several possible ways to fix this issue.

Reviewer #2 (Public Review):

Davies et al combine TurboID with conditional mutagenesis to reveal how a perturbing event alters the accessibility of a sub-cellular proteome to proximity biotinylation. The approach builds on established techniques for antibody-mediated enrichment of biotinylated peptides (rather than purification of whole biotinylated proteins by avidin) to enable mapping of the specific lysines that are biotinylated by TurboID and how access to these sites changes between conditions. The insights gained have a range of potential implications touching on protein trafficking/localization, complex dynamics and membrane topology. The authors apply this strategy to study trafficking of the key P. falciparum adhesin PfEMP1 to the infected erythrocyte surface. This group has previously shown that the exported parasite kinase FIKK4.1 is important for this process but the specific mechanism is unknown. In the first part of the present study, the authors develop PerTurboID and analyze the altered biotinylation patterns upon FIKK4.1 deletion in parasite lines bearing TurboID tags on PTP4 or KAHRP, two proteins required for this pathway and likely direct substrates of FIKK4.1. Numerous changes in site-specific biotinylation are quantitatively assessed on hundreds of proteins and possible implications for these changes are discussed, including topology of parasite integral membrane proteins exported into the RBC compartment as well as how the conformation of the RhopH complex might be altered upon RBC membrane integration. In a final set of experiments, the authors show that among 18 exported FIKK kinases, FIKK4.1 is uniquely important to PfEMP1 surface display but not to the distinct RIFIN class of parasite proteins that are also trafficked to the RBC surface. On the whole, the data are compelling and provide an important new approach that advances the proximity labeling toolkit.

While the resolution of PerTurboID captures the site-specific changes in biotinylation abundance and position that occur upon loss of FIKK4.1, a limitation of the study is that these observations do not necessarily clarify the model for how FIKK4.1 is controlling the PfEMP1 trafficking pathway. The authors convincingly show that FIKK4.1 uniquely supports PfEMP1 surface presentation and cytoadhesion. However, this is not connected to the PerTurboID data in a way that provides a mechanism for how this is achieved by FIKK4.1 activity and in my opinion doesn't deliver on the title claim to "reveal the impact of kinase deletion on cytoadhesion". Certainly the changes in biotinylation suggest a range of interesting possibilities related to the accessibility and topology of proteins within and beyond the PfEMP1 trafficking pathway; however, it is hard to interpret the relationship of these changes to the process in view. For instance, deletion of FIKK4.1 increases biotinylation of several Maurer's clefts proteins in both the PTP4- and KAHRP-TurboID experiments but why this is or whether it is significant for PfEMP1 transport is unclear.

Reviewer #2 (Public Review):

This study follows up on a previous study by the group (Sibille et al Nature Communications 2022) in which high density Neuropixel probes were inserted tangentially through the superficial layers of the superior colliculus (SC) to record the activity of retinocollicular axons and postsynaptic collicular neurons in anesthetized mice. By correlating spike patterns, connected pairs could be identified which allowed the authors to demonstrate that functionally similar retinal axon-SC neuron pairs were strongly connected.

In the current study, the authors use similar techniques in vGAT-ChR2 mice and add a fiber optic to identify light-activated GABAergic and non-light-activated nonGABAergic neurons. Using their previously verified techniques to identify connected pairs, within regions of optogenetic activation they identified 214 connected pairs of retinal axons and nonGABAergic neurons and 91 pairs of connected retinal axons and GABAergic neurons. The main conclusion is that retinal activity contributed more to the activity of postsynaptic nonGABAergic SC neurons than to the activity of postsynaptic GABAergic SC neurons.

The study is very well done. The figures are well laid out and clearly establish the conclusions. My main comments are related to the comparison to other circuits and further questions that might be addressed in the SC.

It is stated several times that the superior colliculus and the visual cortex are the two major brain areas for visual processing and these areas are compared throughout the manuscript. However, since both the dorsal lateral geniculate nucleus (dLGN) and SC include similar synaptic motifs, including triadic arrangements of retinal boutons with GABAergic and nonGABAergic neurons, it might be more relevant to compare and contrast retinal convergence and other features in these structures.

The GABAergic and nonGABAergic neurons showed a wide range of firing rates. It might be interesting to sort the cells by firing rates to see if they exhibit different properties. For example, since the SC contains both GABAergic interneurons and projection neurons it would be interesting to examine whether GABAergic neurons with higher firing rates exhibit narrower spikes, similar to cortical fast spiking interneurons. Similarly, it might be of interest to sort the neurons by their receptive field sizes since this is associated with different SC neuron types.

The recording techniques allowed for the identification of the distance between connected retinocollicular fibers and postsynaptic neurons. It might also be interesting to compare the properties of connected pairs recorded at dorsal versus ventral locations since neurons with different genetic identities and response properties are located in different dorsal/ventral locations (e.g. Liu et al. Neuron 2023). Also, regarding the strength of connections, previous electron microscopy studies have shown that the retinocollicular terminals differ in density and size in the dorsal/ventral dimension (e.g Carter et al JCN 1991).

Was optogenetic activation of GABAergic neurons ever paired with visual activation? It would be interesting to examine the receptive fields of the nonGABAergic neurons before and after activation of the GABAergic neurons (as in Gale and Murphy J Neurosci 2016).

Reviewer #2 (Public Review):

The authors compare their single-cell data of the self-forming brain-eye centroids with the published single-cell data from human fetal retinas and brain/optic organoids. This analysis further supports the similarity of their centroids with the human fetal retinal cell clusters, including the detection of the VSX2+/PAX2+ cells. The new findings further support the presented centroids' applicability for future studies on human RGC development and axon guidance mechanisms.

Reviewer #2 (Public Review):

The authors wanted to address the differential processing of GSDME by caspase 3 and 7, finding that while in humans GSDME is only processed by CASP3, Takifugu GSDME, and other mammalian can be processed by CASP3 and 7. This is due to a change in a residue in the human CAPS7 active site that abrogates GSDME cleavage. This phenomenon is present in humans and other primates, but not in other mammals such as cats or rodents. This study sheds light on the evolutionary changes inside CASP7, using sequences from different species. Although the study is somehow interesting and elegantly provides strong evidence of this observation, it lacks the physiological relevance of this finding, i.e. on human side, mouse side, and fish what are the consequences of CASP3/7 vs CASP3 cleavage of GSDME.

Fish also present a duplication of GSDME gene and Takifugu present GSDMEa and GSDMEb. It is not clear in the whole study if when referring to TrGSDME is the a or b. This should be stated in the text and discussed in the differential function of both GSDME in fish physiology (i.e. PMIDs: 34252476, 32111733 or 36685536).

Reviewer #2 (Public Review):

In this study, Abele et al. present evidence to suggest that two different forms of regulated cell death, pyroptosis and apoptosis, are not equivalent in their ability to clear infection with recombinant Salmonella strains engineered to express the pro-pyroptotic NLRC4 agonist, FliC ("FliC-ON"), or the pro-apoptotic protein, BID ("BID-ON"). In general, individual experiments are well-controlled, and most conclusions are justified. However, the cohesion between different types of experiments could be strengthened and the overall impact and significance of the study could be articulated better.

Reviewer #2 (Public Review):

This work started with transcriptomic profiling of ductal cells to identify the upregulation of calcineurin in the zebrafish after beta-cell ablation. By suppressing calcineurin with its chemical inhibitor cyclosporin A and expressing a constitutively active form of calcineurin ubiquitously or specifically in ductal cells, the authors found that inhibited calcineurin activity promoted beta-cell regeneration transiently while ectopic calcineurin activity hindered beta-cell regeneration in the pancreatic tail. They also showed similar effects in the basal state but only when it was within a particular permissive window of Notch activity. To further investigate the roles of calcineurin in the ductal cells, the authors demonstrated that calcineurin inhibition additionally induced the proliferation of the ductal cells in the regenerative context or under a limited level of Notch activity. Interestingly, the enhanced proliferation was followed by a depletion of ductal cells, suggesting that calcineurin inhibition would exhaust the ductal cells. Based on the data, the authors proposed a very attractive and intriguing model of the role of calcineurin in maintaining the balance of the progenitor proliferation and the endocrine differentiation. However, the conclusions of this paper are only partially supported by the data as some evidence from the data remains suggestive.

1. In the transcriptomic profiling, genes differentially regulated in the ablated adults could be solely due to the chemical effects of metronidazole instead of the beta-cell ablation. A control group without ins:NTR-mCherry but treated with metronidazole is necessary to exclude the side effects of metronidazole.

2. Although it has been shown that the pancreatic duct is a major source of the secondary islets in the pancreatic tail in previous studies, there is no direct evidence showing the cyclosporin A-induced cells share the source in this manuscript. Without any proper lineage tracing work, the origin of those cyclosporin A-induced cells cannot be concluded.

3. It is interesting to see an increase of beta cells in the primary islet after cyclosporin A treatment (Supplemental Fig 2B). However, it remains unclear if their formation shares the same mechanism with the newly formed beta cells in the pancreatic tail.

4. The conclusion of the effect of cyclosporin A on the endocrine progenitors (Line 175) is not convincing because the data cannot distinguish the endocrine progenitors from the insulin-expressing cells. Indeed, Figure 2E shows that neurod1+ cells are fewer than ins+ cells (Figure 2D) in the pancreatic tail at 10 dpt, suggesting that all or at least the majority of neurod1+ cells are already ins+.

5. Figure 5D shows a significant loss of nkx6.1+ cells in the combined treatment group but there is no direct evidence showing this was a result of differentiation as the authors suggested. This cell loss also outnumbered the increase in ins+ cells (Figure 4D). The cell fates of these lost cells are still undetermined, and the authors did not demonstrate if apoptosis could be a reason of the cell loss.

Reviewer #2 (Public Review):

Novelty: The concept that capillary stalls occur in the ischemic penumbra is not new. However, there are several interesting findings in the current study.<br /> 1- Flow reversal, 2- the effect of flow disturbances on oxygenation, and 3- capillary pericytes do not affect the hemodynamics in the penumbra.<br /> However, more in-depth analysis is needed and the underlying mechanism of flow reversal and the link between flow reversal and pericytes is unclear.

Strengths:<br /> 1. The study employs a combination of techniques including Laser speckle imaging, two photon microscopy and biophysical modelling to specifically examine hemodynamic and metabolic changes in the penumbra following experimental stroke.<br /> 2. The importance of following microvascular flow changes during hours after stroke.<br /> 3. The authors used a rat model of stroke and confirmed previous work that has been performed in mice about capillary stalls and flow disturbance in the ischemic penumbra.

Weaknesses:<br /> 1- The reliance on laser speckle to define the ischemic core and penumbra is not convincing.<br /> 2- The mechanisms behind microvascular flow disturbance are poorly defined.<br /> 3- The inability to measure capillary flow simultaneously in the regions of interest: e.g, Bessel beam imaging or volumetric imaging.<br /> 4- Lack of baseline measurements.

Reviewer #2 (Public Review):

In the manuscript by Salmani et al., the authors explore the transcriptomic characterization of dopamine neurons in order to explore which neurons are particularly vulnerable to 6-OHDA-induced toxicity. To do this they perform single nucleus RNA sequencing of a large number of cells in the mouse midbrain in control animals and those exposed to 6-OHDA. This manuscript provides a detailed atlas of the transcriptome of various types of ventral midbrain cells - though the focus here is on dopaminergic cells, the data can be mined by other groups interested in other cell types as well. The results in terms of cell type classification are largely consistent with previous studies, though a more nuanced picture of cellular subtypes is portrayed here, a unique advantage of the large dataset obtained. The major advance here is exploring the transcriptional profile in the ventral midbrain of animals treated with 6-OHDA, highlighting potential candidate genes that may influence vulnerability. This approach could be generalizable to investigate how various experiences and insults alter unique cell subtypes in the midbrain, providing valuable information about how these stimuli impact DA cell biology and which cells may be the most strongly affected.

Overall, the manuscript is relatively heavy on characterization and comparatively light on functional interpretation of findings. This limits the impact of the proposed work. It also isn't clear what the vulnerability factors may be in the neurons that die. Beyond the characterization of which neurons die - what is the reason that these neurons are susceptible to lesion? Also, the interpretation of these findings is going to be limited by the fact that 6-OHDA is an injectable, and the effects depend on the accuracy of injection targeting and the equal access of the toxin to access all cell populations. Though the site of injection (MFB) should hit most/all of the forebrain-projecting DA cells, the injection sites for each animal were not characterized (and since the cells from animals were pooled, the effects of injection targeting on the group data would be hard to determine in any case).

I am also not clear why the authors don't explore more about what the genes/pathways are that differentiate these conditions and why some cells are particularly vulnerable or resilient. For example, one could run GO analyses, weighted gene co-expression network analysis, or any one of a number of analysis packages to highlight which genes/pathways may give rise to vulnerability or resilience. Since the manuscript is focused on identifying cells and gene expression profiles that define vulnerability and resilience, there is much more that could have been done with this based on the data that the authors collected.

Another limitation of this study as presented is the missed opportunity to integrate it with the rich literature on midbrain dopamine (and non-dopamine) neuron subtypes. Many subtypes have been explored, with divergent functions, and can usually be distinguished by either their projection site, neurotransmitter identity, or both. Unfortunately, the projection site does not seem to track particularly well with transcriptomic identities, aside from a few genes such as DAT or the DRD2 receptor. However, this could have been more thoroughly explored in this manuscript, either by introducing AAVretro barcodes through injection into downstream brain sites, or through existing evidence within their sequencing dataset. There are likely clear interpretations from some of that literature, some of which may be more exciting than others. For example, the authors note that vGluT2-expressing cells were part of the resilient territory. This might be because this is expressed in medially-located DA cells and not laterally-located ones, which tends to track which cells die and which don't.

It is not immediately clear why the authors used a relaxed gate for mCherry fluorescence in Figure 1. This makes it difficult to definitively isolate dopaminergic neurons - or at least, neurons with a DAT-Cre expression history. While the expression of TH/DAT should be able to give a fairly reliable identification of these cells, the reason for this decision is not made clear in the text.

Reviewer #2 (Public Review):

In their study, Zaman et al. demonstrate that deletion of either the receptor tyrosine kinase Kit from cerebellar interneurons or the kit ligand (KL) from Purkinje cells reduces the inhibition of Purkinje cells. They delete Kit or KL at different developmental time points, illustrating that Kit-KL interactions are not only required for developmental synapse formation but also for synapse maintenance in adult animals. The study is interesting as it highlights a molecular mechanism for the formation of inhibitory synapses in Purkinje cells.

The tools generated, such as the floxed Kit mouse line and the virus for Kit overexpression, may have broader applications in neuroscience and beyond.

However, to enhance the publication's impact and strengthen its hypotheses, conclusions, and scientific rigor, it would be beneficial to include additional experimental details, data analyses (particularly regarding the quantification of electrophysiology data), as well as methodological and textual clarifications.

One general weakness is that Kit expression is not limited to molecular layer interneurons but also extends to the Purkinje layer and Golgi interneurons. Although this expression may not conflict with the reported results, as Purkinje layer interneurons form few or no synapses onto Purkinje cells, it should be highlighted in the text (introduction and/or discussion).

In summary, the data support the hypothesis that the interaction between Kit and KL between cerebellar Molecular Layer Interneurons and Purkinje Cells plays a crucial role in promoting the formation and maintenance of inhibitory synapses onto PCs. This study provides valuable insights that could inform future investigations on how this mechanism contributes to the dynamic regulation of Purkinje cell inhibition across development and its impact on mouse behavior.

Reviewer #2 (Public Review):

This paper takes a novel look at the protein economy of primary human and mouse T-cells - in both resting and activated state. Their findings in primary human T-cells are that:

1. A large fraction of ribosomes are stalled in resting cultured primary human lymphocytes, and these stalled ribosomes are likely to be monosomes.<br /> 2. Elongation occurs at similar rates for HeLa cells and lymphocytes, with the active ribosomes in resting lymphocytes translating at a similar rate as fully activated lymphocytes.

They then turn their attention to mouse OT-1 lymphocytes, looking at translation rates both in vitro and in vivo. Day 1 resting T-cells also show stalling - which curiously wasn't seen on freshly purified cells - I didn't understand these differences.

In vivo, they show that it is possible to monitor accurate translation and measure rates. Perhaps most interestingly they note a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner.

This was an interesting and provocative paper. Lots of interesting techniques and throwing down challenges to the community - it manages to address a number of important issues without necessarily providing answers.

Reviewer #2 (Public Review):

The authors introduce the notions of "variant vulnerability" and "drug applicability" as metrics quantifying the sensitivity of a given target variant across a panel of drugs and the effectiveness of a drug across variants, respectively. Given a data set comprising a measure of drug effect (such as growth rate suppression) for pairs of variants and drugs, the vulnerability of a variant is obtained by averaging this measure across drugs, whereas the applicability of a drug is obtained by averaging the measure across variants.

The authors apply the methodology to a data set that was published by Mira et al. in 2015. The data consist of growth rate measurements for a combinatorially complete set of 16 genetic variants of the antibiotic resistance enzyme beta-lactamase across 10 drugs and drug combinations at 3 different drug concentrations, comprising a total of 30 different environmental conditions. For reasons that did not become clear to me, the present authors select only 7 out of 30 environments for their analysis. In particular, for each chosen drug or drug combination, they choose the data set corresponding to the highest drug concentration. As a consequence, they cannot assess to what extent their metrics depend on drug concentration. This is a major concern since Mira et al. concluded in their study that the differences between growth rate landscapes measured at different concentrations were comparable to the differences between drugs. If the new metrics display a significant dependence on drug concentration, this would considerably limit their usefulness.

As a consequence of the small number of variant-drug combinations that are used, the conclusions that the authors draw from their analysis are mostly tentative with weak statistical support. For example, the authors argue that drug combinations tend to have higher drug applicability than single drugs, because a drug combination ranks highest in their panel of 7. However, the effect profile of the single drug cefprozil is almost indistinguishable from that of the top-ranking combination, and the second drug combination in the data set ranks only 5th out of 7.

To assess the environment-dependent epistasis among the genetic mutations comprising the variants under study, the authors decompose the data of Mira et al. into epistatic interactions of different orders. This part of the analysis is incomplete in two ways. First, in their study, Mira et al. pointed out that a fairly large fraction of the fitness differences between variants that they measured were not statistically significant, which means that the resulting fitness landscapes have large statistical uncertainties. These uncertainties should be reflected in the results of the interaction analysis in Figure 4 of the present manuscript. Second, the interpretation of the coefficients obtained from the epistatic decomposition depends strongly on the formalism that is being used (in the jargon of the field, either a Fourier or a Taylor analysis can be applied to fitness landscape data). The authors need to specify which formalism they have employed and phrase their interpretations accordingly.

Reviewer #2 (Public Review):

Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system where immune cells play an important role in disease pathobiology. Increased incidence of disease in individuals carrying certain HLA class-II genes plus studies in animal models suggests that HLA-DRB1*15 restricted CD4 T cells might be responsible for disease initiation, and other immune cells such as B cells, CD8 T cells, monocytes/macrophages, and dendritic cells (DC) also contribute to disease pathology. However, a direct role of human immune cells in disease is lacking to a lag between immune activation and the first sign of clinical disease. Therefore, there is an emphasis on understanding whether immune cells from HLA-DR15+ MS patients differ from HLA-DR15+ healthy controls in their phenotype and pro-inflammatory capacity. To overcome this, authors have used severely immunodeficient B2m-NOG mice that lack B, T cells, and NK cells and have defective innate immune responses and engrafted PBMCs from 3 human donors (HLA-DR15+ MS and HI donors, HLA-DR13+ MS donor) in these B2m-NOG mice to determine whether they can induce CNS inflammation and demyelination like MS.<br /> The study's strength is the use of PBMCs from HLADRB1-typed MS subjects and healthy control, the use of NOG mice, the characterization of immune subsets (revealing some interesting observations), CNS pathology etc. The major weaknesses are i) lack of sufficient sample size (n=1 in each group) to make any conclusion, ii) lack of phenotype in mice, iii) no disease phenotype even in humanized mice immunized for disease using standard disease induction protocol employed in an animal model of MS, and iv) mechanistic data on why CD8 T cells are more enriched than CD4+ T cells. The last point is very important as postmortem human MS patients' brain tissue had been shown to have more CD8+ T cells than CD4+ T cells.

Thus, this work is an important step in the right direction as previous humanized studies have not used HLA-DRB1 typed PBMCs however the weaknesses as highlighted above make the findings incremental to the field.

Reviewer #2 (Public Review):

Place cells fire sequentially during hippocampal theta oscillations, forming a spatial representation of behavioral experiences in a temporally-compressed manner. The firing sequences during theta cycles are widely considered as essential assemblies for learning, memory, and planning. Many theoretical studies have investigated the mechanism of hippocampal theta firing sequences; however, they are either entirely extrinsic or intrinsic. In other words, they attribute the theta sequences to external sensorimotor drives or focus exclusively on the inherent firing patterns facilitated by the recurrent network architectures. Both types of theories are inadequate for explaining the complexity of the phenomena, particularly considering the observations in a previous paper by the authors: theta sequences independent of animal movement trajectories may occur simultaneously with sensorimotor inputs (Yiu et al., 2022).

In this manuscript, the authors concentrate on the CA3 area of the hippocampus and develop a model that accounts for both mechanisms. Specifically, the model generates extrinsic sequences through the short-term facilitation of CA3 cell activities, and intrinsic sequences via recurrent projections from the dentate gyrus. The model demonstrates how the phase precession of place cells in theta sequences is modulated by running direction and the recurrent DG-CA3 network architecture. To evaluate the extent to which firing sequences are induced by sensorimotor inputs and recurrent network architecture, the authors use the Pearson correlation coefficient to measure the "intrinsicity" and "extrinsicity" of spike pairs in their simulations.

I find this research topic to be both important and interesting, and I appreciate the clarity of the paper. The idea of combining intrinsic and extrinsic mechanisms for theta sequences is novel, and the model effectively incorporates two crucial phenomena: phase precession and directionality of theta sequences. I particularly commend the authors' efforts to integrate previous theories into their model and conduct a systematic comparison. This is exactly what our community needs: not only the development of new models, but also understanding the critical relationships between different models.

Reviewer #2 (Public Review):

The manuscript by Elfstrom et al describes the impact of implementing self-sampling as the primary screening test in Sweden to address decreases in coverage following the COVID pandemic. The authors have a very rich dataset including all records of invitations to screen and screening results in the Stockholm area. A limitation is that there is no individual record linkage to allow investigation of the profile of the individuals who chose to screen using the self-sample.

The conclusions are generally well supported by the authors with the following exceptions:

1) There was not enough evidence presented in the manuscript to conclude 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."

2) The authors state there is no evidence that delays in screening have impacted cervical cancer rates however they present no data to this effect in the manuscript.

Reviewer #2 (Public Review):

In this article, Moses and Harel present genetic knock-out and partial rescue of the phenotypes of neuropeptides gh1 and fshb, and tshb in a short-lived vertebrate African turquoise killifish Nothobranchius furzeri. Neuropeptides are among the key regulators of growth, reproduction, and metabolism. Understanding their mechanisms of action has important implications for vertebrate physiology.

The authors first characterize the loss of function phenotypes of gh1, fshb, and tshb in killifish, followed by attempts to rescue the loss of function phenotypes through ectopic expression of two of the neuropeptides. The primary strength and innovation of this work are partially rescuing the phenotypes by muscular injection of plasmids followed by electroporation, including a doxycycline-inducible system for tunable expression control. The techniques for tunable expression control and rescue of knock-out phenotypes have not been established for killifish and will be useful to expand the technical repertoire of this emerging model organism. Once established, these techniques can be extended to other categories of genes to rapidly evaluate their function and the impact of their loss or gain of function on killifish and other fish models.

However, the phenotypes discussed need further characterization, many technical details are unclear, and it seems that appropriate controls are missing for some of the experiments. The rescued phenotypes also need more validation.

Reviewer #2 (Public Review):

This article focuses on drug resistance acquired by Plasmodium falciparum malaria parasites that have been pressured with different inhibitors of the essential enzyme DHODH (dihydroorotate dehydrogenase). The study focuses on collateral sensitivity between DSM265, which has been evaluated in a human clinical trial and found to select for resistance via the point mutation C276Y (C276F and G181S were also implicated; PMID 29909069), and the GSK compound TMCDC-125334, against which a panel of DHODH mutant parasites (including C276Y) were found to have increased sensitivity. The authors herein explore this case of "collateral sensitivity" by examining whether these two inhibitors, when used simultaneously, might preclude the selection of resistant parasites. The answer, in this case, is no; collateral sensitivity did not prevent parasites from acquiring a novel mutation (V532A) that mediated resistance to both. Culture competition assays provide evidence that this mutant retains normal fitness. The authors conclude that for this target the idea of combining these inhibitors is not a viable therapeutic strategy. The authors also illustrate how TMCDC-125334 can select for resistance via a separate mutation (I263S) or amplification of a chromosomal segment containing dhodh. They also present modeling data to examine binding poses and how mutations could impact drug binding, which is allosteric to the enzyme's substrates (orotate and FMN). The data are thorough and provide convincing evidence that in this case collateral sensitization by distinct chemotypes does not translate into a viable strategy to inhibit DHODH in a way that can preclude mutations that confer cross-resistance.

Reviewer #2 (Public Review):

In their recent manuscript, Broca-Brisson et al. deliver a multidisciplinary approach to investigate creatine transporter deficiency (CTD) using human-derived brain organoids. The authors have provided a compelling CTD human brain organoid model using induced pluripotent stem cells (iPSCs) derived from individuals with CTD. This model shows distinct differences in creatine uptake between organoids originating from CTD patients and their healthy counterparts. Furthermore, the researchers effectively restored creatine uptake by reintroducing the wild-type CRT in the iPSCs.

The team used advanced molecular biology techniques and sophisticated mass spectrometry to identify changes in protein regulation within these CTD brain organoids. They propose an intriguing theory linking reduced creatine uptake to abnormalities in the GSK3β kinase pathway and mitochondrial function, which might underlie intellectual disability seen in CTD patients.<br /> This study is well-structured and easy to follow, with clear and concise explanations of the experiments. The authors present an important idea: a dysfunction in just one protein transporter (CRT) can cause significant biochemical changes in the brain. Their findings are well-presented and backed by high-quality figures and comprehensive data analysis.

There are only minor suggestions for improvement in this manuscript. The authors strongly link creatine uptake, the GSK3β pathway, and intellectual disability. Enhancing this claim with data on phosphorylation differences between organoids derived from healthy individuals and those from CTD patients could solidify this foundation and facilitate a more holistic understanding of the disease. In addition, the in vitro model based on organoids might be closer than other experimental setups; however, proving that those differences are also present in vivo would greatly benefit the story.

There is also some uncertainty around the rescue experiment using the exogenous SLC6A8 gene. Could the difference in creatine uptake between the rescue iPSCs and the healthy control be due to CRT overexpression? Higher levels of the transporter may explain the elevated levels of intracellular creatine. Thus, a comparison using Western blotting experiments could be a valuable addition to evaluating the expression levels of this protein.

Overall, this study provides valuable insights into CTD and potential therapeutic targets. It enriches our understanding of CTD and opens up new avenues for future research in this field.

Reviewer #2 (Public Review):

The authors used cutting-edge bio-telemetry technology to decipher the roles of wind speed and wave height on the take-off of albatrosses from the water surface. They revealed that each of these factors contributes to take-off in a unique way with interesting interactions of the two factors. The authors achieved their objectives and their results support their conclusions. This work will set new standards in integrating information about bird movement and environmental conditions experienced by the bird in a comprehensive, integrative and hypothesis-driven framework. The approach of the authors is highly advanced, providing heuristic insights for many additional systems where organisms are influenced by, and respond to small-scale environmental conditions.

Reviewer #2 (Public Review):

The manuscript by Seah and Saranathan investigates the cell-based growth mechanism of so called honeycomb-structures in the upper lamina of papilionid wing scales by investigating a number of different species. The authors chose Parides eurimedes as a focus species with the developmental pathway of five other papilionid as a comparative backup. Through state-of-the-art microscopy images of different developmental steps, the author find that the intricate f-actin filaments reorganise, support cuticular discs that template the air holes that form the honeycomb lattice. The manuscript is well written and easy to follow, yet based on a somewhat limited sample size for their focus species, limiting attempts to suppress expression and alter structure shape.

The fact that the authors find a novel reorganisation mechanism is exciting and warrants further research, e.g. into the formation of other microscale features or smaller scale structures (e.g. the mentioned gyroid networks).<br /> The authors place their results in the discussion in the light of current literature (although the references could be expanded further to include the breadth of the field). However, the mechanistic explanation completely ignores the mechanical properties of the membranes as an origin of some of the observed phenomena (see McDougal's work for example) and places the occurence of some features into Turing patterns and Ostwald ripening, which I find somewhat unlikely and I suggest that the authors discover this aspects further in the discussion.

I have little concerns regarding the experimental approach beyond the somewhat limited sample size. One thing the authors should more clearly mention are the pupation periods for all investigated species as only the periods for two species are named.