Reviewer #1 (Public Review):

This paper describes the role of WRNIP1 AAA+ ATPase, particularly its UBZ domain for ubiquitin-binding, but not ATPase, to prevent the formation of the R-loop when DNA replication is mildly perturbated. By combining cytological analysis for DNA damage, R-loop and chromosome aberration with the proximity ligation assay for colocalization of various proteins involved in DNA replication and transcription, the authors provide solid evidence to support the claim. The authors also revealed a distinct role of WRNIP1 in the prevention of R-loop-induced DNA damage from FANCD2, which is inconsistent with the known relationship between WRNIP1 and FANCD2 in the repair of crosslinks.

Reviewer #2 (Public Review):

This paper aims at establishing the role of WRN-interacting protein 1 (WRNIP1) and its UBZ domain (an N-terminal ubiquitin-binding zinc finger domain) on genome instability caused by mild inhibition of DNA synthesis by aphidicolin. The authors used human MRC5 fibroblasts investigated with standard methods in the field. The results clearly showed that WRNIP1 silencing and UBZ-mutation (D37A) increased DNA damage, chromosome aberrations, and transcription-replication conflicts caused by aphidicolin.

The conclusions of the paper are overall well supported by results, however, aspects of some data analyses would need to be clarified and/or extended.

(1) The methods (immunofluorescence microscopy and dot-blots) to determine R-loop levels can lack sensitivity and specificity. In particular, since the S9.6 antibody can bind to other structures besides heteroduplex, dot-blot analyses only grossly assess R-loop levels in cellular samples of purified nucleic acids, which are constituted by many different types of DNA/RNA structures.

(2) Experimental plan has analyzed the impact of WRNIP1 lack or mutations at steady-state conditions. Thus, the possible role of WRNIP1 at an early step of the mechanism would require some sort of kinetics analysis of the molecular process, therefore not at steady-state conditions. The findings of a co-localization of R-loops and WRNIP1 have been obtained with the S9.6 antibody, which recognizes DNA-RNA heteroduplexes. Since WRNIP1 is known to be recruited at stalled forks and DNA cleavage sites, it is not surprising that WRNIP1 is very close to heteroduplexes, abundant structures at replication forks and cleavage sites. Similar interpretations may also be valid for Rad51/S9.6 co-localization findings.

(3) Determination of DNA damage, chromosome aberration, and co-localization data are reported as means of measurements with appropriate statistics. However, the fold-change values relative to corresponding untreated samples are not reported. In some instances, it seems that WRNIP1 silencing or mutations actually reduce or do not affect aphidicolin effects. That leaves open the interpretation of specific results.

Reviewer #1 (Public Review):

In chicken embryos, the counter-rotating migration of epiblast cells on both sides of the forming primitive streak (PS), a process referred to as polonaise movements, has attracted longstanding interest as a paradigm of morphogenetic cell movements. However, the association between these cell movements and PS development is still controversial. This study investigated PS development and polonaise movements separately at their initial stage, showing that both could be uncoupled (at least at the initial phase), being activated via Vg1 signaling.

Strengths of this study

Polonaise movements, i.e., the circular cell migration of epiblast cells on both sides of the forming PS in avian embryos, have been the subject of research through live imaging and promoted the development of new tools to analyze quantitatively such movements. However, conclusions from previous studies remain controversial, at least partly due to the nature of perturbations to PS development and polonaise movements.

This study performed the challenging technique of electroporation to successfully mark and manipulate Wnt/PCP pathways in unincubated chicken embryo cells at the initiation phase of these two processes. In addition, the authors separately altered PS development and polonaise movements: PS development was perturbed by inhibiting either the Wnt/PCP pathway or DNA synthesis using aphidicolin, while polonaise movements were modified by the development of a second PS after engrafting Vg1-expressing COS cells located at the opposite end of the blastoderm. The study concluded that Vg1 elicits both PS development and polonaise movements, which occur in a parallel and are not inter-dependent.

To support these conclusions, particle image velocimetry (PIV) of cell trajectories captured by live imaging was performed. These tools delineated visually appealing cell movements and gave rise to vorticity profiles, adding more value to this study.

Weaknesses of this study

Engrafted Vg1-expressing COS cells located at the anterior end of the blastoderm elicited both the development of a second PS and marked bilateral polonaise movements while perturbing these movements along the original PS. How do polonaise movements along the second PS dominate over those along the normal PS? The authors suggested a model in which Vg1 acts in a graded or dose-dependent manner since engrafted COS cells over-expressed Vg1. This model can be tested by reducing the mass of engrafted COS cells. Although the authors propose performing this analysis in further investigations, it would be preferable to incorporate into this study for better consistency.

Thank you for indicating that this will be a focus of future studies.

Reviewer #1 (Public Review):

As a pathogen, S. aureus has evolved strategies to evade the host's immune system. It effectively remains 'under the radar' in the host until it reaches high population densities, at which point it triggers virulence mechanisms, enabling it to spread within the host. The agr quorum sensing system is central to this process, as it coordinates the pathogen's virulence in response to its cell density.

In this study, Podkowik and colleagues suggest that cells activating agr signaling also benefit from protection against H2O2 stress, whereas inactivation of agr increases cell death. The underlying cause of this lack of protection is tied to an ATP deficit in the agr mutant, leading to increased glucose consumption and NADH production, ultimately resulting in a redox imbalance. In response to this imbalance, the agr mutant increases respiration, resulting in the endogenous production of ROS which synergizes with H2O2 to mediate killing of the agr mutant. Suppressing respiration in the agr mutant restored protection against H2O2 stress.

Additionally, the authors establish that agr-dependent protection against oxidative stress is also linked to RNAIII activation, and the subsequent block of Rot translation. However, the specific protective genes regulated by Rot remain unidentified. Thus, according to the evidence provided, agr triggers intrinsic mechanisms that not only decrease harmful ROS production within the cell but also alleviate its detrimental effects.

Interestingly, these protective mechanisms are long-lived, and guard the cells against external oxidative stressors such as H2O2, even after the agr system has been 'turned off' in the population.

While the study offers valuable insight into how agr signaling protects cells against H2O2 stress, a reevaluation of the interpretation of redox imbalance is warranted.

Reviewer #2 (Public Review):

In their study, Podkowik et al. elucidate the protective role of the accessory gene regulator (agr) system in Staphylococcus aureus against hydrogen peroxide (H2O2) stress. Their findings demonstrate that agr safeguards the bacterium by controlling the accumulation of reactive oxygen species (ROS), independent of agr activation kinetics. This protection is facilitated through a regulatory interaction between RNAIII and Rot, impacting virulence factor production and metabolism, thereby influencing ROS levels. Notably, the study highlights the remarkable adaptive capabilities of S. aureus conferred by agr. The protective effects of agr extend beyond the peak of agr transcription at high cell density, persisting even during the early log-phase. This indicates the significance of agr-mediated protection throughout the infection process. The absence of agr has profound consequences, as observed by the upregulation of respiration and fermentation genes, leading to increased ROS generation and subsequent cellular demise. Interestingly, the study also reveals divergent effects of agr deficiency on susceptibility to hydrogen peroxide compared to ciprofloxacin. While agr deficiency heightens vulnerability to H2O2, it also upregulates the expression of bsaA, countering the endogenous ROS induced by ciprofloxacin. These findings underscore the complex and context-dependent nature of agr-mediated protection. Furthermore, in vivo investigations using murine models provide valuable insights into the importance of agr in promoting S. aureus fitness, particularly in the context of neutrophil-mediated clearance, with notable emphasis on the pulmonary milieu. Overall, this study significantly advances our understanding of agr-mediated protection in S. aureus and sheds light on the sophisticated adaptive mechanisms employed by the bacterium to fortify itself against oxidative stress encountered during infection.

The conclusions drawn in this paper are generally well-supported by the data. To enhance the clarity of the study, it is recommended that the authors consider refraining from combining the data for lactate production during microaerobic growth with the remaining data obtained for aerobic growth. Different aeration conditions can significantly impact the metabolic status of the cells.

In this regard, the statement, "Collectively, these data suggest that Δagr increases respiration and aerobic fermentation to compensate for low metabolic efficiency," might be potentially misleading and could benefit from a revision to accurately reflect the nuances of the experimental conditions.

Additionally, the authors' statement, 'The tendency of Δagr cells to forgo the additional ATP yield from acetate production in favor of NAD+-generating lactate (23, 24) underscores the importance of redox balance in Δagr cells,' appears contradictory to the data presented in Fig 5, where the Δagr mutant demonstrates an approximately threefold increase in acetate production during exponential growth compared to the wild-type strain. A clarification or adjustment in the manuscript may be necessary to ensure consistency and accurate interpretation.

Furthermore, the authors' statement, 'Collectively, these observations suggest that a surge in NADH consumption and reductive stress in the Δagr strain induces a burst in respiration, but levels of NADH are saturating, thereby driving fermentation in the presence of oxygen,' may need revision. Data presented in Figure 5 suggest the opposite - a surge in NADH accumulation leading to a decrease in the NAD/NADH ratio, rather than a surge in the 'consumption' of NADH. Clarifying this point in the manuscript would ensure accurate representation of the findings.

The authors attention to these matters would greatly contribute to the precision and clarity of the findings.

Reviewer #2 (Public Review):

Summary

Song et al investigate the role of the frontal eye field (FEF) and the intraparietal sulcus (IPS) in mediating the shift in ocular dominance (OD) observed after a period of dichoptic stimulation during which attention is selectively directed to one eye. This manipulation has been previously found to transiently shift OD in favor of the unattended eye, similar to the effect of short-term monocular deprivation. To this aim, the authors combine psychophysics, fMRI, and transcranial magnetic stimulation (TMS). In the first experiment, the authors determine the regions of interest (ROIs) based on the responses recorded by fMRI during either dichoptic or binocular stimulation, showing selective recruitment of the right FEF and IPS during the dichoptic condition, in line with the involvement of eye-based attention. In a second experiment, the authors investigate the causal role of these two ROIs in mediating the OD shift observed after a period of dichoptic stimulation by selectively inhibiting with TMS (using continuous theta burst stimulation, cTBS), before the adaptation period (50 min exposure to dichoptic stimulation). They show that, when cTBS is delivered on the FEF, but not the IPS or the vertex, the shift in OD induced by dichoptic stimulation is reduced, indicating a causal involvement of the FEF in mediating this form of short-term plasticity. A third control experiment rules out the possibility that TMS interferes with the OD task (binocular rivalry), rather than with the plasticity mechanisms. From this evidence, the authors conclude that the FEF is one of the areas mediating the OD shift induced by eye-selective attention.

The authors have addressed the issues that I raised during the first round of review.<br /> While the results of the new experiment (Experiment 4), leave some unresolved isssues (addressed in the discussion section), they provide a very important replication of the main result, showing that even if the observed effect is small, it is robust.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Reviewer #2 (Public Review):

In a study by Shen et al.. 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 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. The authors extrapolated and suggested that PNRC1 expression 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 #1 (Public Review):

In this study, the authors investigate the role of triglycerides in spermatogenesis. This work is based on their previous study (PMID: 31961851) on triglyceride sex differences in which they showed that somatic testicular cells play a role in whole body triglyceride homeostasis. In the current study, they show that lipid droplets (LDs) are significantly higher in the stem and progenitor cell (pre-meiotic) zone of the adult testis than in the meiotic spermatocyte stages. The distribution of LDs anti-correlates with the expression of the triglyceride lipase Brummer (Bmm), which has higher expression in spermatocytes than early germline stages. Analysis of a bmm mutant (bmm[1]) - a P-element insertion that is likely a hypomorphic - and its revertant (bmm[rev]) as a control shows that bmm acts autonomously in the germline to regulate LDs. In particular, the number of LDs is significantly higher in spermatocytes from bmm[1] mutants than from bmm[rev] controls. Testes from males with global loss of bmm (bmm[1]) are shorter than controls and have fewer differentiated spermatids. The zone of bam expression, typically close to the niche/hub in WT, is now many cell diameters away from the hub in bmm[1] mutants. There is an increase in the number of GSCs in bmm[1] homozygotes, but this phenotype is probably due to the enlarged hub. However, clonal analyses of GSCs lacking bmm indicate that a greater percentage of the GSC pool is composed of bmm[1]-mutant clones than of bmm[rev]-clones. This suggests that loss of bmm could impart a competitive advantage to GSCs, but this is not explored in greater detail. Despite the increase in number of GSCs that are bmm[1]-mutant clones, there is a significant reduction in the number of bmm[1]-mutant spermatocyte and post-meiotic clones. This suggests that fewer bmm[1]-mutant germ cells differentiate than controls. To gain insights into triglyceride homeostasis in the absence of bmm, they perform mass spec-based lipidomic profiling. Analyses of these data support their model that triglycerides are the class of lipid most affected by loss of bmm, supporting their model that excess triglycerides are the cause of spermatogenetic defects in bmm[1]. Consistent with their model, a double mutant of bmm[1] and a diacylglycerol O-acyltransferase 1 called midway (mdy) reverts the bmm-mutant germline phenotypes.

There are numerous strengths of this paper. First, the authors report rigorous measurements and statistical analyses throughout the study. Second, the authors utilize robust genetic analyses with loss-of-function mutants and lineage-specific knockdown. Third, they demonstrate the appropriate use of controls and markers. Fourth, they show rigorous lipidomic profiling. Lastly, their conclusions are appropriate for the results. In other words, they don't over-state the results. Overall, the rigorously quantified results support the major aim that appropriate regulation of triglycerides are needed in a germline cell-autonomous manner for spermatogenesis.

This paper should have a positive impact on the field. First and foremost, there is limited knowledge about the role of lipid metabolism in spermatogenesis. The lipidomic data will be useful to researchers in the field who study various lipid species. Going forward, it will be very interesting to determine what triglycerides regulate in germline biology. In other words, what functions/pathways/processes in germ cells are negatively impacted by elevated triglycerides. And as the authors point out in the discussion, it will be important to determine what regulates bmm expression such that bmm is higher in later stages of germline differentiation.

Reviewer #2 (Public Review):

Summary:

Here, the authors show that neutral lipids play a role in spermatogenesis. Neutral lipids are components of lipid droplets, which are known to maintain lipid homeostasis, and to be involved in non-gonadal differentiation, survival, and energy. Lipid droplets are present in the testis in mice and Drosophila, but not much is known about the role of lipid droplets during spermatogenesis. The authors show that lipid droplets are present in early differentiating germ cells, and absent in spermatocytes. They further show a cell autonomous role for the lipase brummer in regulating lipid droplets and, in turn, spermatogenesis in the Drosophila testis. The data presented show that a relationship between lipid metabolism and spermatogenesis is congruous in mammals and flies, supporting Drosophila spermatogenesis as an effective model to uncover the role lipid droplets play in the testis.

Strengths and weaknesses:

The authors do a commendably thorough characterization of where lipid droplets are detected in normal testes: located in young somatic cells, and early differentiating germ cells. They use multiple control backgrounds in their analysis, including w[1118], Canton S, and Oregon R, which adds rigor to their interpretations. The authors employ markers that identify which lipid droplets are in somatic cells, and which are in germ cells. The authors use these markers to present measured distances of somatic and germ cell-derived lipid droplets from the hub. Because they can also measure the distance of somatic and germ cells with age-specific markers from the hub, these results allow the authors to correlate position of lipid droplets with the age of cells in which they are present. This analysis is clearly shown and well quantified.

The quantification of lipid droplet distance from the hub is applied well in comparing brummer mutant testes to wild type controls. The authors measure the number of lipid droplets of specific diameters, and the spatial distribution of lipid droplets as a function of distance from the hub. These measurements quantitatively support their findings that lipid droplets are present in an expanded population of cells further from the hub in brummer mutants. The authors further quantify lipid droplets in germline clones of specified ages; the quantitative analysis here is displayed clearly and supports a cell autonomous role for brummer in regulating lipid droplets in spermatocytes.

Data examining testis size and number of spermatids in brummer mutants clearly indicates the importance of regulating lipid droplets to spermatogenesis. The authors show beautiful images supported by rigorous quantification supporting their findings that brummer mutants have both smaller testes with fewer spermatids at both 29 and 25C. There is also significant data supporting defects in testis size, but not spermatid number, in 14-day-old brummer mutant animals compared to controls. Their analysis clearly shows an expanded region beyond the testis apex that includes younger germ cells, supporting a role for lipid droplets influencing germ cell differentiation during spermatogenesis.

The authors present a series of data exploring a cell autonomous role for brummer in the germline, including clonal analysis and tissue specific manipulations. The clonal data indicating increased lipid droplets in spermatocyte clones, and a higher proportion of brummer mutant GSCs at the hub are convincing and supported by quantitation. The authors also show a tissue specific rescue of the brummer testis size phenotype by knocking down mdy specifically in germ cells, which is also supported by statistically significant quantitation. The authors present data examining the number of spermatocyte and post-meiotic clones 14 days after clonal induction. Their finding is significant with a p-value of 0.0496, which they acknowledge is less robust than their other data reported in this study, and could be a result of a low sample size. They indicate that future studies might validate these results with additional samples.

The authors do a beautiful job of validating where they detect brummer-GFP by presenting their own pseudotime analysis of publicly available single cell RNA sequencing data. Their data is presented very clearly, and supports expression of brummer in older somatic and germline cells of the age when lipid droplets are normally not detected. The authors also present a thorough lipidomic analysis of animals lacking brummer to identify triglycerides as an important lipid droplet component regulating spermatogenesis.

Impact:

The authors present data supporting the broad significance of their findings across phyla. This data represents a key strength of this manuscript. The authors show that loss of a conserved triglyceride lipase impacts testis development and spermatogenesis, and that these impacts can be rescued by supplementing diet with medium-chain triglycerides. The authors point out that these findings represent a biological similarity between Drosophila and mice, supporting the relevance of the Drosophila testis as a model for understanding the role of lipid droplets in spermatogenesis. The connection buttresses the relevance of these findings and this model to a broad scientific community.

Reviewer #2 (Public Review):

Summary:

This study looks into the complex dominance patterns of S-allele incompatibilities in Brassicaceae, through which it attempts to learn more about the sheltering of deleterious load. I found several weak points in the analyses that diminished my excitement about the results. In particular, the way in which deleterious mutations were classified lacked the ability to distinguish the severity of the mutations and thus their expected associated dominance. Furthermore, the simulation approach could have provided this exact sort of insight but was not designed to do so, making this comparison to the empirical data also less than exciting for me.

Major and minor comments:

I think the introduction (or somewhere before we dive into it in the results) of the dominance hierarchy for the S-alleles needs a more in-depth explanation. Not being familiar with this beforehand really made this paper inaccessible to me until I then went to find out more before continuing. I would expect this paper to be broad enough that self-contained information makes it accessible to all readers. For example, lines 110-115 could be in the Introduction.

Along with my above comment, perhaps it is not my place to comment, but I find the paper not of a broad enough scope to be of interest to a broad readership. This S-allele dominance system is more than simple balancing selection, it is a very complex and specific form of dominance between several haplotypes, and the mechanism of dominance does not seem to be genetic. I am not sure that it thus extrapolates to broad comments on general dominance and balancing selection, e.g. it would not be the same as considering inversions and this form of balancing selection where we also expect recessive deleterious mutations to accumulate.

It would have been particularly interesting, or a nice addition, to see deleterious mutations classed by something like SNPeff or GERP where you can have different classes of moderate to severe deleterious variants, which we would expect also to be more recessive the more deleterious they are. In line with my next comment on the simulations, I think relative differences between mutations expected to be more or less dominant may be even more insightful into the process of sheltering which may or may not be going on here.

In the simulations, h=0 and s=0.01 (as in Figure 5) for all deleterious mutations seems overly simplistic, and at the convenient end for realistic dominance. I think besides recessive lethals which we expect to be close to h=0 would have a much larger selection coefficient, and other deleterious mutations would only be partially recessive at such an s value. I expect this would change some of the simulation results seen, though to what degree I am not certain. It would be nice to at least check the same exact results for h=0.3 or 0.2 (or additionally also for recessive lethals, e.g. h=0 and s=-0.9). I would also disagree with the statement in line 677, many studies have shown, particularly those on balancing selection, that partially recessive deleterious mutations are not eliminated by natural selection and do play a role in population genetic dynamics. I am also not surprised that extinction was found for higher s values when the mutation rate for such mutations was very high and the distribution of s values was constant. An influx of such highly deleterious mutations is unlikely to ever let a population survive, yet that does NOT mean that in nature, the rare influx of such mutations does lead to them being sheltered. I find overall that the simulation results contribute very little, to none, to this paper, as without something more realistic, like a simultaneous distribution of s and h values, you cannot say which, if any class of these mutations are the ones expected to accumulate because of S-allele dominance. Rather they only show the disappointing or less exciting result that fully recessive, weakly deleterious mutations (which I again think do not even exist in nature as I said above) have minor, to no effect across the classes of S-allele dominance. They provide no insight into whether any type of recessive deleterious mutation can accumulate under the S-allele dominance hierarchy, and that is the interesting question at hand. I would either remove these simulations or redo them in another approach. The authors never mention what simulation approach was used, so I can only assume this is custom, in-house code. Yet I do not find that code provided on the github page. I do not know if the lack of a distribution for h and s values is then a choice or a programming limitation, but I see it as one that should be overcome if these simulations are meant to be meaningful to the results of the study.

Reviewer #2 (Public Review):

Summary:

In the manuscript, Yu et al reported a two-sample Mendelian randomization study to evaluate the causation between polyunsaturated fatty acids (PUFA) and cerebral aneurysm, based on summary statistics from published genome-wide association studies. The authors identified that omega-3 fatty acids and Docosahexaenoic acid decreased the risk for intracranial aneurysm (IA) and aneurysmal subarachnoid hemorrhage (aSAH). COLOC analysis suggested that the acids and IA, aSAH likely share causal variants in gene fatty acid desaturase 2.

Strengths:

The methodology is sound, with appropriate sensitivity analysis.

Weaknesses:

The results did not provide significant novel findings. The interpretation of the results is not sound.

Reviewer #1 (Public Review):

Summary:

The authors performed two-sample MR combined with sensitivity analyses and colocalization to test the effect of PUFA on cerebral aneurysms. They found that genetically predicted omega-3 and DHA decreased the risk for intracranial aneurysm (IA) and subarachnoid haemorrhage (SAH) but not for unruptured IA (uIA).

Strengths:

PUFA on the risk of cerebral aneurysms is of clinical importance; the authors performed multiple sensitivity analyses to ensure MR fulfills its assumptions.

Weakness:

In my opinion, the major weakness is the selection of IVs, the same IVs should be used for each exposure, especially when the outcomes (IA, SAH, and uIA) are closely related. The removal of IVs was inconsistent, for example, why was LPA rs10455872 removed for SAH but not for uIA? (significantly more IVs were used for uIA). The authors should provide more details for the justification of the removal of IVs other than only indicating "confounder" in supplementary tables. The authors should also perform additional analyses including all IVs and IVs from other PUFA GWAS.

In addition, it seems that the SNPs in the FADS locus were driving the MR association, while FADS is a very pleiotropic locus associated with many lipid traits, removing FADS could attenuate the MR effect. The authors should perform a sensitivity analysis to remove this locus.

Instead of removing multiple "confounder" IVs which I think may bias the MR results due to very closely related lipid traits, the authors should perform multivariable MR to identify independent effects of PUFAs to IA, conditioning on other PUFAs and/or other lipids.

Colocalization was not well described, the authors should include the colocalization results for each locus in a supplementary table. They also mentioned "a large PP for H4 (PP.H4 above 0.75) strongly supports shared causal variants affecting both gene expression and phenotype". The authors should make sure that the colocalization was performed using the expression data of each gene or using the GWAS summary of each PUFA locus.

Reviewer #2 (Public Review):

Significance of the findings:

In this study, blood donors were assessed using serology and viral neutralization assays to determine the prevalence of SARS-CoV-2 antibodies. S1 and NCP antibodies were used to distinguish between vaccination and natural infection and virus-specific neut titers were used to determine which variants the antibodies respond to. The study reports almost universal antibody prevalence and increases in antibodies against specific variants at different points corresponding to circulating variants identified phylogenetically in neighbouring countries. The authors propose this approach for settings like Bolivia where genetic sequencing is not readily available. Unfortunately, there are significant limitations to this approach that limit its utility - serological data are available after the fact in a fast-moving pandemic and so are a poor alternative to phylogenetic data. Rather, serological information can supplement phylogenetic data and is most useful in estimating population-level immunity.

(1) Considerations in interpreting the results:

a. Serology provides different information to phylogenetic sequencing of the viruses and so both are important. Viral sequencing provides real-time information on circulating variants and indicates the proportion of each variant in circulation at any point as there are almost always multiple variants spreading but it is the fastest spreading variant that comes to dominate. Importantly serology measures asymptomatic infections as well, providing population estimates of infection that are not available through viral gene sequencing.

b. A major concern in the interpretation of serology is that antibody titers vary markedly over time with rapid declines in the first year post-infection or post-vaccination. However, these declines vary depending on whether hybrid immunity is present. Disentangling this retrospectively is a challenge. A low antibody titer could reflect an infection that occurred a few months ago but may be below the threshold for positivity at the time of testing. There is also substantial individual variability in antibody responses.

c. Serology becomes increasingly difficult to untangle when an individual has had doses of vaccine and multiple natural infections with different variants. Due to the importance of hybrid immunity in population risk to new variants, it would be useful for estimates of hybrid immunity to be generated based on anti-S1 and anti-NCP antibodies. From a population immunity perspective, this could be important in guiding future protection and boosting strategies.

d. Since there is cross-neutralization by the antibodies stimulated by each variant, it is important to establish the sensitivity and specificity of each of the neutralization assays in a panel comprising multiple variants. An assessment of the accuracy of the neut assay for each variant is needed to be confident that it is able to distinguish between variants.

e. Blood donors are notoriously poor representations of the general population in many countries, driven partly by whether donation is financially rewarded. For example, in the USA, drug addicts are disproportionately over-represented in blood donor populations as they use it as a source of money. The authors provide no information on whether the blood donor population in Bolivia is representative of the entire population. Comparison of the prevalence of specific disease markers in the general population and in blood donors could provide a signal of their comparability.

(2) Please provide the sensitivity and specificity of each of the assays so that the reader can assess the degree of accuracy in the assay that claims that the prevalent antibodies are due to, for example, omicron.

(3) Please provide an assessment of the representativity of the blood donor population eg. Is the prevalence of hepatitis B serological markers in the blood donor population comparable with the prevalence of hepatitis B serological markers in the general population from community-based studies?

Reviewer #1 (Public Review):

Summary:

This study provides valuable and comprehensive information about the SARS-CoV-2 seroprevalence during 2021 and 2022 in different regions of Bolivia. Moreover, data on immune responses against the SARS-CoV-2 variants based on neutralization tests denotes the presence of several virus variants circulating in the Bolivian population. Evidence for seroprevalence data provided by the authors is solid, across the study period, while data regarding variant circulation is limited to the early stages of the pandemic.

Strengths:

The major strength of this study is that it provided nationwide seroprevalence estimates from infection and/or vaccination based on antibodies against both spike and the nucleocapsid protein in a large representative sample of sera collected at two time-points from all departments of Bolivia, gaining insight into COVID-19 epidemiology. On the other hand, data from virus neutralization assays inferred the circulation during the study period of four SARS-CoV-2 variants in the population. Overall, the study results provide an overview of the level of viral transmission and vaccination and insights into the spread across the country of SARS-CoV-2 variants.

Weaknesses:

The assessment of a Lambda variant that circulated in several neighboring countries (Peru, Chile, and Argentina), which had a significant impact on the COVID-19 pandemic in the region, may have strengthened the study to contrast Gamma spread. In addition, even though neutralizing antibodies can certainly reveal previous infections of SARSCOV2 variants in the population, it is of limited value to infer from this information some potential timing estimates of specific variant circulation, considering the heterogeneous effects that past infections, vaccinations, or a combination of both could have on the level of variant-specific neutralizing antibodies and/or their cross-neutralization capacity.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The conclusions of this paper are well supported by data, particularly regarding seroprevalence that reliably reflects the epidemiology of COVID-19 in Bolivia, and seroprevalence trends in other low- and middle-income countries.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

Since this is the first study that has been conducted to assess indicators of immunity against SARS-CoV-2 in the population of Bolivia at a nationwide scale, seroprevalence data provided by geographic regions at two time-points can be useful as a reference for potential retrospective global meta-analysis and further explore and compare the risk factors for infection, variant distribution, and the impact on infection and vaccination, gaining deeper insights into understanding the evolution of the COVID-19 pandemic in Bolivia and in the region.

Reviewer #1 (Public Review):

Summary:<br /> This research used cell-based signaling assay and Gaussian-accelerated molecular dynamics (GaMD) to study peptide-mediated signaling activation of Polycystin-1 (PC1), which is responsible for the majority of autosomal dominant polycystic kidney disease (ADPKD) cases. Synthetic peptides of various lengths derived from the N-terminal portion of the PC1 C-terminal fragment (CTF) were applied to HEK293T cells transfected with stalkless mouse CTF expression construct. It was shown that peptides including the first 7, 9, and 17 residues of the N-terminal portion could activate signaling to the NFAT reporter. To further understand the underlying mechanism, docking and peptide-GaMD simulations of peptides composed of the first 9, 17, and 21 residues from the N-terminal portion of the human PC1 CTF were performed. These simulations revealed the correlation between peptide-CTF binding and PC1 CTF activation characterized by the close contact (salt bridge interaction) between residues R3848 and E4078. Finally, a Potts statistical model was inferred from diverged PC1 homologs to identify strong/conserved interacting pairs within PC1 CTF, some of which are highly relevant to the findings from the peptide GaMD simulations. The peptide binding pockets identified in the GaMD simulations may serve as novel targets for the design of therapeutic approaches for treating ADPKD.

Strengths:<br /> (1) The experimental and computational parts of this study complement and mostly support each other, thus increasing the overall confidence in the claims made by the authors.

(2) The use of exogenous peptides and a stalkless CTF in the GaMD is a step forward compared to earlier simulations using the full CTF, CTF mutants, or the stalkless CTF alone. And it led to findings of novel binding pockets.

(3) Since the PC1 shares characteristics with the Adhesion class of GPCRs, the approaches used in this work may be extended to other similar systems.

Weaknesses:<br /> (1) The GaMD simulations all include the exogenous peptides, thus lacking a control where no such peptide is present (and only stalkless CTF). An earlier study (PNAS 2022 Vol. 119 No. 19 e2113786119) covered this already but it should be mentioned here that there was no observation of close/activation for the stalkless CTF.

(2) Although 5 independent trajectories were generated for each peptide, the authors did not provide sufficient details regarding the convergence of the simulation. This leaves some uncertainties in their results. Given that the binding poses changed relative to the starting docked poses for all three peptides, it is possible that some other binding pockets and/or poses were not explored.

(3) The free energy profiles (Figures 2 to 4) based on the selected coordinates provide important information regarding binding and CTF conformational change. However, it is a coarse-grained representation and complementary analysis such as RDFs, and/or contact maps between the peptide and CTF residues might be helpful to understand the details of their interactions. These details are currently only available in the text.

(4) The use of a stalkless CTF is necessary for studying the functions of the exogenous peptides. However, the biological relevance of the stalkless CTF to ADPKD was not clearly explained, if any.

Reviewer #2 (Public Review):

Summary:<br /> The autosomal dominant polycystic kidney disease (ADPKD) is a major form of polycystic kidney disease (PKD). To provide better treatment and avoid side effects associated with currently available options, the authors investigated an interesting GPCR, polycystin-1 (PC1), as a potential therapeutic target. In vitro and in silico studies were combined to identify peptide agonists for PC1 and to elucidate their roles in PC1 signaling. Overall, regarding the significance of the findings, this work described valuable peptide agonists for PC1 and the combined in vitro and in silico approach can be useful to study a complex system like PC1. However, the strength of the evidence is incomplete, as more experiments are needed as controls to validate the computational observations. The work appears premature.

Strengths:<br /> (1) This work first described the experimental discovery of short peptides designed to mimic the stalk region of PC1, followed by computational investigation using docking and MD simulations. PC1 is a complex membrane protein and an emerging target for ADPKD, but it can be challenging to study. The knowledge and the peptide discovery can be valuable and useful to understand the mechanism and potential modulation of PC1.

(2) The authors published the mechanistic study of PC1 and identified key interacting residues such as N3074-S3585 and R3848-E4078, using very similar techniques (PNAS 2022, 119(19), e2113786119). This work furthers this research by identifying peptides that are stalk mimics for PC1 activation.

(3) Eight peptides were designed and tested experimentally first; three were computationally studied with docking and GaMD simulations to understand their mechanism (s).

Weaknesses:<br /> (1) The therapeutic potential of PC1 peptide agonists is unclear in the introduction. For example, while the FDA-approved drug Jynarque was mentioned, the text was misleading as it sounded like Jynarque targeted PC1. In fact, it targets another GPCR, the vasopressin receptor 2 (V2). A clear comparison of targeting PC1 over V2 pathways and their therapeutic relevance can help the readers better understand the importance of this work. Importantly, a clear background on the relationship between PC1 agonism and treatments for ADPKD is necessary.

(2) PC1 is a complex membrane protein, and most figures focus on the peptide-binding site. For general readers (or readers that did not read the previous PNAS publication), it is hard to imagine the overall structure and understand where the key interactions (e.g., R3848-E4078) are in the protein and how peptide binding affects locally and globally. I suggest enhancing the illustrations.

(3) The authors used the mouse construct for the cellular assays and the peptide designs in preparation for future in vivo assays. This is helpful in understanding biology, but the relevance of drug discovery is weakened. Related to Point 1, the therapeutic potential of PC1 peptide agonist is largely missing.

(4) More control experiments are needed. For example, a 7-residue hydrophilic sequence (GGKKKKK) is attached to the peptide design to increase solubility. This 7-residue peptide should be tested for PC1 activation as a control. Second, there is no justification for why the peptide design must begin with residue T3041. Can other segments of the stalk also be agonists?

(5) There are some major concerns about the simulations: The GaMD simulations showed different binding sites of p-21, p-17, and p-9, and the results report the simulated conformations as "active conformational states". However, these are only computational findings without structural biology or mutagenesis data to validate. Further, neither docking nor the simulation data can explain the peptide SAR. Finally, it will be interesting if the authors can use docking or GaMD and explain why some peptide designs (like P11-P15) are less active (as control simulations).

Reviewer #3 (Public Review):

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

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

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

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

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

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

Reviewer #1 (Public Review):

Summary:

The authors show that upon treatment with Doxorubicin (Doxo), there is an increase in senescence and inflammatory markers in the muscles. They also show these genes get upregulated in C2C12 myoblasts when treated with conditioned media or 15d-PGJ2. 15dPGJ2 induces cell death in the myoblasts, decreases proliferation (measured by cell numbers), and decreases differentiation and fusion. 15d-PGJ2 modified Cys184 of HRas, which is required for its activation as indicated by the FRET analysis with RAF RBD. They also showed that 15d-PGJ2 activates ERK signaling, but not Akt signaling, through the electrophilic center. 15d-PGJ2 inhibits Golgi localization of HRAS (only WT, not C181 or C184 mutant). They also showed that expressing the WT HRas followed by 15d-PGJ2 treatment led to a decrease in the levels of MHC mRNA and protein, and this defect is dependent on C184. This is a well-written manuscript with interesting insights into the mechanism of action of 15d-PGJ2. However, some clarification and experiments will help the paper advance the field significantly.

Strengths:

The data clearly shows that 15d-PGJ2 has a negative role in the myoblast cells and that it leads to modification of HRas protein. Moreover, the induction of biosynthetic enzymes in the PGD2 pathway also supports the induction of 15d-PGJ2 in Doxorubicin-treated cells. Both conditioned media experiments and the 15d-PGJ2 experiments show that 15d-PGJ2 could be the active component secreted by the senescent myoblasts.

Weaknesses:

The genes that are upregulated in the muscles upon injection with Doxo are also markers for inflammation. Since Doxo is also known to induce systemic inflammation, it is important to delineate these two effects (inflammatory cells vs senescent cells). The expression of beta Gal and other markers of senescence in the tissue sections will help to delineate these.

In Figure 2, where the defect in the differentiation of myoblasts upon treatment with 15d-PGJ2 is shown, most of the cells die within 48 hours at higher concentrations, making it difficult to perform the experiments. This also shows that 15d-PGJ2 was toxic to these cells. Lower concentrations show a decrease in the differentiation based on the lower number of nuclei in fibers and low expression of MyoD, MyoG, and MHC. However, it is unclear if this is due to increased cell death or defective differentiation. It would be a lot more informative if the cell count, cell division, and cell death could be plotted for these concentrations of the drug during the experiment. Also, in the myoblast experiments, are the effects of treatment with Dox reversible?

In Figure 3, most of the experiments are done at a high concentration, which induces almost complete cell death within 48 hours. Even at such a high concentration of 15dPGJ2, the increase in ERK phosphorylation is minimal.

The experiment Figure 4C shows that C181 and C84 mutants of the HRas show higher levels in Golgi compared with WT. However, this could very well be due to the defect in palmitoylation rather than the modification with 15d-PGJ2. Though the authors allude to the possibility that intracellular redistribution of HRas by 15d-PGJ2 requires C181 palmitoylation, the direct influence of C184 modification on C181 palmitoylation is not shown. To have a meaningful conclusion, the authors need to compare the palmitoylation and modification with 15d-PGJ2.

To test if the inhibition of myoblast differentiation depends on HRas, they overexpressed the HRas and mutants in the C2C12 lines. However, this experiment does not take the endogenous HRAs into consideration, especially when interpreting the C184 mutant. An appropriate experiment to test this would be to knock down or knock out HRas (or make knock-in mutations of C184) and show that the effect of 15d-PGJ2 disappears. Moreover, in this specific experiment, it is difficult to interpret without a control with no HRas construct and another without the 15d-PGJ2 treatment.

Moreover, the overall study does not delineate the toxic effects of 15d-PGJ2 from its effect on the differentiation.

Reviewer #2 (Public Review):

Summary:

In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.

Strengths:

The regulation of HRas by 15d-PGJ2 is well controlled.

Weaknesses:

The novelty of the study is compromised as the activation of PGD and 15d-PGJ2, as well as the regulation of HRas and cell proliferation, have been previously reported. Additionally, there are major technical concerns related to the senescence models, limiting data interpretation regarding the relevance to senescent cells.

Major concerns:<br /> (1) The C2C12 cell line is not an ideal model for senescence study due to its immortalized nature and lack of normal p16 expression. A more suitable myoblasts model is recommended, with a more comprehensive characterization of senescence features.

(2) The source of increased PGD or its metabolites in the conditioned medium is unclear. Including other senescence models, such as replicative or oncogene-induced senescence, would strengthen the study. Again, C2C12 is not suitable for replicative senescence due to its immortalized status.

(3) In the in vivo part, it's unclear whether the increased expression of PTGS1, PTGS2, and PTGDS is due to senescence or other side effects of DOXO.

(4) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of a conditioned medium. There is no explanation of how differentiation was quantified or how the fusion index was calculated.

Reviewer #1 (Public Review):

This study makes an interesting finding: a polyunsaturated fatty acid, Lin-Glycine, increases the conductance of KCNQ1/KCNE1 channels by stabilizing a state of the selectivity filter that allows K+ conduction. The stabilization of a conducting state appears well supported by single-channel analysis, though some method details are missing. The linkage to PUFA action through the selectivity filter is supported by the disruption of PUFA effects by mutation of residues which change conformation in two KCNQ1 structures from the literature. Claims about differences in Lin-Glycine binding to these two structural conformations seem to lack clear support, thus the claim seems speculative that PUFAs increase Gmax by binding to a crevice in the pore domain. A potentially definitive functional experiment is conducted by single-channel recordings with selectivity filter domain mutation Y315F which ablates the Lin-Glycine effect on Gmax. However, this appears to be an n=1 experiment. Overall, the major claim of the abstract is supported: "... that the selectivity filter in KCNQ1 is normally unstable ... and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state." However, the claim in the abstract that selectivity filter instability "explains the low open probability" seems too general.

Reviewer #2 (Public Review):

Summary:

Golluscio et al. address one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by polyunsaturated fatty acids (PUFA). PUFA is known to upregulate KCNQ1 and KCNQ1/KCNE1 channels by two mechanisms: one shifts the voltage dependence to the negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium by charge effect, the second mechanism is less known. By applying the single-channel recordings and mutagenesis on the putative binding sites (most of them related to the selectivity filter), they concluded that the selectivity filter is stabilized to a conductive state by PUFA binding.

Strengths:<br /> They mainly used single-channel recordings and directly assessed the behavior of the selectivity filter. The method is straightforward and convincing enough to support their claims.

Weaknesses:<br /> The structural model they used is the KCNQ1 channel without KCNE1 because KCNQ1/KCNE1 channel complex is not available yet. As the binding site of PUFAs might overlap with KCNE1, it is not very clear how PUFA binds to the KCNQ1 channel in the presence of KCNE1.

Using other previous PUFA-related KCNQ1 mutants will strengthen their conclusions. For example, the Gmax of the K326E mutant is reduced by PUFA binding. Examining whether K326E shows reduced numbers of non-empty sweeps in the single-channel recordings will be a good addition.

Reviewer #3 (Public Review):

Summary:

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

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

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

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

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

Reviewer #1 (Public Review):

Summary:

This study provides the detailed molecular mechanism of how OGT, an O-GlcNac transferase, promotes cancer progression. Using loss-of-function OGT models, the authors demonstrated that OGT cleaves HCF-1, an important guardian of genomic stability. The resulting genomic instability in OGT-knockout tumors leads to cytosolic DNA accumulation, the activation of cGAS-mediated type I IFN responses, and increased CD8+ T cell infiltration into the tumors. Moreover, treatment with OGT inhibitor synergized with anti-PDL1 immune-checkpoint blockade.

Strengths:

Novel findings of how OGT promotes tumor progression.

Weaknesses:

(1) Some of the data is problematic and does not always support the authors' conclusions.<br /> (2) The writing needs significant improvement. In places, it is hard to understand or could mislead the readers.<br /> (3) Figure legends are minimalistic and do not provide sufficient information.<br /> (4) Discussion does not put the findings of this study into a broader context of the field but merely restates them.

Reviewer #2 (Public Review):

Summary:

In this study, the author demonstrates that deficiency or pharmacological inhibition of O-glcNac transferase (OGT) enhances tumor immunity in colorectal cancer models. The authors propose that OGT deficiency triggers a DNA damage response, activating the cGAS-STING innate immunity pathway and promoting a Type I interferon response. They suggest that OGT-mediated processing of HSF1 is crucial in maintaining genomic integrity. This research is significant as it identifies OGT inhibition as a potential immunomodulatory target in cancer treatment.

Strengths:

The strength of the paper lies primarily in the in vivo data, demonstrating the impact of OGT deficiency or inhibition on modulating tumor growth and anti-tumor immunity. The experiments are well-controlled. However, there are several unresolved questions:

Weaknesses:

The mechanisms of how OGT deficiency can trigger DNA damage and the role of this response in promoting immunity are only partially addressed in the manuscript.

Reviewer #1 (Public Review):

The manuscript by Wu et al. explores the role of the histone reader protein SntB in Aspergillus flavus, claiming it to be a key regulator of development and aflatoxin biosynthesis. While the study incorporates various techniques, including gene deletion, ChIP-seq, and RNA-seq, several concerns and omissions in the paper raise questions about the validity and completeness of the presented findings.

(1) Omissions of Prior Work:<br /> The authors fail to acknowledge and integrate prior research by Pfannenstiel et al. (2018) on the sntB gene in A. flavus, which covered phenotypic changes, RNA-seq data, and histone modifications. This omission raises concerns about the transparency and completeness of the current study.

The absence of reference to studies by Karahoda et al. (2022, 2023) revealing SntB's involvement in the KERS complex in A. flavus and A. nidulans is a major oversight. This raises questions about the specificity of SntB's regulatory functions, as it may be part of a larger complex. The authors should clarify why these studies were omitted and how they ensure that SntB alone, and not the entire KERS complex, is responsible for the observed effects.

(2) Transparency and Accessibility of Data:<br /> The lack of accessibility and visualization tools for ChIP-seq and RNA-seq data poses a challenge for independent verification and in-depth analysis. The authors should address this issue by providing more accessible data or explaining the limitations of data availability. A critical component missing from the paper is a detailed presentation of ChIP-seq data, specifically demonstrating SntB binding patterns on key promoters. This omission weakens the link between SntB and the mentioned regulatory genes. The authors should include these crucial data visualizations to strengthen their claims.

(3) SntB Binding Sites and Consensus Sequence:<br /> The study mentions several genes upregulated in the sntB mutant without demonstrating SntB binding sites on their promoters. A detailed analysis of SntB binding maps is necessary to establish a direct link between SntB and these regulatory genes.

(4) Mechanistic Insight into Peroxisome Biogenesis:<br /> If SntB indeed regulates peroxisome biogenesis, the absence of markers for peroxisomes and the localization of peroxisomes in the sntB mutant vs. WT strains is a significant gap. Providing evidence for peroxisome regulation is crucial for understanding the proposed mechanism and validating the study's claims.

In summary, while the manuscript presents intriguing findings regarding SntB's role in A. flavus, the omissions of prior work, lack of transparency in data accessibility, and insufficient mechanistic insights call for revisions and additional experimental evidence to strengthen the validity and impact of the study. Addressing these concerns will enhance the manuscript's contribution to the field.

Additionally, the way the English language is used could be improved.

Reviewer #2 (Public Review):

Summary:<br /> This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control. Overall, this is generally an excellent manuscript.

Strengths:<br /> The data in this manuscript is robust and the experiments conducted are appropriate.

Weaknesses:<br /> (1) The authors found that SntB played key roles in the oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, the authors have to better measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.

(2) Why did the authors only study the function of catC among the 7 genes related to an oxidative response listed in Table S14?

Reviewer #1 (Public Review):

Summary:

In their manuscript, Yu et al. describe the chemotactic gradient formation for CCL5 bound to - i.e. released from - glycosaminoglycans. The authors provide evidence for phase separation as the driving mechanism behind chemotactic gradient formation. A conclusion towards a general principle behind the finding cannot be drawn since the work focuses on one chemokine only, which is particularly prone to glycan-induced oligomerisation.

Strengths:

The principle of phase separation as a driving force behind and thus as an analytical tool for investigating protein interactions with strongly charged biomolecules was originally introduced for protein-nucleic acid interactions. Yu et al. have applied this in their work for the first time for chemokine-heparan sulfate interactions. This opens a novel way to investigate chemokine-glycosaminoglycan interactions in general.

Weaknesses:

As mentioned above, one of the weaknesses of the current work is the exemplification of the phase separation principle by applying it only to CCL5-heparan sulfate interactions. CCL5 is known to form higher oligomers/aggregates in the presence of glycosaminoglycans, much more than other chemokines. It would therefore have been very interesting to see, if similar results in vitro, in situ, and in vivo could have been obtained by other chemokines of the same class (e.g. CCL2) or another class (like CXCL8).

In addition, the authors have used variously labelled CCL5 (like with the organic dye Cy3 or with EGFP) for various reasons (detection and immobilisation). In the view of this reviewer, it would have been necessary to show that all the labelled chemokines yield identical/similar molecular characteristics as the unlabelled wildtype chemokine (such as heparan sulfate binding and chemotaxis). It is well known that labelling proteins either by chemical tags or by fusion to GFPs can lead to manifestly different molecular and functional characteristics.

Reviewer #2 (Public Review):

Although the study by Xiaolin Yu et al is largely limited to in vitro data, the results of this study convincingly improve our current understanding of leukocyte migration.

(1) The conclusions of the paper are mostly supported by the data although some clarification is warranted concerning the exact CCL5 forms (without or with a fluorescent label or His-tag) and amounts/concentrations that were used in the individual experiments. This is important since it is known that modification of CCL5 at the N-terminus affects the interactions of CCL5 with the GPCRs CCR1, CCR3, and CCR5 and random labeling using monosuccinimidyl esters (as done by the authors with Cy-3) is targeting lysines. Since lysines are important for the GAG-binding properties of CCL5, knowledge of the number and location of the Cy-3 labels on CCL5 is important information for the interpretation of the experimental results with the fluorescently labeled CCL5. Was the His-tag attached to the N- or C-terminus of CCL5? Indicate this for each individual experiment and consider/discuss also potential effects of the modifications on CCL5 in the results and discussion sections.

(2) In general, the authors appear to use high concentrations of CCL5 in their experiments. The reason for this is not clear. Is it because of the effects of the labels on the activity of the protein? In most biological tests (e.g. chemotaxis assays), unmodified CCL5 is active already at low nM concentrations.

(3) For the statistical analyses of the results, the authors use t-tests. Was it confirmed that data follow a normal distribution prior to using the t-test? If not a non-parametric test should be used and it may affect the conclusions of some experiments.

Reviewer #3 (Public Review)

Summary:

The authors are trying to find out whether the levels of omega-6 and omega-3 fatty acids in the blood are linked to the likelihood of dying from anything, of dying from cancer and of dying from cardiovascular disease. They use a large dataset called UKBiobank where fatty acid levels were measured in blood at the start of the study and what happened to the participants over the following years (average of 12.7 years) was followed. They find that both omega-6 AND omega-3 fatty acids were linked with less likelihood of dying from anything, from cancer and from cardiovascular disease. The effects of omega-3s were stronger. They then made a ratio of omega-6 to omega-3 fatty acids and found that as that ratio increased risk of dying also increased. This supports the idea that omega-3s have stronger effects than omega-6s.

Strengths:

This is a large study (over 85,000 participants) with a good follow up period (average 12.7 years). Using blood levels of fatty acids is superior to using estimated dietary intakes. The authors take account of many variables that could interfere with the findings (confounding variables) - they do this using statistical methods.

Weaknesses:

UKBioBank is not entirely representative of the UK population.

Reviewer #1 (Public Review):

Summary:<br /> Honzejkova K., et al. resolved the structure of one of the MAP3K proteins. Apoptosis signal-regulating kinase 1 (ASK1) is one of the main crucial stress sensors, which directs cells toward differentiation, and apoptosis. As a result, ASK1 dysregulation has been associated with a multitude of diseases like neurodegenerative, cardiovascular, and cancer. Understanding the structural-functional interplay of ASK1 would help researchers target this member of the MAP3K proteins to develop therapeutic interventions for these disorders.

Strengths:

Major strengths:<br /> • Structure of the C-terminal truncated ASK1 protein.

Weaknesses:<br /> • Lack of ASK1:TRX1 complex structure. The authors used instead SV AUC and HDX-MS techniques to compensate for the inability to get a sufficiently stable ASK1:TRX complex.<br /> • There is not enough information about Cryo-EM data processing like 2D classification averages, local resolution of the EM map, or FSC figures.<br /> • You can't reliably report the presence of a hydrogen bond with a 3.7Å resolution.

Reviewer #2 (Public Review):

Summary:<br /> The authors attempted to solve the 3D structure of ASK1 by Cryo-EM.

Strengths:<br /> The authors solved the 3D structure of N-terminal domain s of ASK1 complexed with TRX. They found TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. The conclusions drawn from this paper are convincing and will greatly contribute to the development of new drugs targeting ASK1.

Weaknesses:<br /> To study the ASK1 structure, C-terminally truncated ASK1 was used in the study, but not the full-length form of ASK1.

Reviewer #1 (Public Review):

The manuscript by Lu et al aims to study the effects of tubulin post-translational modification in C. elegans touch receptor neurons. Authors use gene editing to engineer various predicted PTM mutations in a-tubulin MEC-12 and b-tubulin MEC-7. Authors generate and analyze an impressive battery of mutants in predicted phosphorylation site and acetylation site of b-tubulin MEC-7, K40 acetylation site in a-tubulin MEC-12, enzymatic site of the a-tubulin acetyltransferase MEC-17, and PTM sites in the MEC-12 and MEC-7 C-tails (glutamylation, detyrosination, delta-tubulin). This represents a lot of work, and will appeal to a readership interested in C. elegans touch receptor neurons. The major concern/criticism of this manuscript is whether the introduced mutation(s) directly affects a specific PTM or whether the mutation affects gene expression, protein expression/stability/localization, etc. As such, this work does convincingly demonstrate, as stated in the title, that "Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration."

For example, the authors manipulate the C-terminal tail of MEC-12 and MEC-7, to test the idea that polyglutamylation may be an important PTM. These mutants displayed subtle phenotypes. The authors show that branch point GT335 and polyglutamyation polyE recognizing antibodies stain cultured embryonic touch receptor neurons (TRNs), but did not examine staining in C. elegans TRNs in situ. To my knowledge, these antibodies have not been shown to stain the TRNs in any published papers, raising the question of how these "glutamylation" mutations are affecting mec-12 and -7. The rationale for using cultured embryonic TRNs and the relevance of the data and its interpretation are not clear.

The final paragraph of the discussion is factually incorrect. The C. elegans homologs of the CCP carboxypeptidases are called CCPP-1 and CCPP-6. There are several publications on their functions in C. elegans.

Reviewer #1 (Public Review):

In their revised manuscript Hijaze et al. adequately addressed the majority of my previous concerns in a satisfactory manner. In particular, they validated their morpholino knock-down experiments by explaining how they determined the optimal concentrations and provided an immunohistological evidence for the reduction in ROCK protein abundance. The authors also added new antibody stainings providing evidence that ROCK and F-actin do not interact directly but likely through other kinases that modulate f-actin, and that the localization of f-actin at the spicule tips remains unaffected by the knock-down. In addition, the authors revised their discussion to not overstate their observations, and by focusing on the potential mechanisms by which ROCK may affect biomineralization (i.e. mechano sensing and exocytosis of vesicles). Here I would like to add, that f-actin mediated exocytosis does not necessarily target mineral baring vesicles but may also promote the exocytosis of matrix proteins that are essential for the normal formation of the spicules and that are an integral component of other biominerals, as well. I strongly encourage the authors to continue on this exciting research, including the development of methods to analyze the molecular mechanisms that control vesicular trafficking in mineralizing systems.

Reviewer #2 (Public Review):

This project is on the role of ROCK in skeletogenesis during sea urchin development. That skeleton is produced by a small number of cells in the embryo with signaling inputs from the ectoderm providing patterning cues. The skeleton is built from secretion of CaCO3 by the skeletogenic cells. The authors conclude that ROCK is involved in the regulation of skeletogenesis with a role both in regulating actomyosin in the process, and in the gene regulatory network (GRN) underlying the entire sequence of events.

The strength of the paper is that they show in detail how perturbations of ROCK results in abnormal actomyosin activity in the skeletogenic cells, and they show alterations both in expression of transcription factors of the GRN, and expression of genes involved in assembly of the skeletal matrix. Two different approaches lead to this conclusion: morpholino perturbations and the actions of a selective inhibitor of the kinase activity. Thus, they achieved their goal which was to test the hypothesis that ROCK is involved in the process of skeletogenesis. Those tests support the hypothesis with data that was quantitatively significant.

The discussion was transparent regarding where the analysis ended and where the next phase of work should begin. While actomyosin involvement was altered when ROCK was perturbed, it isn't known how direct or indirect the role of ROCK might be. Also, while the regulatory input to spicule initiation and growth is affected when ROCK is inhibited, it isn't clear exactly where ROCK is involved.

Reviewer #1 (Public Review):

Summary:

The OSCA/TMEM63 channels have recently been identified as mechanosensitve channels. In a previous study, the authors found that OSCA subtypes (1, 2, and 3) respond differently to stretch and poke stimuli. For example, OSCA1.2 is activated by both poke and stretch, while OSCA3.1, responds strongly to stretch but poorly to poke stimuli. In this study the authors use cryo-EM, mutagenesis, and electrophysiology to dissect the mechanistic determinants that underlie the channels' ability to respond to poke and stretch stimuli.

The starting hypothesis of the study is that the mechanical activation of OSCA channels relies on the interactions between the protein and the lipid bilayer and that the differential responses to poke and stretch might stem from variations in the lipid-interacting regions of OSCA proteins. The authors specifically identify the amphipathic helix (AH), the fenestration, and the Beam Like Domain (BLD) as elements that might play a role in mechanosensing.

The authors use solid methodology to show that poke and stretch responses likely use different mechanisms in OSCA channels and that the poke response can be uncoupled from the stretch response in OSCA1.2 by mutations in the AH and the positively charged residues in the fenestration. However, the study falls short of explaining why OSCA3.1 does not respond efficiently to poke stimuli. This question is particularly important as the AH residues that are important for the poke response in OSCA1.2 are present in OSCA3.1.

Unfortuntately, due to staffing issues, the authors were unable to perform additional experiments that would address some of the critical issues that were brought up during peer review. Nevertheless, the structural and functional data presented is of high quality and the findings on OSCA1.2 will be of interest to anyone working in the fields of mechanosensation, sensory biology, and ion channels.

Reviewer #2 (Public Review):

Jojoa-Cruz et al. have submitted a revised manuscript and their responses to reviewers' comments on the major weaknesses of the paper and recommendations. The authors have made minimal changes to the manuscript itself, which highly resembles the initial submission. Most concerningly, the authors appeared to agree with reviewers' comments, but did not and are not going to carry out any of the recommended experiments, including electrophysiology [Reviewer 2- major point 3), recommended point 5; Reviewer 3- recommended point 4] and western blot [Reviewer 3- recommended point 3], by explaining that they have left the lab. The major weakness and issues raised in the previous review process therefore remain in the current version of the manuscript.

Moreover, in the public review major weakness, the reviewer pointed out issues on the inadequacy of the functional validation on the structural domains based on mutagenesis of OSCA1.2 vs. OSCA3.1 and using poke and stretch assays, as well as weakness in the corresponding mechanistic interpretation of the functional data. These issues need to be addressed or improved to a certain extent through revised study design and execution of experiments.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public Review):

Summary:

In the current manuscript, the authors find distinct roles for the calcium sensors Syt7 and Doc2alpha in the regulation of asynchronous release and calcium-dependent synaptic vesicle docking in hippocampal neurons. The authors data indicate that Doc2 functions in activating a component of asynchronous release beginning with the initial stimulus, while Syt7 does not appear to have a role at this early stage. A role for Syt7 in supporting both synchronous and asynchronous release appears during stimulation trains, where Syt7 is proposed to promote synaptic vesicle docking or capture during stimulation. Doc2 mutants show facilitation initially during a train and display higher levels of synchronous release initially, before reaching a similar plateau to controls later in the train. The authors contribute the increased synchronous release in Doc2 mutants to Syt1 having access to more SVs that can fuse synchronously. In contrast, Syt7 mutants show depression during a train, and continue to decline during stimulation. The authors contribute this to a role for Syt7 in promoting calcium-dependent SV docking and capture that feeds SVs to both synchronous and asynchronous fusion pathways. Importantly, phenotypes of a double Doc2/Syt7 mutant collapse onto the Doc2 phenotype, suggesting the two proteins are not additive in their role in supporting distinct aspects of SV release. Rapid freeze EM after stimulation provides support for a role for Syt7 in SV docking/capture at release sites, as they display less docked SVs after stimulation. In the case of Doc2, EM reveals fewer SVs fusion pits later during a stimulation, consistent with fewer asynchronous fusion events. The authors also provide modeling that supports aspects of their conclusions from the experimental data. I cannot evaluate the modeling data or the specific experimental subtlities of the GluSnFR quantification approach, as these are outside of my reviewer expertise.

Strengths:

The use of multiple approaches (optical imaging, physiology, rapid freeze EM, modeling, double mutant analysis) provides compelling support for distinct roles of the two proteins in regulating SV release.

Weaknesses:

Some of the phenotypes for both Doc2 and Syt7 mutants have been reported in the authors' prior publications. It is not clear how well the GluSnFR approach is for accurately separating synchronous versus asynchronous release kinetics. The authors also tend to overstate the significance of the two proteins for asynchronous release in general, as a significant fraction of this release component is still intact in the double mutant, indicating these two proteins are only part of the asynchronous release mechanism.

Reviewer #2 (Public Review):

Summary:

The goal of this study is to provide a deeper understanding of the roles of syt7 and Doc2 in synaptic vesicle fusion. Depending on the system studied, and the nature of the preparation, it appears that syt7 functions as a sensor for asynchronous release, synaptic facilitation, both processes, or neither. The perspective offered by Chapman, Watanabe, and colleagues varies from those previously published, and is therefore novel and interesting.

Strengths:

The strengths of the study include the complementary imaging and electrophysiology approaches for assessing the function of syt7, and the use of appropriate knockout lines. High resolution imaging approaches to measure synaptic activity is also a strength.

Weaknesses:

It is not clear to this reviewer that the computational modeling effort is important or even necessary. The study also attempts to derive kinetic information (on the ms time scale) from EM. While the interpretations are not unreasonable, they should be taken with some caution.

Overall, the study does a good job of attempting to resolve the various ambiguities existing in the field regarding the potential roles of syt7 and Doc2 in membrane fusion. There are, of course, a great number of proteins which have been identified to act at fusion sites to drive or otherwise modify release phenotypes. Efforts such as this are going to become increasingly important as we work to attribute discrete roles to each one.

Reviewer #1 (Public Review):

This fascinating paper by A.L. Schneider et al. describes voyAGEr, a shiny-based interface for easy exploration of the GTEx dataset by non- or novice programmers. Importantly, voyAGEr is open source and available from github, which could greatly accelerate additional development and further uses of this interesting tool.

The authors developed a pipeline for modeling age-related changes in gene expression in the GTEx data called ShARP-LM, fitting a linear model for age, sex and age&sex interaction terms. This pipeline underlies the later analyses that can be applied within voyAGEr. These analyses are labeled by tissue so that users can easily begin a query based on a tissue or a gene of possible interest.

voyAGEr implements many kinds of interesting R-based tools such as pathway overrepresentation analysis and gene co-expression module analysis, in a way that akes these approaches accessible to non-bioinformaticist aging researchers.

As the tidal wave of publicly available large, high-dimensional datasets such as transcriptomes continues to grow exponentially, the usefulness of tools such as voyAGEr will only increase. While test users may be able to imagine features or refinements they wish were already present, due to the open source approach they or anyone else including but not limited to the present authors can implement additional features in the future. I look forward to using this tool and to staying abreast of its future development.

Overall, this study describes a new tool of interest to the field. The manuscript is clearly written overall, with a few minor suggested corrections, as noted below. The figures and supplementary information are all clear and all add to the manuscript.

Reviewer #2 (Public Review):

The purpose of this study is to develop a tool that serves as a starting point for investigating and uncovering genes and pathways associated with aging. The tool utilizes information from the GTEx public database, which contains post-mortem human data. It focuses on identifying age-related gene expression changes across different age range, biological sexes, and medical histories, with a focus on specific tissues.

Additionally, the authors envision the platform as continuously evolving, with ongoing development and expansion to include new data and features, ensuring it remains a cutting-edge resource for researchers studying aging.

voyAGEr presents a tool for exploring gene expression changes across multiple tissues in the context of aging. One of the main strengths of the tool is its intuitive and user-friendly interface, which allows for easy navigation and exploration of gene expression patterns for biologists. Users can explore changes in gene expression of single genes across multiple tissues, enabling them to identify genes of interest that can be further investigated.

A particularly noteworthy strength of the tool is its ability to show tissue-specific gene expression patterns. This feature is essential for elucidating the paradigm of tissue-specific asynchronous aging and provides a unique and valuable resource for the aging community.

However, the choice of the R shiny platform for visualization may not be the most conducive to extensibility and open-source collaboration, owing to its lack of modularity. Alternatives like Flask or FastAPI, which are more production-oriented, could be more appropriate. Additionally, despite using preprocessed data and functioning primarily as a visualization platform, the tool occasionally experiences lag, indicating room for performance improvement. These aspects are worth considering for future versions of the tool.

Overall, voyAGEr offers an entry point for further investigation of genes involved in aging, and its ability to show tissue-specific gene expression patterns provides a unique and valuable resource for the scientific community.

Finally, the tool is complemented by a comprehensive tutorial that elucidates each functionality and includes examples. The authors have shared the code for preprocessing and the tool itself. They also acknowledge the limitations of the statistical inference tests and their interpretation in the manuscript, contributing to its transparency.

Reviewer #3 (Public Review):

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

Reviewer #2 (Public Review):

Summary: In the revised manuscript, the authors aim to investigate brain-wide activation patterns following administration of the anesthetics ketamine and isoflurane, and conduct comparative analysis of these patterns to understand shared and distinct mechanisms of these two anesthetics. To this end, they perform Fos immunohistochemistry in perfused brain sections to label active nuclei, use a custom pipeline to register images to the ABA framework and quantify Fos+ nuclei, and perform multiple complementary analyses to compare activation patterns across groups.

In the latest revision, I am happy to say that the authors have greatly improved their manuscript. The data are now well analyzed and the experiments fully described. They addressed all of my concerns. It is an interesting study.

Reviewer #3 (Public Review):

The present study presents a comprehensive exploration of the distinct impacts of Isoflurane and Ketamine on c-Fos expression throughout the brain. To understand the varying responses across individual brain regions to each anesthetic, the researchers employ principal component analysis (PCA) and c-Fos-based functional network analysis. The methodology employed in this research is both methodical and expansive. Notably, the utilization of a custom software package to align and analyze brain images for c-Fos positive cells stands out as an impressive addition to their approach. This innovative technique enables effective quantification of neural activity and enhances our understanding of how anesthetic drugs influence brain networks as a whole.

The primary novelty of this paper lies in the comparative analysis of two anesthetics, Ketamine and Isoflurane, and their respective impacts on brain-wide c-Fos expression. The study reveals the distinct pathways through which these anesthetics induce loss of consciousness. Ketamine primarily influences the cerebral cortex, while Isoflurane targets subcortical brain regions. This finding highlights the differing mechanisms of action employed by these two anesthetics-a top-down approach for Ketamine and a bottom-up mechanism for Isoflurane. Furthermore, this study uncovers commonly activated brain regions under both anesthetics, advancing our knowledge about the mechanisms underlying general anesthesia.

Reviewer #1 (Public Review):

Summary:

In the paper "Disentangling the relationship between cancer mortality and COVID-19", the authors study whether the number of deaths in cancer patients in the USA went up or down during the first year (2020) of the COVID-19 pandemic. They found that the number of deaths with cancer mentioned on the death certificate went up, but only moderately. In fact, the excess with-cancer mortality was smaller than expected if cancer had no influence on the COVID mortality rate and all cancer patients got COVID with the same frequency as in the general population. The authors conclude that the data show no evidence of cancer being a risk factor for COVID and that the cancer patients were likely actively shielding themselves from COVID infections.

Strengths:

The paper studies an important topic and uses sound statistical and modeling methodology. It analyzes both, deaths with cancer listed as the primary cause of death, as well as deaths with cancer listed as one of the contributing causes. The authors argue, correctly, that the latter is a more important and reliable indicator to study relationships between cancer and COVID. The authors supplement their US-wide analysis by analysing three states separately.

Weaknesses:

The main findings of the paper can be summarized as six numbers. Nationally, in 2022, multiple-cause cancer deaths went up by 2%, Alzheimer's deaths by 31%, and diabetes deaths by 39%. At the same time, assuming no relationship between these diseases and either Covid infection risk or Covid mortality risk, the deaths should have gone up by 7%, 46%, and 28%. The authors focus on cancer deaths and as 2% < 7%, conclude that cancer is not a risk factor for COVID and that cancer patients must have "shielded" themselves against Covid infections.

However, I did not find any discussion of the other two diseases. For diabetes, the observed excess was 39% instead of "predicted by the null model" 28%. I assume this should be interpreted as diabetes being a risk factor for Covid deaths. I think this should be spelled out, and also compared to existing estimates of increased Covid IFR associated with diabetes.

And what about Alzheimer's? Why was the observed excess 31% vs the predicted 46%? Is this also a shielding effect? Does the spring wave in NY provide some evidence here? Why/how would Alzheimer's patients be shielded? In any case, this needs to be discussed and currently, it is not.

Reviewer #2 (Public Review):

The article is very well written, and the approach is quite novel. I have two major methodological comments, that if addressed will add to the robustness of the results.

(1) Model for estimating expected mortality. There is a large literature using a different model to predict expected mortality during the pandemic. Different models come with different caveats, see the example of the WHO estimates in Germany and the performance of splines (Msemburi et al Nature 2023 and Ferenci BMC Medical Research Methodology 2023). In addition, it is a common practice to include covariates to help the predictions (e.g., temperature and national holidays, see Kontis et al Nature Medicine 2020). Last, fitting the model-independent for each region, neglects potential correlation patterns in the neighbouring regions, see Blangiardo et al 2020 PlosONE.

Based on the above:<br /> a. I believe that the authors need to run a cross-validation to justify model performance. I would suggest training the data leaving out the last year for which they have mortality and assessing how the model predicts forward. Important metrics for the prediction performance include mean square error and coverage probability, see Konstantinoudis et al Nature Communications 2023. The authors need to provide metrics for all regions and health outcomes.

b. In the context of validating the estimates, I think the authors need to carefully address the Alzheimer case, see Figure 2. It seems that the long-term trends pick an inverse U-shape relationship which could be an overfit. In general, polynomials tend to overfit (in this case the authors use a polynomial of second degree). It would be interesting to see how the results change if they also include a cubic term in a sensitivity analysis.

c. The authors can help with the predictions using temperature and national holidays, but if they show in the cross-validation that the model performs adequately, this would be fine.

d. It would be nice to see a model across the US, accounting for geography and spatial correlation. If the authors don't want to fit conditional autoregressive models in the Bayesian framework, they could just use a random intercept per region.

(2) I think the demographic model needs further elaboration. It would be nice to show more details, the mathematical formula of this model in the supplement, and explain the assumptions.

Reviewer #3 (Public Review):

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

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

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

Reviewer #1 (Public Review):

Summary:<br /> This manuscript from Mukherjee et al examines potential connections between telomere length and tumor immune responses. This examination is based on the premise that telomeres and tumor immunity have each been shown to play separate, but important, roles in cancer progression and prognosis as well as prior correlative findings between telomere length and immunity. In keeping with a potential connection between telomere length and tumor immunity, the authors find that long telomere length is associated with reduced expression of the cytokine receptor IL1R1. Long telomere length is also associated with reduced TRF2 occupancy at the putative IL1R1 promoter. These observations lead the authors towards a model in which reduced telomere occupancy of TRF2 - due to telomere shortening - promotes IL1R1 transcription via recruitment of the p300 histone acetyltransferase. This model is based on earlier studies from this group (i.e. Mukherjee et al., 2019) which first proposed that telomere length can influence gene expression by enabling TRF2 binding and gene transactivation at telomere-distal sites. Further mechanistic work suggests that G-quadruplexes are important for TRF2 binding to IL1R1 promoter and that TRF2 acetylation is necessary for p300 recruitment. Complementary studies in human triple-negative breast cancer cells add potential clinical relevance but do not possess a direct connection to the proposed model. Overall, the article presents several interesting observations, but disconnection across central elements of the model and the marginal degree of the data leave open significant uncertainty regarding the conclusions.

Strengths:<br /> Many of the key results are examined across multiple cell models.

The authors propose a highly innovative model to explain their results.

Weaknesses:<br /> Although the authors attempt to replicate most key results across multiple models, the results are often marginal or appear to lack statistical significance. For example, the reduction in IL1R1 protein levels observed in HT1080 cells that possess long telomeres relative to HT1080 short telomere cells appears to be modest (Supplementary Figure 1I). Associated changes in IL1R1 mRNA levels are similarly modest.

Related to the point above, a lack of strong functional studies leaves an open question as to whether observed changes in IL1R1 expression across telomere short/long cancer cells are biologically meaningful.

Statistical significance is described sporadically throughout the paper. Most major trends hold, but the statistical significance of the results is often unclear. For example, Figure 1A uses a statistical test to show statistically significant increases in TRF2 occupancy at the IL1R1 promoter in short telomere HT1080 relative to long telomere HT1080. However, similar experiments (i.e. Figure 2B, Figure 4A - D) lack statistical tests.

Reviewer #2 (Public Review):

This study highlights the role of telomeres in modulating IL-1 signaling and tumor immunity. The authors demonstrate a strong correlation between telomere length and IL-1 signaling by analyzing TNBC patient samples and tumor-derived organoids. Mechanistic insights revealed non-telomeric TRF2 binding at the IL-1R1. The observed effects on NF-kB signaling and subsequent alterations in cytokine expression contribute significantly to our understanding of the complex interplay between telomeres and the tumor microenvironment. Furthermore, the study reports that the length of telomeres and IL-1R1 expression is associated with TAM enrichment. However, the manuscript lacks in-depth mechanistic insights into how telomere length affects IL-1R1 expression. Overall, this work broadens our understanding of telomere biology.

Reviewer #2 (Public Review):

In this study, the authors address discrepancies in determining the local bacterial burden in osteomyelitis between that determined by culture and enumeration by DNA-directed assay. Discrepancies between culture and other means of bacterial enumeration are long established and highlighted by Staley and Konopka's classic, "The great plate count anomaly" (1985). Here, the authors first present data demonstrating the emergence of discrepancies between CFU counts and genome copy numbers detected by PCR in S. aureus strains infecting osteocyte-like cells. They go on to demonstrate PCR evidence that S. aureus can be detected in bone samples from sites meeting a widely accepted clinicopathological definition of osteomyelitis. They conclude their approach offers advantages in quantifying intracellular bacterial load in their in vitro "co-culture" system.

Weaknesses<br /> - My main concern here is the significance of these results outside the model osteocyte system used by this group. Although they carefully avoid over-interpreting their results, there is a strong undercurrent suggesting their approach could enhance aetiologic diagnosis in osteomyelitis and that enumeration of the infecting pathogen might have clinical value. In the first place, molecular diagnostics such as 16S rDNA-directed PCR are well established in identifying pathogens that don't grow. Secondly, it is hard to see how enumeration could have value beyond in vitro and animal model studies since serial samples will rarely be available from clinical cases.

- I have further concerns regarding the interpretation of the combined bacterial and host cell-directed PCRs against the CFU results. Significance is attached to the relatively sustained genome counts against CFU declines. On the one hand, it must be clearly recognised that the detection of bacterial genomes does not equate to viable bacterial cells with the potential for further replication or production of pathogenic factors. Of equal importance is the potential contribution of extracellular DNA from lysed bacteria and host cells to these results. The authors must clarify what steps, if any, they have taken to eliminate such contributions for both bacteria and host cells. Even the treatment with lysotaphin may have coated their osteocyte cultures with bacterial DNA, contributing downstream to the ddPCR results presented.

Strengths<br /> - On the positive side, the authors provide clear evidence for the value of the direct buffer extraction system they used as well as confirming the utility of ddPCR for quantification. In addition, the successful application of MinION technology to sequence the EF-Tu amplicons from clinical samples is of interest.

- Moreover, the phenomenology of the infection studies indicating greater DNA than CFU persistence and differences between the strains and the different MOI inoculations are interesting and well-described, although I have concerns regarding interpretation.

Reviewer #1 (Public Review):

Summary:<br /> This work shows, based on basic laboratory investigations of in-vitro-grown bacteria as well as human bone samples, that conventional bacterial culture can substantially underrepresent the quantity of bacteria in infected tissues. This has often been mentioned in the literature, however, relatively limited data has been provided to date. This manuscript compares culture to a digital droplet PCR approach, which consistently showed greater levels of bacteria across the experiments (and for two different strains).

Strengths:<br /> Consistency of findings across in vitro experiments and clinical biopsies. There are real-world clinical implications for the findings of this study.

Weaknesses:<br /> No major weaknesses. Only three human samples were analyzed, although the results are compelling.

Reviewer #1 (Public Review):

This manuscript presents an extremely exciting and very timely analysis of the role that the nucleosome acidic patch plays in SWR1-catalyzed histone exchange. Intriguingly, SWR1 loses activity almost completely if any of the acidic patches are absent. To my knowledge, this makes SWR1 the first remodeler with such a unique and pronounced requirement for the acidic patch. The authors demonstrate that SWR1 affinity is dramatically reduced if at least one of the acidic patches is absent, pointing to a key role of the acidic patch in SWR1 binding to the nucleosome. The authors also pinpoint a specific subunit - Swc5 - that can bind nucleosomes, engage the acidic patch, and obtain a cryo-EM structure of Swc5 bound to a nucleosome. They also identify a conserved arginine-rich motif in this subunit that is critical for nucleosome binding and histone exchange in vitro and for SWR1 function in vivo. The authors provide evidence that suggests a direct interaction between this motif and the acidic patch.

Strengths:<br /> The manuscript is well-written and the experimental data are of outstanding quality and importance for the field. This manuscript significantly expands our understanding of the fundamentally important and complex process of H2A.Z deposition by SWR1 and would be of great interest to a broad readership.

Reviewer #2 (Public Review):

Summary:<br /> In this study, Baier et al. investigated the mechanism by which SWR1C recognizes nucleosomal substrates for the deposition of H2A.Z. Their data convincingly demonstrate that the nucleosome's acidic patch plays a crucial role in the substrate recognition by SWR1C. The authors presented clear evidence showing that Swc5 is a pivotal subunit involved in the interaction between SWR1C and the acidic patch. They pared down the specific region within Swc5 responsible for this interaction. However, two central assertions of the paper are less convincing. First, the data supporting the claim that the insertion of one Z-B dimer into the canonical nucleosome can stimulate SWR1C to insert the second Z-B dimer is somewhat questionable (see below). Given that this claim contradicts previous observations made by other groups, this hypothesis needs further testing to eliminate potential artifacts. Secondly, the claim that SWR1C simultaneously recognizes the acidic patch on both sides of the nucleosome also needs further investigation, as the assay used to establish this claim lacks the sensitivity necessary to distinguish any difference between nucleosomal substrates containing one or two intact acidic patches.

Strengths:<br /> As mentioned in the summary, the authors presented clear evidence demonstrating the role of Swc5 in recognition of the nucleosome acidic patch. The identification of the specific region in Swc5 responsible for this interaction is important.

Weaknesses:

Major comments:

(1) Figure 1B: It is unclear how much of the decrease in FRET is caused by the bleaching of fluorophores. The authors should include a negative control in which Z-B dimers are omitted from the reaction. In the absence of ZB dimers, SWR1C will not exchange histones. Therefore, any decrease in FRET should represent the bleaching of fluorophores on the nucleosomal substrate, allowing normalization of the FRET signal related to A-B eviction.

(2) Figure S3: The authors use the decrease in FRET signal as a metric of histone eviction. However, Figure S3 suggests that the FRET signal decrease could be due to DNA unwrapping. Histone exchange should not occur when SWR1C is incubated with AMP-PNP, as histone exchange requires ATP hydrolysis (10.7554/eLife.77352). And since the insertion of Z-B dimer and the eviction of A-B dimer are coupled, the decrease of FRET in the presence of AMP-PNP is unlikely due to histone eviction or exchange. Instead, the FRET decrease is likely due to DNA unwrapping (10.7554/eLife.77352). The authors should explicitly state what the loss of FRET means.

(3) Related to point 2. One way to distinguish nucleosomal DNA unwrapping from histone dimer eviction is that unwrapping is reversible, whereas A-B eviction is not. Therefore, if the authors remove AMP-PNP from the reaction chamber and a FRET signal reappears, then the initial loss of FRET was due to reversible DNA unwrapping. However, if the removal of AMP-PNP did not regain FRET, it means that the loss of FRET was likely due to A-B eviction. The authors should perform an AMP-PNP and/or ATP removal experiment to make sure the interpretation of the data is correct.

(4) The nature of the error bars in Figure 1C is undefined; therefore, the statistical significance of the data is not interpretable.

(5) The authors claim that the SWR1C requires intact acidic patches on both sides of the nucleosomes to exchange histone. This claim was based on the experiment in Figure 1C where they showed mutation of one of two acidic patches in the nucleosomal substrate is sufficient to inhibit SWR1C-mediated histone exchange activity. However, one could argue that the sensitivity of this assay is too low to distinguish any difference between nucleosomes with one (i.e., AB/AB-apm) versus two mutated acidic patches (i.e., AB-apm/AB-apm). The lack of sensitivity of the eviction assay can be seen when Figure 1B is taken into consideration. In the gel-shift assay, the AB-apm/AB-apm nucleosome exhibited a 10% SWR1C-mediated histone exchange activity compared to WT. However, in the eviction assay, the single AB/AB-apm mutant has no detectable activity. Therefore, to test their hypothesis, the authors should use the more sensitive in-gel histone exchange assay to see if the single AB/AB-apm mutant is more or equally active compared to the double AB-apm/AB-apm mutant.

(6) The authors claim that the AZ nucleosome is a better substrate than the AA nucleosome. This is a surprising result as previous studies showed that the two insertion steps of the two Z-B dimers are not cooperative (10.7554/eLife.77352 and 10.1016/J.CELREP.2019.12.006). The authors' claim was based on the eviction assay shown in Fig 1C. However, I am not sure how much variation in the eviction assay is contributed by different preparations of nucleosomes. The authors should use the in-gel assay to independently test this hypothesis.

Minor comments:

(1) Abstract line 4: To say 'Numerous' studies have shown acidic patch impact chromatin remodeling enzymes activity may be too strong.

(2) Page 15, line 15: The authors claim that swc5∆ was inviable on formamide media. However, the data in Figure 8 shows cell growth in column 1 of swc5∆.

(3) The authors should use standard yeast nomenclature when describing yeast genes and proteins. For example, for Figure 8 and legend, Swc5∆ was used to describe the yeast strain BY4741; MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YBR231c::kanMX4. Instead, the authors should describe the swc5∆ mutant strain as BY4741 MAT a his3∆1 leu2∆0 met15∆0 ura3∆0 swc5∆::kanMX4. Exogenous plasmid should also be indicated in italics and inside brackets, such as [SWC5-URA3] or [swc5(R219A)-URA3].

(4) According to Lin et al. 2017 NAR (doi: 10.1093/nar/gkx414), there is only one Swc5 subunit per SWR1C. Therefore, the pincher model proposed by the authors would suggest that there is a missing subunit that recognizes the second acidic patch. The authors should point out this fact in the discussion. However, as mentioned in Major comment 6, I am not sure if the pincer model is substantiated.

Reviewer #2 (Public Review):

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

Comments on revised version:

I have nothing to add and am satisfied with the rebuttal.

Reviewer #1 (Public Review):

This paper is of importance to scientists interested in molecular mechanisms by which actin point mutations affect its function to ultimately lead to disease states. This work thoroughly characterizes the effect of the E334Q mutation in cytoplasmic gamma-actin on two binding partners: cofilin and myosin (non-muscle myosin 2 and myosin 5). Overall, the data showing effects on cofilin function and myosin binding are convincing and the experiments performed expertly using state-of-the art approaches. Additional binding partners of actin that were not examined here may also have altered function when interacting with the mutant actin.

Comments on revised version:

The authors seem to have done a pretty thorough job with the rebuttal.

Reviewer #1 (Public Review):

Summary:

This study, titled "Enhancing Bone Regeneration and Osseointegration using rhPTH(1-34) and Dimeric R25CPTH(1-34) in an Osteoporotic Beagle Model," provides valuable insights into the therapeutic effects of two parathyroid hormone (PTH) analogs on bone regeneration and osseointegration. The research is methodologically sound, employing a robust animal model and a comprehensive array of analytical techniques, including micro-CT, histological/histomorphometric analyses, and serum biochemical analysis.

Strengths:

The use of a large animal model, which closely mimics postmenopausal osteoporosis in humans, enhances the study's relevance to clinical applications. The study is well-structured, with clear objectives, detailed methods, and a logical flow from introduction to conclusion. The findings are significant, demonstrating the potential of rhPTH(1-34) and dimeric R25CPTH(1-34) in enhancing bone regeneration, particularly in the context of osteoporosis.

Weaknesses:

There are no major weaknesses.

Reviewer #2 (Public Review):

Summary:

This article explores the regenerative effects of recombinant PTH analogues on osteogenesis.

Strengths:

Although PTH has known to induce the activity of osteoclasts, accelerating bone resorption, paradoxically its intermittent use has become a common treatment for osteoporosis. Previous studies successfully demonstrated this phenomenon in vivo, but most of them used rodent animal models, inevitably having a limitation. In this article, the authors tried to address this, using a beagle model, and assessed the osseointegrative effect of recombinant PTH analogues. As a result, the authors clearly observed the regenerative effects of PTH analogues, and compared the efficacy, using histologic, biochemical, and radiologic measurement for surgical-endocrinal combined large animal models. The data seem to be solid, and has potential clinical implications.

Weaknesses:

As PTH's mechanism has already been widely accepted, and the main focus of this article was to compare the preclinical efficacy of PTH analogues, the lack of detail biologic mechanism could be allowed. However, there are some suggestions to enhance the readability of the article:

First, the authors should clarify why they compared the effects of rhPTH(1-34) and of dimeric R25C2 PTH(1-34)? In most of the parameters, rhPTH(1-34) seems to be superior to dimeric R25C2 PTH(1-34). Why did the authors insist that the anabolic effects of dimer were prominent? Even though implication of dimeric R25C2 PTH(1-34) was drawn from genetic mutation studies, the authors should describe more clearly in the discussion the potential clinical benefits of the dimeric R25C2 PTH(1-34) compared to rhPTH(1-34), especially if dimeric R25C2 PTH(1-34) has just partial agonistic effect in pharmacodynamics.

Second, please describe the intermittent and continuous application of PTH analogues. Many of the readers may misunderstand that the authors' daily injection of PTHs were actually to mimic the clinical intermittent application or continuous one. Incorporation of the author's intention for experimental design would be more helpful for readers.

Third, please unify the nomenclature. Ensure consistency in the nomenclature throughout the article. Unify the naming conventions for PTH analogues, such as rhPTH(1-34) vs teriparatide and (Cys25)PTH(1-84) vs R25CPTH(1-34) vs R25CPTH(1-34) vs (1-84). Choose one nomenclature for each analogue and use it consistently throughout the article.

Overall, this paper is well-written, but these suggestions aim to improve clarity and consistency for a broader readership.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public Review):

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

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

Reviewer #1 (Public Review):

In the present study, the authors carefully evaluated the metabolic effects of intermittent fasting on normal chow and HFD fed mice and reported that intermittent fasting induces beiging of subcutaneous white adipose tissue. By employing complementary mouse models, the authors provided compelling evidence to support a mechanism through ILC3/IL-22/IL22R pathway. They further performed comprehensive single-cell sequencing analyses of intestinal immune cells from lean, obese, obese undergone intermittent fasting mice and revealed altered interactome in intestinal myeloid cells and ILC3s by intermittent fasting via activating AhR. Overall, this is a very interesting and timely study uncovering a novel connection between intestine and adipose tissue in the context of executing metabolic benefits of intermittent fasting.

(1) The authors showed increased plasma IL-22 and its expression in intestine. Are intestinal ILC3s the main source of plasma IL-22?

(2) The authors transplanted intestinal ILC3s from NCD mice to DIO mice and showed significant metabolic improvements. However, in Fig. 1, intermittent fasting increased IL-22-positive ILC3s proportion rather than changing the total number. Please clarify whether this transplantation is due to increasing ILC3s number or introducing more IL-22 positive ILC3s (which are decreased in DIO). Are these transplanted ILC3s by default homing to intestine rather than to other tissues?

(3) The authors adopted cold challenge at 4 degree for 6 hours to assess beiging in subcutaneous WAT and showed difference in core temperature. However, thermogenesis in this acute cold challenge is mainly by brown adipose tissue. Beiging is a chronic and adaptive response. Based on the data in WAT, there is a beiging phenotype, but the core body temperature in acute cold challenge is not an accurate readout. It would be a missed opportunity by not evaluating thermogenic activity in BAT.<br /> More browning genes should be included to strengthen the beiging phenotype of WAT. Moreover, inflammation in WAT can be examined to provide a whole picture of adipose tissue remodeling through this pathway.

(4) For the SVF beige adipocyte differentiation, 100 ng/mL IL-22 was used. This is highly above the physiological concentration at ~5 pg/mL. Please justify this high concentration used.

The authors showed increased Ucp1 and Cidea expression by IL-22 treatment in SVFs. Please be aware that these increases are likely due to boosted adipogenesis as told by the morphology. Please examine more adipogenic markers to confirm. Is this higher adipogenesis caused by the high concentration of IL-22?<br /> In line 201, the authors drew the conclusion that IL-22 increased SVF beige differentiation. To fully support this conclusion, the authors should assure adipogenesis at the same baseline and then compare beiging, or examine the effect of IL-22 on normal adipogenesis to compare with beige differentiation.

Reviewer #2 (Public Review):

Summary:<br /> This study aims to investigate the mediatory role of intestinal ILC3-derived IL-22 in intermittent fasting-elicited metabolic benefits.

Strengths:<br /> The observation of induction of IL-22 production by intestinal ILC3 is significant, and the scRNAseq provides new information into intestine-resident immune cell profiling in response to repeated fasting and refeeding.

Weaknesses:<br /> The experimental design for some studies needs to be improved to enhance the rigor of overall study. There is a lack of direct evidence showing that the metabolically beneficial effects of IF are mediated by intestinal ILC3 and their derived IL-22. The mechanism by which IL-22 induces thermogenic program is unknown. The browning effect induced by IF may involve constitutive activation of lipolysis, which was not considered.

Majority of weaknesses have been addressed in the revision. Based on the analysis of thermogenic genes in addition to Ucp1 (Fig. 4D and S6F), the alteration on thermogenesis induced by IL-22 is dependent on UCP1 but not other markers such as PGC1a, PPARg, and Cidea. The data need to be discussed in the Section of Discussion.

Reviewer #1 (Public Review):

• A summary of what the authors were trying to achieve.

The authors cultured pre- and Post-vaccine PBMCs with overlapping peptides encoding S protein in the presence of IL-2, IL-7, and IL-15 for 10 days, and extensively analyzed the T cells expanded during the culture; by including scRNAseq, scTCRseq, and examination of reporter cell lines expressing the dominant TCRs. They were able to identify 78 S epitopes with HLA restrictions (by itself represents a major achievement) together with their subset, based on their transcriptional profiling. By comparing T cell clonotypes between pre- and post-vaccination samples, they showed that a majority of pre-existing S-reactive CD4+ T cell clones did not expand by vaccinations. Thus, the authors concluded that highly-responding S-reactive T cells were established by vaccination from rare clonotypes.

• An account of the major strengths and weaknesses of the methods and results.

Strengths:

• Selection of 4 "Ab sustainers" and 4 "Ab decliners" from 43 subjects who received two shots of mRNA vaccinations.<br /> • Identification of S epitopes of T cells together with their transcriptional profiling. This allowed the authors to compare the dominant subsets between sustainers and decliners.

Weaknesses were properly addressed in the revised manuscript, and I do not have any additional concerns.

Reviewer #3 (Public Review):

Summary: The paper aims to investigate the relationship between anti-S protein antibody titers with the phenotypes&clonotypes of S-protein-specific T cells, in people who receive SARS-CoV2 mRNA vaccines. To do this, the paper recruited a cohort of Covid-19 naive individuals that receives the SARS-CoV2 mRNA vaccines and collect sera and PBMCs samples on different timepoints. Then they mainly generate three sets of data: 1). Anti-S protein antibody titers on all timepoints. 2) Single-cell RNAseq/TCRseq dataset for divided T cells after stimulation by S-protein for 10 days. 3) Corresponding epitopes for each expanded TCR clones. After analyzing these result, the paper reports two major findings&claims: A) Individuals having sustained anti-S protein antibody response also have more so-called Tfh cells in their single-cell dataset. B). S-reactive T cells do exist before the vaccination, but they seems to be unable to response to Covid-19 vaccination properly.

The paper's strength is it uses a very systemic and thorough strategy trying to dissect the relationship between antibody titers, T cell phenotypes, TCR clonotypes and corresponding epitopes, and indeed it reports several interesting findings about the relationship of Tfh clonotypes/sustained antibody and about the S-reactive clones that exist before the vaccination. The conclusion is solid in general but some claims are overstated. My suggestion is the authors should further limit their claims in abstract, for example,

"Even before vaccination, S-reactive CD4+ T cell clonotypes did exist, most of which (MAY) cross-reacted with environmental or symbiotic bacteria" -- The paper don't have experimental evidence to show these TCR clones respond to these epitopes.

"These results suggest that de novo acquisition of memory Tfh-like cells upon vaccination (LIKELY) contributes to the longevity of anti-S antibody titers." --Given the small sample size and the statistical analysis was not significant, this claim was overstated.

"S-reactive T cell clonotypes detected immediately after 2nd vaccination polarized to follicular helper T (Tfh)-like cells (UNDER IN VITRO CULTURE)". -- the conclusion was based on vitro cultured cells, which had limitation.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors induced large doxorubicin-resistant (L-DOXR) cells by generating DOX gradients using their Cancer Drug Resistance Accelerator (CDRA) chip. The L-DOXR cells showed enhanced proliferation rates, migration capacity, and carcinogenesis. Then the authors identified that the chemoresistance of L-DOXR cells is caused by failed epigenetic control of NUPR1/HDAC11 axis.

Strengths:

- Chemoresistant cancer cells were generated using a novel technique and their oncogenic properties were clearly demonstrated using both in vivo and in vitro analysis.<br /> - The mechanisms of chemoresistance of the L-DOXR cells could be elucidated using in vivo chemoresistant xenograft models, an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis.<br /> - This technique has great capability to be used for understanding the chemoresistant mechanisms of tumor cells.

Reviewer #1 (Public Review):

Lim W et al. investigated the mechanisms underlying doxorubicin resistance in triple negative breast cancer cells (TNBC). They use a new multifluidic cell culture chamber to grow MB-231 TNBC cells in the presence of doxorubicin and identify a cell population of large, resistant MB-231 cells they term L-DOXR cells. These cells maintain resistance when grown as a xenograft model, and patient tissues also display evidence for having cells with large nuclei and extra genomic content. RNA-seq analysis comparing L-DOXR cells to WT MB-231 cells revealed upregulation of NUPR1. Inhibition or knockdown of NUPR1 resulted in increased sensitivity to doxorubicin. NUPR1 expression was determined to be regulated via HDAC11 via promoter acetylation. The data presented could be used as a platform to understand resistance mechanisms to a variety of cancer therapeutics.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public Review):

Summary:<br /> This is a very well-written manuscript by Saenz de Meira and colleagues on a careful study reporting on the key role of glutamate transporter vGlut2 expression in the neurons of the ventral perimammillary nucleus (PMv) of the hypothalamus expressing the leptin receptor LepRb in energy homeostasis, puberty, and estrous cyclicity. The authors first show using cre-dependent chemogenetic viral tools that the selective activation of the PMv LepRb induces luteinizing hormone (LH) release. Then the authors demonstrate that the selective invalidation of vGlut2 in LepRb-expressing cells in the all body induces obesity and mild alteration of sexual maturation in both sexes and blunted estrous cyclicity in females. Finally, the authors knock out vGlut2 in PMv neurons in which they reintroduce LepRb expression in an otherwise LepRb-null background using an AAV Cre approach. This latter very elegant experiment shows that while the sole re-expression of LepRb in PMv neurons in LepRb-null mice was shown before to restore puberty onset, deleting vGlut2 in LepRb-expressing PMv neurons blunts this effect.

Strengths:<br /> The authors employ state-of-the-art methods and their conclusions are robustly supported by the results.

Weaknesses:<br /> None identified. Only minor comments have been formulated.

Reviewer #1 (Public Review):

Summary:<br /> In previous work, the Elias group has shown that leptin-sensing PMv neurons make connections with the neuroendocrine reproductive axis and are involved in reproductive function/s. Sáenz de Miera et al. build on this body of work to investigate the sufficiency of leptin sensing PMv neurons to evoke the release of luteinizing hormone. The team further investigates how glutamate signaling from leptin-sensing neurons can influence pubertal timing in females, along with mature estrous cycles. Genetic ablation of Slc17a6 (Vglut2) from LepRb-expressing cells resulted in a delay of the first estrus cycle post-pubertal transition, along with a significantly lengthened estrous cycle in mature females. However, this deficit did not lengthen the latency to the birth of the first litter in experimental dams. Restoration of leptin signaling in LepRb PMv neurons was previously shown to induce puberty and instate reproductive function in LepRb knock-out female mice (Mahany et al., 2018). Here, Sáenz de Miera et al. use a combined genetic and viral strategy to demonstrate that glutamate signaling in LepRb PMv neurons is required for sexual maturation in LepRb knock-out female mice.

Strengths:<br /> Most of the experiments performed in this manuscript are well-justified and rigorously tested. The genetic method to simultaneously remove glutamate signaling and restore the leptin receptor in LepRb PMv neurons was well executed and showed that glutamate signaling in LepRb PMv neurons is necessary for leptin-dependent fertility.

Weaknesses:<br /> Analysis of experimentally induced luteinizing hormone release could be confounded by spontaneous pulses of luteinizing hormone that are independent of LepRb PMv neurons.

Reviewer #3 (Public Review):

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

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

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

Reviewer #1 (Public Review):

Summary:<br /> In this study, Maestri et al. use an integrative framework to study the evolutionary history of coronaviruses. They find that coronaviruses arose recently rather than having undergone ancient codivergences with their mammalian hosts. Furthermore, recent host switching has occurred extensively, but typically between closely related species. Humans have acted as an intermediate host, especially between bats and other mammal species.

Strengths:<br /> The study draws on a range of data sources to reconstruct the history of virus-host codivergence and host switching. The analyses include various tests of robustness and evaluations through simulation.

Weaknesses:<br /> The analyses are limited to a single genetic marker (RdRp) from coronaviruses, but using other sections of the genome might lead to different conclusions. The genetic marker also lacks resolution for recent divergences, which precludes the detailed examination of recent host switches. Careful and detailed reconstruction of the timescale would be helpful for clarifying the evolutionary history of coronaviruses alongside their hosts.

Reviewer #2 (Public Review):

Summary:<br /> In their study titled "Recent evolutionary origin and localized diversity hotspots of mammalian coronaviruses," authors Benoît Perez-Lamarque, Renan Maestri, Anna Zhukova, and Hélène Morlon investigate the complex evolutionary history of coronaviruses, particularly those affecting mammals, including humans. The study focuses on unraveling the evolutionary trajectory of these viruses, which have shown a high propensity for causing pandemics, as evidenced by the SARS-CoV2 outbreak.

The research addresses a significant gap in our understanding of the evolutionary dynamics of coronaviruses, particularly their history, patterns of host-to-host transmission, and geographical spread. These aspects are important for predicting and managing future pandemic scenarios.

Historically, studies have employed cophylogenetic tests to explore virus-host relationships within the Coronaviridae family, often suggesting a long history of virus-host codiversification spanning millions of years. However, the team led by Perez-Lamarque proposes a novel phylogenetic framework that contrasts this traditional view. Their approach, which involves adapting gene tree-species tree reconciliation, is designed to robustly test the validity of two competing scenarios: an ancient origination and codiversification versus a more recent emergence and diversification through host switching.

Upon applying this innovative framework to the study of coronaviruses and their mammalian hosts, the authors' findings challenge the prevailing notion of a deep evolutionary history. Instead, their results strongly support a scenario where coronaviruses have a more recent origin, likely in bat populations, followed by diversification predominantly through host-switching events. This diversification, interestingly, seems to occur preferentially within mammalian orders.

A critical aspect of their findings is the identification of hotspots of coronavirus diversity, particularly in East Asia and Europe. These regions align with the proposed scenario of a relatively recent origin and subsequent localized host-switching events. The study also highlights the rarity of spillovers from bats to other species, yet underscores the relatively higher likelihood of such spillovers occurring towards humans, suggesting a significant role for humans as an intermediate host in the evolutionary journey of these viruses.

The research also points out the high rates of host-switching within mammalian orders, including between humans, domesticated animals, and non-flying wild mammals.

In conclusion, the study by Perez-Lamarque and colleagues presents an important quantitative advance in our understanding of the evolutionary history of mammalian coronaviruses. It suggests that the long-held belief in extensive virus-host codiversification may have been substantially overestimated, paving the way for a reevaluation of how we understand, predict, and potentially control the spread of these viruses.

Strengths:<br /> The study is conceptually robust, and its conclusions are convincing.

Weaknesses:<br /> Despite the availability of a dated host tree the authors were only able to use the "undated" model in ALE, with the dated method (which only allows time-consistent transfers) failing on their dataset (possibly due to dataset size?). Further exploration of the question would be potentially valuable.

Reviewer #3 (Public Review):

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

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

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

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

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

Reviewer #3 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

Summary:<br /> This paper presents valuable findings that gustation and feeding state influence the preferred environmental temperature preference in flies. Interestingly, the authors showed that by refeeding starved animals with the non-nutritive sugar sucralose, they are able to tune their preference towards a higher temperature in addition to nutrient-dependent warm preference. The authors show that temperature-sensing and sweet-sensing gustatory neurons (SGNs) are involved in the former but not the latter. In addition, their data indicate that peptidergic signals involved in internal state and clock genes are required for taste-dependent warm preference behavior.

The authors made an analogy of their results to the cephalic phase response (CPR) in mammals where the thought, sight, and taste of food prepare the animal for the consumption of food and nutrients. They further linked this behavior to core regulatory genes and peptides controlling hunger and sleep in flies having homologues in mammals. These valuable behavioral results can be further investigated in flies with the advantage of being able to dissect the neural circuitry underlying CPR and nutrient homeostasis.

Strengths:<br /> (1) The authors convincingly showed that tasting is sufficient to drive warm temperature preference behavior in starved flies and that it is independent of nutrient-driven warm preference.

(2) By using the genetic manipulation of key internal sensors and genes controlling internal feeding and sleep states such as DH44 neurons and the per genes for example, the authors linked gustation and temperature preference behavior control to the internal state of the animal.

Weaknesses:<br /> (1) The title is somewhat misleading, as the term homeostatic temperature control linked to gustation only applies to starved flies.

(2) The authors used a temperature preference assay and refeeding for 5 minutes, 10 minutes, and 1 hour. Experimentally, it makes a difference if the flies are tested immediately after 10 minutes or at the same time point as flies allowed to feed for 1 hour. Is 10 minutes enough to change the internal state in a nutrition-dependent manner? Some of the authors' data hint at it (e.g. refeeding with fly food for 10 minutes), but it might be relevant to feed for 5/10 minutes and wait for 55/50min to do the assays at comparable time points.

(3) A figure depicting the temperature preference assay in Figure 1 would help illustrate the experimental approach. It is also not clear why Figure 1E is shown instead of full statistics on the individual panels shown above (the data is the same).

(4) The authors state that feeding rate and amount were not changed with sucralose and glucose. However, the FLIC assay they employed does not measure consumption, so this statement is not correct, and it is unclear if the intake of sucralose and glucose is indeed comparable. This limits some of the conclusions.

(5) The authors make a distinction between taste-induced and nutrient-induced warm preference. Yet the statistics in most figures only show the significance between the starved and refed flies, not the fed controls. As the recovery is in many cases incomplete and used as a distinction of nutritive vs non-nutritive signals (see Figure 1E) it will be important to also show these additional statistics to allow conclusions about how complete the recovery is.

(6) The starvation period used is ranging from 1 to 3 days, as in some cases no effect was seen upon 1 day of starvation (e.g. with clock genes or temperature sensing neurons). While the authors do provide a comparison between 18-21 and 26-29 hours old flies in Figure S1, a comparison for 42-49 and 66-69 hours of starvation is missing. This also limits the conclusion as the "state" of the animal is likely quite different after 1 day vs. 3 days of starvation and, as stated by the authors, many flies die under these conditions.

(7) In Figure 2, glucose-induced refeeding was not tested in Gr mutants or silenced animals, which would hint at post-ingestive recovery mechanisms related to nutritional intake. This is only shown later (in Figure S3) but I think it would be more fitting to address this point here. The data presented in Figure S3 regarding the taste-evoked vs nutrient-dependent warm preference is quite important while in some parts preliminary. It would nonetheless be justified to put this data in the main figures. However, some of the conclusions here are not fully supported, in part due to different and low n numbers, which due to the inherent variability of the behavior do not allow statistically sound conclusions. The authors claim that sweet GRNs are only involved in taste-induced warm preference, however, glucose is also nutritive but, in several cases, does not rescue warm preference at all upon removal of GRN function (see Figures S3A-C). This indicates that the Gal4 lines and also the involved GRs are potentially expressed in tissues/neurons required for internal nutrient sensing.

(8) In Figure 4, fly food and glucose refeeding do not fully recover temperature preference after refeeding. With the statistical comparison to the fed control missing, this result is not consistent with the statement made in line 252. I feel this is an important point to distinguish between state-dependent and taste/nutrition-dependent changes.

(9) The conclusion that clock genes are required for taste-evoked warm preference is limited by the observation that they ingest less sucralose. In addition, the FLIC assay does not allow conclusions about the feeding amount, only the number of food interactions. Therefore, I think these results do not allow clear-cut conclusions about the impact of clock genes in this assay.

(10) CPR is known to be influenced by taste, thought, smell, and sight of food. As the discussion focused extensively on the CPR link to flies it would be interesting to find out whether the smell and sight of food also influence temperature preference behavior in animals with different feeding states.

(11) In the discussion in line 410ff the authors claim that "internal state is more likely to be associated with taste-evoked warm preference than nutrient-induced warm preference." This statement is not clear to me, as neuropeptides are involved in mediating internal state signals, both in the brain itself as well as from gut to brain. Thus, neuropeptidergic signals are also involved in nutrient-dependent state changes, the authors might just not have identified the peptides involved here. The global and developmental removal of these signals also limits the conclusions that can be drawn from the experiments, as many of these signals affect different states, circuits, and developmental progression.

Reviewer #2 (Public Review):

Animals constantly adjust their behavior and physiology based on internal states. Hungry animals, desperate for food, exhibit physiological changes immediately upon sensing, smelling, or chewing food, known as the cephalic phase response (CPR), involving processes like increased saliva and gastrointestinal secretions. While starvation lowers body temperature, the mechanisms underlying how the sensation of food without nutrients induces behavioral responses remain unclear. Hunger stress induces changes in both behavior and physiological responses, which in flies (or at least in Drosophila melanogaster) leads to a preference for lower temperatures, analogous to the hunger-driven lower body temperature observed in mammals. In this manuscript, the authors have used Drosophila melanogaster to investigate the issue of whether taste cues can robustly trigger behavioral recovery of temperature preference in starving animals. The authors find that food detection triggers a warm preference in flies. Starved flies recover their temperature preference after food intake, with a distinction between partial and full recovery based on the duration of refeeding. Sucralose, an artificial sweetener, induces a warm preference, suggesting the importance of food-sensing cues. The paper compares the effects of sucralose and glucose refeeding, indicating that both taste cues and nutrients contribute to temperature preference recovery. The authors show that sweet gustatory receptors (Grs) and sweet GRNs (Gustatory Receptor Neurons) play a crucial role in taste-evoked warm preference. Optogenetic experiments with CsChrimson support the idea that the excitation of sweet GRNs leads to a warm preference. The authors then examine the internal state's influence on taste-evoked warm preference, focusing on neuropeptide F (NPF) and small neuropeptide F (sNPF), analogous to mammalian neuropeptide Y. Mutations in NPF and sNPF result in a failure to exhibit taste-evoked warm preference, emphasizing their role in this process. However, these neuropeptides appear not to be critical for nutrient-induced warm preference, as indicated by increased temperature preference during glucose and fly food refeeding in mutant flies. The authors also explore the role of hunger-related factors in regulating taste-evoked warm preference. Hunger signals, including diuretic hormone (DH44) and adipokinetic hormone (AKH) neurons, are found to be essential for taste-evoked warm preference but not for nutrient-induced warm preference. Additionally, insulin-like peptides 6 (Ilp6) and Unpaired3 (Upd3), related to nutritional stress, are identified as crucial for taste-evoked warm preference. The investigation then extends into circadian rhythms, revealing that taste-evoked warm preference does not align with the feeding rhythm. While flies exhibit a rhythmic feeding pattern, taste-evoked warm preference occurs consistently, suggesting a lack of parallel coordination. Clock genes, crucial for circadian rhythms, are found to be necessary for taste-evoked warm preference but not for nutrient-induced warm preference.

Strengths:<br /> A well-written and interesting study, investigating an intriguing issue. The claims, none of which to the best of my knowledge controversial, are backed by a substantial number of experiments.

Weakness:<br /> The experimental setup used and the procedures for assessing the temperature preferences of flies are rather sparingly described. Additional details and data presentation would enhance the clarity and replicability of the study. I kindly request the authors to consider the following points: i) A schematic drawing or diagram illustrating the experimental setup for the temperature preference assay would greatly aid readers in understanding the spatial arrangement of the apparatus, temperature points, and the positioning of flies during the assay. The drawing should also be accompanied by specific details about the setup (dimensions, material, etc). ii) It would be beneficial to include a visual representation of the distribution of flies within the temperature gradient on the apparatus. A graphical representation, such as a heatmaps or histograms, showing the percentage of flies within each one-degree temperature bin, would offer insights into the preferences and behaviors of the flies during the assay. In addition to the detailed description of the assay and data analysis, the inclusion of actual data plots, especially for key findings or representative trials, would provide readers with a more direct visualization of the experimental outcomes. These additions will not only enhance the clarity of the presented information but also provide the reader with a more comprehensive understanding of the experimental setup and results. I appreciate the authors' attention to these points and look forward to the potential inclusion of these elements in the revised manuscript.

Reviewer #1 (Public Review):

Xie and Colleagues propose here to investigate the mechanism by which exercise inhibits bone metastasis progression. The authors describe that osteocyte, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing sEVs with tumor suppressor microRNAs. Furthermore, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Interestingly, exercise preconditioning effectively suppressed bone metastasis progression.

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 markers Ki-67 and CCND3 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) In Figure 1, 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. The data displayed in supplementary figures showing CCND3 as a marker of proliferation ,and GFP as a marker of cancer cells, is much more robust and should be moved to the main figures.

(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.

(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) Some conclusions of the paper are not entirely 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. While blocking miR-99b3p with antagomiRs rescued the decreases in proliferation, it is unclear whether this effect is mediated by osteocytes or other cells that express this miRNA. In vivo experiments demonstrating a direct role of osteocytes are needed to support the notion that osteocytes maintain tumor dormancy in NSCLC bone metastasis. In vivo, studies assessing tumor dormancy directly would be needed to confirm osteocytes promote cancer cell dormancy.

Reviewer #1 (Public Review):

Summary:<br /> TRIP13/Pch2 is a conserved essential regulator of meiotic recombination from yeast to humans. In this manuscript, the authors generated TRIP13 null mice and Flag-tagged TRIP13 knock-in mice to study its role in meiosis. They demonstrate that TRIP13 regulates MORMA domain proteins and is essential for meiotic completion and fertility. The main impact of this manuscript is its clarification of the in vivo function of TRIP13 during mouse meiosis and previously unrecognized role as a dose-sensitive regulator of meiosis.

Strengths:<br /> Two previously reported Trip13 mutations in mice are both hypomorphic alleles with distinct phenotypes, precluding a conclusion on its function. This study for the first time generated the TRIP13 null mice, definitively revealed the function of TRIP13 in meiosis. The authors also show novel localization of TRIP13 at SC and its independence from the axial element components. The finding of dose-sensitive regulation of meiosis by TRIP13 has implication in understanding human meiosis and disease phenotypes.

The results support the main conclusions and advance the understand of meiosis in the germline.

Reviewer #2 (Public Review):

Summary and Strengths:<br /> In this manuscript, Chotiner and colleagues demonstrated the localization of TRIP13 and clarified the phenotypes of Trip13-null mice in mouse meiosis. The meiotic phenotypes of Trip13 have been well characterized using the hypomorph alleles in the literature. However, the null phenotypes have not been examined, and the localization of TRIP13 was not clearly demonstrated. The study fills these important knowledge gaps in the field. The demonstration of TRIP13 localization to SC in mice provides an explanation of how HOMRA domain proteins are evicted from SC in diverse organisms. This conclusion was confirmed in both IF and TRIP13-tagged Tg mice. Further, the phenotypes of Trip13-null mice are very clear. The manuscript is well crafted, and the discussion section is well organized and comprehends the topic in the field. All in all, the manuscript will provide important knowledge in the field of meiosis.

Weaknesses:<br /> The heterozygous phenotypes demonstrate that TRIP13 is a dosage-sensitive regulator of meiosis. In relation to this conclusion, as summarized in the discussion section, other mutants defective in meiotic recombination showed dosage-sensitive phenotypes. However, the authors did not examine meiotic recombination in the Trip13-null mice.

Reviewer #1 (Public Review):

Summary:<br /> The authors employed a combinatorial CRISPR-Cas9 knockout screen to uncover synthetically lethal kinase genes that could play a role in drug resistance to kinase inhibitors in triple-negative breast cancer. The study successfully reveals FYN as a mediator of resistance to depletion and inhibition of various tyrosine kinases, notably EGFR, IGF-1R, and ABL, in triple-negative breast cancer cells and xenografts. Mechanistically, they demonstrate that KDM4 contributes to the upregulation of FYN and thereby is an important mediator of drug resistance. All together, these findings suggest FYN and KDM4A as potential targets for combination therapy with kinase inhibitors in triple-negative breast cancer. Moreover, the study may also have important implications for other cancer types and other inhibitors, as the authors suggest that FYN could be a general feature of drug-tolerant persister cells.

Strengths:<br /> (1) The authors used a large combination matrix of druggable tyrosine kinase gene knockouts, enabling studying of co-dependence of kinase genes. This approach mitigates off-target effects typically associated with kinase inhibitors, enhancing the precision of the findings.

(2) The authors demonstrate the importance of FYN in drug resistance in multiple ways. They demonstrate synergistic interactions using both knockouts and inhibitors, while also revealing its transcriptional upregulation upon treatment, strengthening the conclusion that FYN plays a role in the resistance.

(3) The study extends its impact by demonstrating the potent in vivo efficacy of certain combination treatments, underscoring the clinical relevance of the identified strategies.

Weaknesses:<br /> (1) The methods and figure legends are incomplete, posing a barrier to the reproducibility of the study and hindering a comprehensive understanding and accurate interpretation of the results.

(2) The authors make use of a large quantity of public data (Fig. 2D/E, Fig. 3F/L/M, Fig 4C, Fig 5B/H/I), whereas it would have strengthened the paper to perform these experiments themselves. While some of this data would be hard to generate (e.g. patient data) other data could have been generated by the authors. The disadvantage of the use of public data is that it merely comprises associations, but does not have causal/functional results (e.g. FYN inhibition in the different cancer models with various drugs). Moreover, by cherry-picking the data from public sources, the context of these sources is not clear to the reader, and thus harder to interpret correctly. For example, it is not directly clear whether the upregulation of FYN in these models is a very selective event or whether it is part of a very large epigenetic re-programming, where other genes may be more critical. While some of the used data are from well-known curated databases, others are from individual papers that the reader should assess critically in order to interpret the data. Sometimes the public data was redundant, as the authors did do the experiments themselves (e.g. lung cancer drug-tolerant persisters), in this case, the public data could also be left out.

More importantly, the original sources are not properly cited. While the GEO accession numbers are shown in a supplementary table, the articles corresponding to this data should be cited in the main text, and preferably also in the figure legend, to clarify that this data is from public sources, which is now not always the case (e.g. line 224-226). If these original papers do already mention the upregulation of FYN, and the findings from the authors are thus not original, these findings should be discussed in the Discussion section instead of shown in the Results.

(3) The claim in the abstract (and discussion) that the study "highlights FYN as broadly applicable mediator of therapy resistance and persistence", is not sufficiently supported by the results. The current study only shows functional evidence for this for an EGFR, IGF1R, and Abl inhibitor in TNBC cells. Further, it demonstrates (to a limited extent) the role of FYN in gefitinib and osimertinib resistance (also EGFR inhibitors) in lung cancer cells. Thus, the causal evidence provided is only limited to a select subset of tyrosine kinase inhibitors in two cancer types. While the authors show associations between FYN and drug resistance in other cancer types and after other treatments, these associations are not solid evidence for a causal connection as mentioned in this statement. Epigenetic reprogramming causing drug resistance can be accompanied by altered gene expression of many genes, and the upregulation of FYN may be a consequence, but not a cause of the drug resistance. Therefore, the authors should be more cautious in making such statements about the broad applicability of FYN as a mediator of therapy resistance.

(4) The rationale for picking and validating FYN as the main candidate gene over other genes such as FGFR2, FRK2, and TEK is not clear.<br /> a. While gene pairs containing FGFR2 knockouts seemed to be equally effective as FYN gene pairs in the primary screening, these could not be validated in the validation experiment. It is unclear whether multiple individual or a pool of gRNAs were used for this validation, or whether only 1 gRNA sequence was picked per gene for this validation. If only 1 gRNA per gene was used, this likely would have resulted in variable knockout efficiencies. Moreover, the T7 endonuclease assay may not have been the best method to check knockout efficiency, as it only implies endonuclease activity around a gene (but not to the extent of indels that can cause frameshifts, such as by TIDE analysis, or extent of reduction in protein levels by western blot).<br /> b. Moreover, FRK2 and TEK, also demonstrated many synergistic gene pairs in the primary screen. However, many of these gene pairs were not included in the validation screening. The selection criteria of candidate gene pairs for validation screening is not clear. Still, TEK-ABL2 was also validated as a strong hit in the validation screen. The authors should better explain the choice of FYN over other hits, and/or mention that TEK and FRK2 may also be important targets for combination treatment that can be further elucidated.

(5) On several occasions, the right controls (individual treatments, performed in parallel) are not included in the figures. The authors should include the responses to each of the single treatments, and/or better explain the normalization that might explain why the controls are not shown.<br /> a. Figure 2G: The effect of PP2 treatment, without combined treatment, is not shown.<br /> b. Figure 2H/3G: The effect of the knockouts on growth alone, compared to sgGFP, is not demonstrated. It is unclear whether the viability of knockouts is normalized to sgGFP, or to each untreated knockout.<br /> c. Figure 2L: The effect of SB203580 as a single treatment is not shown.

(6) The study examines the effects at a single, relatively late time point after treatment with inhibitors, without confirming the sequential impact on KDM4A and FYN. The proposed sequence of transcriptional upregulation of KDM4A followed by epigenetic modifications leading to FYN upregulation would be more compellingly supported by demonstrating a consecutive, rather than simultaneous, occurrence of these events. Furthermore, the protein level assessment at 48 hours (for RNA levels not clearly described), raises concerns about potential confounding factors. At this late time point, reduced cell viability due to the combination treatment could contribute to observed effects such as altered FYN expression and P38 MAPK phosphorylation, making it challenging to attribute these changes solely to the specific and selective reduction of FYN expression by KDM4A.

(7) The cut-off for considering interactions "synergistic" is quite low. The manual of the used "SynergyFinder" tool itself recommends values above >10 as synergistic and between -10 and 10 as additive (https://synergyfinder.fimm.fi/synergy/synfin_docs/). Here, values between 5-10 are also considered synergistic. Caution should be taken when discussing those results. Showing the actual dose response (including responses to each single treatment) may be required to enable the reader to critically assess the synergy, along with its standard deviation.

(8) As the effect size on Western blots is quite limited and sometimes accompanied by differences in loading control, these data should be further supported by quantifications of signal intensities of at least 3 biological replicates (e.g. especially Figure 3A/5A). The figure legends should also state how many independent experiments the blots are representative of.

(9) While the article provides mechanistic insights into the likely upregulation of FYN by KDM4A, this constitutes only a fragment of the broader mechanism underlying drug resistance associated with FYN. The study falls short in investigating the causes of KDM4A upregulation and fails to explore the downstream effects (except for p38 MAPK phosphorylation, which may not be complete) of FYN upregulation that could potentially drive sustained cell proliferation and survival. These omissions limit the comprehensive understanding of the complete molecular pathway, and the discussion section does not address potential implications or pathways beyond the identified KDM4A-FYN axis. A more thorough exploration of these aspects would enhance the study's contribution to the field.

(10) FYN has been implied in drug resistance previously, and other mechanisms of its upregulation, as well as downstream consequences, have been described previously. These were not evaluated in this paper, and are also not discussed in the discussion section. Moreover, the authors did not investigate whether any of the many other mechanisms of drug resistance to EGFR, IGF1R, and Abl inhibitors that have been described, could be related to FYN as well. A more comprehensive examination of existing literature and consideration of alternative or parallel mechanisms in the discussion would enhance the paper's contribution to understanding FYN's involvement in drug resistance.

Reviewer #2 (Public Review):

Summary:<br /> Kim et al. conducted a study in which they selected 76 tyrosine kinases and performed CRISPR/Cas9 combinatorial screening to target 3003 genes in Triple-negative breast cancer (TNBC) cells. Their investigation revealed a significant correlation between the FYN gene and the proliferation and death of breast cancer cells. The authors demonstrated that depleting FYN and using FYN inhibitors, in combination with TKIs, synergistically suppressed the growth of breast cancer tumor cells. They observed that TKIs upregulate the levels of FYN and the histone demethylase family, particularly KDM4, promoting FYN expression. The authors further showed that KDM4 weakens the H3K9me3 mark in the FYN enhancer region, and the inhibitor QC6352 effectively inhibits this process, leading to a synergistic induction of apoptosis in breast cancer cells along with TKIs. Additionally, the authors discovered that FYN is upregulated in various drug-resistant cancer cells, and inhibitors targeting FYN, such as PP2, sensitize drug-resistant cells to EGFR inhibitors.

Strengths:<br /> This study provides new insights into the roles and mechanisms of FYN and KDM4 in tumor cell resistance.

Weaknesses:<br /> It is important to note that previous studies have also implicated FYN as a potential key factor in drug resistance of tumor cells, including breast cancer cells. While the current study is comprehensive and provides a rich dataset, certain experiments could be refined, and the logical structure could be more rigorous. For instance, the rationale behind selecting FYN, KDM4, and KDM4A as the focus of the study could be more thoroughly justified.

Reviewer #1 (Public Review):

In this study, Li et al., report that FBXO24 contributes to sperm development by modulating alternative mRNA splicing and MIWI degradation during spermiogenesis. The authors demonstrated that FBXO24 deficiency impairs sperm head formation, midpiece compartmentalization, and axonemal/peri-axonemal organization in mature sperm, which causes sperm motility defects and male infertility. In addition, FBXO24 interacts with various mRNA splicing factors, which causes altered splicing events in Fbxo24-null round spermatids. Interestingly, FBXO24 also modulates MIWI levels via its polyubiquitination in round spermatids. Thus, the authors address that FBXO24 modulates global mRNA levels by regulating piRNA-mediated MIWI function and splicing events in testicular haploid germ cells.

This study is performed with various experimental approaches to explore and elucidate underlying molecular mechanisms for the FBXO24-mediated sperm defects during germ cell development. Overall, the experiments were designed properly and performed well to support the authors' observation in each part. In addition, the findings in this study are useful for understanding the physiological and developmental significance of FBXO24 in the male germ line, which can provide insight into impaired sperm development and male infertility.

In the revised manuscript, the authors address most of the concerns raised in the previous review. The following are representative remaining points.

• Quantification of the defective, vacuolar mitochondria (80%) and missing annulus (30%) was not shown in the figures or described in the results as well as in a few other figures.

Reviewer #2 (Public Review):

Spermatogenesis describes a complex sequence of differentiation events that lead to the development of genetically distinct male germ cells. The final part of spermatogenesis is called spermiogenesis, in which spermatids differentiate into mature sperm by developing an acrosome and a motile flagellum, which are required for reaching and successfully penetrating the oocyte. This process of spermatogenesis is based on a coordinated regulation of gene expressions in round spermatids. In the current study, FBXO24 was identified as a highly expressed protein in human and mouse testis. To define its biological role in vivo, the authors generated genetically engineered Fbxo24 knockout and Fbxo24-HA-labeled transgenic mouse models.

To elucidate the causes of the observed sterility in Fbxo24-KO males, the authors performed molecular and phenotypic analyses that revealed aberrant histone retention, incomplete axonemes, oversized chromatoid bodies (CB), and abnormal mitochondrial coiling along the sperm flagella. These results support the causal role of the FBXO24 gene in sperm motility.

Furthermore, the authors carefully characterized by SEM, TEM and western blot analyses that deletion of FBXO24 leads to incomplete histone-to-protamine exchange and defective chromatin interaction during spermiogenesis. In addition to increased MIWI expression, the authors show that FBXO24 interacts with SCF subunits and mediates the degradation of MIWI via K48-linked polyubiquitination.

This is a solid work demonstrating the role of FBXO24 in modulating alternative mRNA splicing, MIWI degradation and normal spermiogenesis.

Reviewer #3 (Public Review):

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

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

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

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

Reviewer #1 (Public Review):

The authors assess the effectiveness of electroporating mRNA into male germ cells to rescue the expression of proteins required for spermatogenesis progression in individuals where these proteins are mutated or depleted. To set up the methodology, they first evaluated the expression of reporter proteins in wild-type mice, which showed expression in germ cells for over two weeks. Then, they attempted to recover fertility in a model of late spermatogenesis arrest that produces immotile sperm. By electroporating the mutated protein, the authors recovered the motility of ~5% of the sperm, although the sperm regenerated was not able to produce offspring using IVF.

This is a comprehensive evaluation of the mRNA methodology with multiple strengths. First, the authors show that naked synthetic RNA, purchased from a commercial source or generated in the laboratory with simple methods, is enough to express exogenous proteins in testicular germ cells. The authors compared RNA to DNA electroporation and found that germ cells are efficiently electroporated with RNA, but not DNA. The differences between these constructs were evaluated using in vivo imaging to track the reporter signal in individual animals through time. To understand how the reporter proteins affect the results of the experiments, the authors used different reporters: two fluorescent (eGFP and mCherry) and one bioluminescent (Luciferase). Although they observed differences among reporters, in every case expression lasted for at least two weeks.

The authors used a relevant system to study the therapeutic potential of RNA electroporation. The ARMC2-deficient animals have impaired sperm motility phenotype that affects only the later stages of spermatogenesis. The authors showed that sperm motility was recovered to ~5%, which is remarkable due to the small fraction of germ cells electroporated with RNA with the current protocol. The 3D reconstruction of an electroporated testis using state-of-the-art methods to show the electroporated regions is compelling.

The main weakness of the manuscript is that although the authors manage to recover motility in a small fraction of the sperm population, it is unclear whether the increased sperm quality is substantial to improve assisted reproduction outcomes. The quality of the sperm was not systematically evaluated in the manuscript, with the endpoints being sperm morphology and sperm mobility.

Some key results, such as the 3D reconstruction of the testis and the recovery of sperm motility, are qualitative given the low replicate numbers or the small magnitude of the effects. The presentation of the sperm motility data could have been clearer as well. For example, on day 21 after Armc2-mRNA electroporation, only one animal out of the three tested showed increased sperm motility. However, it is unclear from Figure 11A what the percentage of sperm motility for this animal is since the graph shows a value of >5% and the reported aggregate motility is 4.5%. It would have been helpful to show all individual data points in Figure 11A.

The expression of the reporter genes is unambiguous; however, better figures could have been presented to show cell type specificity. The DAPI staining is diffused, and it is challenging to understand where the basement membranes of the tubules are. For example, in Figures 7B3 and 7E3, the spermatogonia seems to be in the middle of the seminiferous tubule. The imaging was better for Figure 8. Suboptimal staining appears to lead to mislabeling of some germ cell populations. For example, in Supplementary Figure 4A3, the round spermatid label appears to be labeling spermatocytes. Also, in some instances, the authors seem to be confusing, elongating spermatids with spermatozoa, such as in the case of Supplementary Figures 4D3 and D4.

The characterization of Armc2 expression could have been improved as well. The authors show a convincing expression of ARMC2 in a few spermatids/sperm using a combination of an anti-ARMC2 antibody and tubules derived from ARMC2 KO animals. At the minimum, one would have liked to see at least one whole tubule of a relevant stage.

Overall, the authors show that electroporating mRNA can improve spermatogenesis as demonstrated by the generation of motile sperm in the ARMC2 KO mouse model.

Reviewer #2 (Public Review):

Summary:

Here, the authors inject naked mRNAs and plasmids into the rete testes of mice to express exogenous proteins - GFP and later ARMC2. This approach has been taken before, as noted in the Discussion to rescue Dmc1 KO infertility. While the concept is exciting, multiple concerns reduce reviewer enthusiasm.

Strengths:

The approach, while not necessarily novel, is timely and interesting.

Weaknesses:

Overall, the writing and text can be improved and standardized - as an example, in some places in vivo is italicized, in others it's not; gene names are italicized in some places, others not; some places have spaces between a number and the units, others not. This lack of attention to detail in the preparation of the manuscript is a significant concern to this reviewer - the presentation of the experimental details does cast some reasonable concern with how the experiments might have been done. While this may be unfair, it is all the reviewers have to judge. Multiple typographical and grammatical errors are present, and vague or misleading statements.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

Reviewer #1 (Public Review):

Summary:

Asymptomatic malaria infections are frequent during the dry season and have been associated with lower cytoadherence of P. falciparum parasites and lower expression of variant surface antigens. The mechanisms underlying parasite adaptation during the low transmission season remain poorly understood. The authors previously established that members of the non-coding RNA RUF6 gene family, transcribed by RNA pol III, are required for expression of the main variant surface antigens in P. falciparum, PfEMP1, which drive parasite cytoadherence and pathogenicity. In this study, the authors investigated the contribution of RNA pol III transcription in the regulation of PfEMP1 expression in different clinical states, either symptomatic malaria cases during the wet season or asymptomatic infections during the dry season.

By reanalyzing RNAseq data from a previous study in Mali, complemented with RT-qPCR on new samples collected in The Gambia, the authors first report the down-regulation of RNA pol III genes (tRNAs, RUF6) in P. falciparum isolates collected from asymptomatic individuals during the dry season, as compared to isolates from symptomatic (wet season) individuals. They also confirm the down-regulation of var (DBLalpha) gene expression in asymptomatic infection as compared to symptomatic malaria. Plasma analysis in the two groups in the Gambian study reveals higher Magnesium levels in the dry season as compared to wet season samples, pointing at a possible role of external factors. The authors tested the effect of MgCl2 supplementation on cultured parasites, as well as three other stimuli (temperature, low glucose, Ile deprivation), and showed that Ile deprivation and MgCl2 both induce down-regulation of RNA pol III transcription but not pol I or pol II (except the active var gene). Using RNAseq, they show that MgCl2 supplementation predominantly inhibits RNA pol III-transcribed genes, including the entire RUF6 family. Conditional depletion of Maf1 leads to the up-regulation of RNA pol III gene transcription, confirming that Maf1 is a RNA pol III inhibitor in P. falciparum, as described in other organisms. Quantitative mass spectrometry shows that Maf1 interacts with RNA pol III complex in the nucleus, and with distinct proteins including two phosphatases in the cytoplasm. Using the Maf1 cKD parasites, the authors document that the down-regulation of RNA pol III by MgCl2 is dependent on Maf1. Finally, they show that MgCl2 results in decreased cytoadherence of infected erythrocytes, associated with reduced PfEMP1 expression.

Strengths:

-The work is very well performed and presented.<br /> -The study uncovers a novel regulatory mechanism relying on RNA pol III-dependent regulation of variant surface antigens in response to external signals, which could contribute to parasite adaptation during the low transmission season.<br /> -Potential regulators of Maf1 were identified by mass spectrometry, including phosphatases, paving the way for future mechanistic studies.

Weaknesses:

-The signaling pathway upstream of Maf1 remains unknown. In eukaryotes, Maf1 is a negative regulator of RNA pol III and is regulated by external signals via the TORC pathway. Since TORC components are absent in the apicomplexan lineage, one central question that remains open is how Maf1 is regulated in P. falciparum. Magnesium is probably not the sole stimulus involved, as suggested by the observation that Ile deprivation also down-regulates RNA pol III activity.<br /> -The study does not address why MgCl2 levels vary depending on the clinical state. It is unclear whether plasma magnesium is increased during asymptomatic malaria or decreased during symptomatic infection, as the study does not include control groups with non-infected individuals. Along the same line, MgCl2 supplementation in parasite cultures was done at 3mM, which is higher than the highest concentrations observed in clinical samples.<br /> -Although the study provides biochemical evidence of Maf1 accumulation in the parasite nuclear fraction upon magnesium addition, this is not fully supported by the immunofluorescence experiments.

Reviewer #2 (Public Review):

Summary:

The study by Diffendall et al. set out to establish a link between the activity of RNA polymerase III (Pol III) and its inhibitor Maf1 and the virulence of Plasmodium falciparum in vivo. Having previously found that knockdown of the ncRNA ruf6 gene family reduces var gene expression in vitro, they now present experimental evidence for the regulation of ruf6 and subsequently, var gene expression by Pol III using a commercially available inhibitor. They confirm their findings with samples from a previously published Gambian cohort study using asymptomatic dry season and mildly symptomatic wet season samples, showing that higher levels of Pol III-dependent transcripts and var transcripts as well as lower MgCl2 plasma concentrations are present in wet season samples. From this, they hypothesize that the external stimuli heat, reduced glucose and essential amino acid supply, and increased MgCl2 levels are sensed by the parasite through the only known Pol III inhibitor Maf1 and result in lower Pol III activity and fewer ruf6 transcripts, which in turn reduces var gene expression, leading to reduced cytoadherence and virulence of P. falciparum. In their in vitro experiments they focus on investigating higher MgCl2 levels and their impact on Pol III and Maf1 activity as well as var gene expression and parasites adherence to purified CD36, thereby successfully confirming their hypothesis for MgCl2. Nicely, MgCl2-induced down-regulation of Pol III activity was shown to be dependent on Maf1 using a knock-down cell line. Additionally, they show that the Maf1-KD cell line displays a slower growth rate with fewer merozoites per schizonts and Maf1 interacts with RNA pol III subunits and some kinases/phosphatases.

Strengths:

Overall, the authors were largely successful in their aims. They provide largely convincing data, and the correlation between Pol III transcription and its inhibition by Maf1 with the expression of ruf6 and var genes is highly interesting. The data provide important insights for researchers investigating the function of Pol III and its inhibitor, non-coding RNAs, and their role in gene regulation, but may also indicate that the parasite senses and responds to its environment, opening up new research directions, particularly in field research.

Weaknesses:

However, most analyses are rather preliminary as only very few (3-5) candidate genes are analyzed by qPCR instead of carrying out comprehensive analyses with a large qPCR panel or RNA-seq experiments with GO term analyses. Data presentation lacks clarity, the number of biological replicates is rather low and the statistical analyses need to be largely revised. Although the in vivo data from wet (mildly symptomatic) and dry (asymptomatic) season parasites with different expression levels of Pol III-regulated genes, var genes, and MgCl2 are interesting, the link between the in vitro data and the in vivo virulence of P. falciparum, which is made in many sections of the manuscript, should be toned down. Especially since (i) the only endothelial receptor studied is CD36, which is associated with parasite binding during mild malaria, and (ii) several studies provide contradictory data on MgCl2 levels during malaria and in different disease states, which is not further discussed, but the authors mainly focused on this external stimulus in their experiments.

Reviewer #3 (Public Review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #1 (Public Review):

Summary:

This manuscript presents the development of a new microscope method termed "open-top two-photon light sheet microscopy (OT-TP-LSM)". While the key aspects of the new approach (open top LSM and Two-photon microscopy) have been demonstrated separately, this is the first system of integrating the two. The integration provides better imaging depth than a single-photon excitation OT-LSM.

Strengths:<br /> - Use of liquid prism to minimize the aberration induced by index mismatching is interesting and potentially helpful to other researchers in the field.<br /> - Use of propidium iodide (PI) provided a deeper imaging depth.

Weaknesses:<br /> -None noted.

Reviewer #2 (Public Review):

In this manuscript, the authors developed an open-top two-photon light sheet microscopy (OT-TP-LSM) that enables high-throughput and high-depth investigation of 3D cell structures. The data presented here shows that OT-T-LSM could be a complementary technique to traditional imaging workflows of human cancer cells.

High-speed and high-depth imaging of human cells in an open-top configuration is the main strength of the presented study. An extended depth of field of 180 µm in 0.9 µm thickness was achieved together with an acquisition of 0.24 mm2/s. This was confirmed by 3D visualization of human cancer cells in the skin, pancreas, and prostate.

Reviewer #1 (Public Review):

This study examines whether the human brain uses a hexagonal grid-like representation to navigate in a non-spatial space constructed by competence and trustworthiness. To test this, the authors asked human participants to learn the levels of competence and trustworthiness for six faces by associating them with specific lengths of bar graphs that indicate their levels in each trait. After learning, participants were asked to extrapolate the location from the partially observed morphing bar graphs. Using fMRI, the authors identified brain areas where activity is modulated by the angles of morphing trajectories in six-fold symmetry. The strength of this paper lies in the question it attempts to address. Specifically, the question of whether and how the human brain uses grid-like representations not only for spatial navigation but also for navigating abstract concepts, such as social space, and guiding everyday decision-making. This question is of emerging importance.

I acknowledge the authors' efforts to address the comments received. However, my concerns persist:

(1) The authors contend that shorter reaction times correlated with increased distances between individuals in social space imply that participants construct and utilize two-dimensional representations. This method is adapted from a previous study by Park et al. Yet, there is a fundamental distinction between the two studies. In the prior work, participants learned relationships between adjacent individuals, receiving feedback on their decisions, akin to learning spatial locations during navigation. This setup leads to two different predictions: If participants rely on memory to infer relationships, recalling more pairs would be necessary for distant individuals than for closer ones. Conversely, if participants can directly gauge distances using a cognitive map, they would estimate distances between far individuals as quickly as for closer ones. Consequently, as the authors suggest, reaction times ought to decrease with increasing decision value, which, in this context, corresponds to distances. However, the current study allowed participants to compare all possible pairs without restricting learning experiences, rendering the application of the same methodology for testing two-dimensional representations inappropriate. In this study, the results could be interpreted as participants not forming and utilizing two-dimensional representations.

(2) The confounding of visual features with the value of social decision-making complicates the interpretation of this study's results. It remains unclear whether the observed grid-like effects are due to visual features or are genuinely indicative of value-based decision-making, as argued by the authors. Contrary to the authors' argument, this issue was not present in the previous study (Constantinescu et al.). In that study, participants associated specific stimuli with the identities of hidden items, but these stimuli were not linked to decision-making values (i.e., no image was considered superior to another). The current study's paradigm is more akin to that of Bao et al., which the authors mention in the context of RSA analysis. Indeed, Bao et al. controlled the length of the bars specifically to address the problem highlighted here. Regrettably, in the current paradigm, this conflation remains inseparable.

(3) While the authors have responded to comments in the public review, my concerns noted in the Recommendation section remain unaddressed. As indicated in my recommendations, there are aspects of the authors' methodology and results that I find difficult to comprehend. Resolving these issues is imperative to facilitate an appropriate review in subsequent stages.

Considering that the issues raised in the previous comments remain unresolved, I have retained my earlier comments below for review.

The weak points of this paper are that its findings are not sufficiently supporting their arguments, and there are several reasons for this:

(1) Does the grid-like activity reflect 'navigation over the social space' or 'navigation in sensory feature space'? The grid-like representation in this study could simply reflect the transition between stimuli (the length of bar graphs). Participants in this study associated each face with a specific length of two bars, and the 'navigation' was only guided by the morphing of a bar graph image. Moreover, any social cognition was not required to perform the task where they estimate the grid-like activity. To make social decision-making that was conducted separately, we do not know if participants needed to navigate between faces in a social space. Instead, they can recall bar graphs associated with faces and compute the decision values by comparing the length of bars. Notably, in the trust game in this study, the competence and trustworthiness are not equally important to make a decision (Equation 1). The expected value is more sensitive to one over the other. This also suggests that the space might not reflect social values but the perceptual differences.

(2) Does the brain have a common representation of faces in a social space? In this study, participants don't need to have a map-like representation of six faces according to their levels of social traits. Instead, they can remember the values of each trait. The evidence of neural representations of the faces in a 2-dimensional social space is lacking. The authors argued the relationship between the reaction times and the distances between faces provides evidence of the formation of internal representations. However, this can be found without the internal representation of the relationships between faces. If the authors seek internal representations of the faces in the brain, it would be important to show that this representation is not simply driven by perceptual differences between bar graphs that participants may recall in association with each face.

Considering these caveats, it is hard for me to agree if the authors provide evidence to support their claims.

Reviewer #2 (Public Review):

Summary:<br /> In this work, Liang et al. investigate whether an abstract social space is neurally represented by a grid-like code. They trained participants to 'navigate' around a two-dimensional space of social agents characterized by the traits warmth and competence, then measured neural activity as participants imagined navigating through this space. The primary neural analysis consisted of three procedures: 1) identifying brain regions exhibiting the hexagonal modulation characteristic of a grid-like code, 2) estimating the orientation of each region's grid, and 3) testing whether the strength of the univariate neural signal increases when a participant is navigating in a direction aligned with the grid, compared to a direction that is misaligned with the grid. From these analyses, the authors find the clearest evidence of a grid-like code in the prefrontal cortex and weaker evidence in the entorhinal cortex.

Strengths:<br /> The work demonstrates the existence of a grid-like neural code for a socially-relevant task, providing evidence that such coding schemes may be relevant for a variety of two-dimensional task spaces.

Weaknesses:<br /> In the revised manuscript, the authors soften their claims about finding a grid code in the entorhinal cortex and provide additional caveats about limitations in their findings. It seems that the authors and reviewers are in agreement about the following weaknesses, which were part of my original review: Claims about a grid code in the entorhinal cortex are not well-supported by the analyses presented. The whole-brain analysis does not suggest that the entorhinal cortex exhibits hexagonal modulation; the strength of the entorhinal BOLD signal does not track the putative alignment of the grid code there; multivariate analyses do not reveal any evidence of a grid-like representational geometry.

In the authors' response to reviews, they provide additional clarification about their exploratory analyses examining whether behavior (i.e., reaction times) and individual difference measures (i.e., social anxiety and avoidance) can be predicted by the hexagonal modulation strength in some region X, conditional on region X having a similar estimated grid alignment with some other region Y. My guess is that readers would find it useful if some of this language were included in the main text, especially with regard to an explanation regarding the rationale for these exploratory studies.

Reviewer #3 (Public Review):

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

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

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

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

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

Reviewer #1 (Public Review):

In this systematic and elegant structure-function analysis study, the authors delve into the intricate involvement of syntaxin 1 in various pivotal stages of synaptic vesicle priming and fusion. The authors use an original and fruitful approach based on the side-by-side comparison of the specific contributions of the two isoforms syntaxin 1 and syntaxin 2, and their respective SNARE domains, in priming, spontaneous and Ca2+-dependent glutamate release. The experimental approach, mastered by the authors, offers an ideal means of unraveling the molecular roles played by syntaxins. Although it is not easy to come up with a model explaining all the observed phenotypes, the authors carefully restrict their conclusions to the role of the C-terminal half of the syntaxin1 C-terminal SNARE domain in the maintenance of the RRP and the clamping of neurotransmitter release. The study is carefully carried out, the conclusions are supported by high quality data and the manuscript is clearly written. In addition, the study clearly set new questions than open new paths for future experimental work.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Salazar-Lázaro et al. systematically dissects out the different functional properties of the SNARE-domains of syntaxin-1 and syntaxin-2. By systematically substituting the SNARE-domain (or its C- or N-terminal half) into the non-cognate counterpart, the authors find that the C-terminal half of the SNARE-complex is especially important for maintaining RRP size and clamping spontaneous release. They also mutate single residues, to further nail down the effect. Overall, this is an interesting manuscript, which sheds light on the functionality of different co-expressed SNARES.

Strengths:<br /> The strength of the manuscript is the systematic dissection, using substitution of either SNARE-domain into the other syntaxin, together with the state-of-the art methods. The authors follow up with a substitution of single and paired residues. This is a large undertaking, which has been very well carried out.

Weaknesses:<br /> No major weaknesses. The large number of experiments paint a somewhat complicated picture because the process under study is complicated.

Reviewer #3 (Public Review):

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

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

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

Reviewer 1 Public Review:

Summary:

The authors set out to clarify the molecular mechanism of endocytosis (re-uptake) of synaptic vesicle (SV) membrane in the presynaptic terminal following release. They have examined the role of presynaptic actin, and of the actin regulatory proteins diaphanous-related formins ( mDia1/3), and Rho and Rac GTPases in controlling the endocytosis. They successfully show that presynaptic membrane-associated actin is required for normal SV endocytosis in the presynaptic terminal, and that the rate of endocytosis is increased by activation of mDia1/3. They show that RhoA activity and Rac1 activity act in a partially redundant and synergistic fashion together with mDia1/3 to regulate the rate of SV endocytosis. The work adds substantially to our understanding of the molecular mechanisms of SV endocytosis in the presynaptic terminal.

Strengths:

The authors use state-of-the-art optical recording of presynaptic endocytosis in primary hippocampal neurons, combined with well-executed genetic and pharmacological perturbations to document effects of alteration of actin polymerization on the rate of SV endocytosis. They show that removal of the short amino-terminal portion of mDia1 that associates with the membrane interrupts the association of mDia1 with membrane actin in the presynaptic terminal. They then use a wide variety of controlled perturbations, including genetic modification of the amount of mDia1/3 by knock-down and knockout, combined with inhibition of activity of RhoA and Rac1 by pharmacological agents, to document the quantitative importance of each agent, and their synergistic relationship in regulation of endocytosis.

The analysis is augmented by ultrastructural analyses that demonstrate the quantitative changes in numbers of synaptic vesicles and in uncoated membrane invaginations that are predicted by the optical recordings.<br /> The manuscript is well-written and the data are clearly explained. Statistical analysis of the data is strengthened by the very large number of data points analyzed for each experiment.

Weaknesses:

There are no major weaknesses.

Reviewer 2 Public Review:

Summary:

This manuscript expands previous work from the Haucke group which demonstrated the role of formins in synaptic vesicle endocytosis. The techniques used to address the research question are state-of-the-art. As stated above there is a significant advance in knowledge, with particular respect to Rho/Rac signalling.

Strengths:

The major strength of the work was to reveal new information regarding the control of both presynaptic actin dynamics and synaptic vesicle endocytosis via Rho/Rac cascades. In addition, there was further mechanistic insight regarding the specific function of mDia1/3. The methods used were state-of-the-art.

Weaknesses:

There are no major weaknesses.

Reviewer #1 (Public Review):

Summary:<br /> Visual Perceptual Learning (VPL) results in varying degrees of generalization to tasks or stimuli not seen during training. The question of which stimulus or task features predict whether learning will transfer to a different perceptual task has long been central in the field of perceptual learning, with numerous theories proposed to address it. This paper introduces a novel framework for understanding generalization in VPL, focusing on the form invariants of the training stimulus. Contrary to a previously proposed theory that task difficulty predicts the extent of generalization - suggesting that more challenging tasks yield less transfer to other tasks or stimuli - this paper offers an alternative perspective. It introduces the concept of task invariants and investigates how the structural stability of these invariants affects VPL and its generalization. The study finds that tasks with high-stability invariants are learned more quickly. However, training with low-stability invariants leads to greater generalization to tasks with higher stability, but not the reverse. This indicates that, at least based on the experiments in this paper, an easier training task results in less generalization, challenging previous theories that focus on task difficulty (or precision). Instead, this paper posits that the structural stability of stimulus or task invariants is the key factor in explaining VPL generalization across different tasks

Strengths:<br /> - The paper effectively demonstrates that the difficulty of a perceptual task does not necessarily correlate with its learning generalization to other tasks, challenging previous theories in the field of Visual Perceptual Learning. Instead, it proposes a significant and novel approach, suggesting that the form invariants of training stimuli are more reliable predictors of learning generalization. The results consistently bolster this theory, underlining the role of invariant stability in forecasting the extent of VPL generalization across different tasks.

- The experiments conducted in the study are thoughtfully designed and provide robust support for the central claim about the significance of form invariants in VPL generalization.

Weaknesses:<br /> - The paper assumes a considerable familiarity with the Erlangen program and the definitions of invariants and their structural stability, potentially alienating readers who are not versed in these concepts. This assumption may hinder the understanding of the paper's theoretical rationale and the selection of stimuli for the experiments, particularly for those unfamiliar with the Erlangen program's application in psychophysics. A brief introduction to these key concepts would greatly enhance the paper's accessibility. The justification for the chosen stimuli and the design of the three experiments could be more thoroughly articulated.

- The paper does not clearly articulate how its proposed theory can be integrated with existing observations in the field of VPL. While it acknowledges previous theories on VPL generalization, the paper falls short in explaining how its framework might apply to classical tasks and stimuli that have been widely used in the VPL literature, such as orientation or motion discrimination with Gabors, vernier acuity, etc. It also does not provide insight into the application of this framework to more naturalistic tasks or stimuli. If the stability of invariants is a key factor in predicting a task's generalization potential, the paper should elucidate how to define the stability of new stimuli or tasks. This issue ties back to the earlier mentioned weakness: namely, the absence of a clear explanation of the Erlangen program and its relevant concepts.

- The paper does not convincingly establish the necessity of its introduced concept of invariant stability for interpreting the presented data. For instance, consider an alternative explanation: performing in the collinearity task requires orientation invariance. Therefore, it's straightforward that learning the collinearity task doesn't aid in performing the other two tasks (parallelism and orientation), which do require orientation estimation. Interestingly, orientation invariance is more characteristic of higher visual areas, which, consistent with the Reverse Hierarchy Theory, are engaged more rapidly in learning compared to lower visual areas. This simpler explanation, grounded in established concepts of VPL and the tuning properties of neurons across the visual cortex, can account for the observed effects, at least in one scenario. This approach has previously been used/proposed to explain VPL generalization, as seen in (Chowdhury and DeAngelis, Neuron, 2008), (Liu and Pack, Neuron, 2017), and (Bakhtiari et al., JoV, 2020). The question then is: how does the concept of invariant stability provide additional insights beyond this simpler explanation?

- While the paper discusses the transfer of learning between tasks with varying levels of invariant stability, the mechanism of this transfer within each invariant condition remains unclear. A more detailed analysis would involve keeping the invariant's stability constant while altering a feature of the stimulus in the test condition. For example, in the VPL literature, one of the primary methods for testing generalization is examining transfer to a new stimulus location. The paper does not address the expected outcomes of location transfer in relation to the stability of the invariant. Moreover, in the affine and Euclidean conditions one could maintain consistent orientations for the distractors and targets during training, then switch them in the testing phase to assess transfer within the same level of invariant structural stability.

- In the section detailing the modeling experiment using deep neural networks (DNN), the takeaway was unclear. While it was interesting to observe that the DNN exhibited a generalization pattern across conditions similar to that seen in the human experiments, the claim made in the abstract and introduction that the model provides a 'mechanistic' explanation for the phenomenon seems overstated. The pattern of weight changes across layers, as depicted in Figure 7, does not conclusively explain the observed variability in generalizations. Furthermore, the substantial weight change observed in the first two layers during the orientation discrimination task is somewhat counterintuitive. Given that neurons in early layers typically have smaller receptive fields and narrower tunings, one would expect this to result in less transfer, not more.

Reviewer #2 (Public Review):

The strengths of this paper are clear: The authors are asking a novel question about geometric representation that would be relevant to a broad audience. Their question has a clear grounding in pre-existing mathematical concepts, that, to my knowledge, have been only minimally explored in cognitive science. Moreover, the data themselves are quite striking, such that my only concern would be that the data seem almost *too* clean. It is hard to know what to make of that, however. From one perspective, this is even more reason the results should be publicly available. Yet I am of the (perhaps unorthodox) opinion that reviewers should voice these gut reactions, even if it does not influence the evaluation otherwise. Below I offer some more concrete comments:

(1) The justification for the designs is not well explained. The authors simply tell the audience in a single sentence that they test projective, affine, and Euclidean geometry. But despite my familiarity with these terms -- familiarity that many readers may not have -- I still had to pause for a very long time to make sense of how these considerations led to the stimuli that were created. I think the authors must, for a point that is so central to the paper, thoroughly explain exactly why the stimuli were designed the way that they were and how these designs map onto the theoretical constructs being tested.

(2) I wondered if the design in Experiment 1 was flawed in one small but critical way. The goal of the parallelism stimuli, I gathered, was to have a set of items that is not parallel to the other set of items. But in doing that, isn't the manipulation effectively the same as the manipulation in the orientation stimuli? Both functionally involve just rotating one set by a fixed amount. (Note: This does not seem to be a problem in Experiment 2, in which the conditions are more clearly delineated.)

(3) I wondered if the results would hold up for stimuli that were more diverse. It seems that a determined experimenter could easily design an "adversarial" version of these experiments for which the results would be unlikely to replicate. For instance: In the orientation group in Experiment 1, what if the odd-one-out was rotated 90 degrees instead of 180 degrees? Intuitively, it seems like this trial type would now be much easier, and the pattern observed here would not hold up. If it did hold up, that would provide stronger support for the authors' theory.

It is not enough, in my opinion, to simply have some confirmatory evidence of this theory. One would have to have thoroughly tested many possible ways that theory could fail. I'm unsure that enough has been done here to convince me that these ideas would hold up across a more diverse set of stimuli.

Reviewer #1 (Public Review):

Summary:<br /> Duan et al analyzed brain imaging data in UKBK and found a pattern in brain structure changes by aging. They identified two patterns and found links that can be differentiated by the categorization.

Strengths:<br /> This discovery harbors a substantial impact on aging and brain structure and function.

Weaknesses:<br /> Therefore, the study requires more validation efforts. Most importantly, data underlying the stratification of the two groups are not obvious and lack further details. Can they also stratified by different methods? i.e. PCA?

Are there any external data that can be used for validation?

Other previous discoveries or claims supporting the results of the study should be explored to support the conclusion.

Sex was merely used as a covariate. Were there sex differences during brain aging? What was the sex ratio difference in groups 1 and 2?

Although statistically significant, Figure 3 shows minimal differences. LTL and phenoAge are displayed in adjusted values but what are the actual values that differ between patterns 1 and 2?

It is not intuitive to link gene expression results shown in Figure 8 and brain structure and functional differences between patterns 1 and 2. Any overlap of genes identified from analyses shown in Figure 6 (GWAS) and 8 (gene expression)?

Reviewer #2 (Public Review):

Summary:<br /> The authors aimed to understand the heterogeneity of brain aging by analyzing brain imaging data. Based on the concept of structural brain aging, they divided participants into two groups based on the volume and rate of decrease of gray matter volume (GMV). The group with rapid brain aging showed accelerated biological aging and cognitive decline and was found to be vulnerable to certain neuropsychiatric disorders. Furthermore, the authors claimed the existence of a "last in, first out" mirroring pattern between brain aging and brain development, which they argued is more pronounced in the group with rapid brain aging. Lastly, the authors identified genetic differences between the two groups and speculated that the cause of rapid brain aging may lie in genetic differences.

Strengths:<br /> The authors supported their claims by analyzing a large amount of data using various statistical techniques. There seems to be no doubt about the quality and quantity of the data. Additionally, they demonstrated their strength in integrating diverse data through various analysis techniques to conclude.

Weaknesses:<br /> There appears to be a lack of connection between the analysis results and their claims. Readers lacking sufficient background knowledge of the brain may find it difficult to understand the paper. It would be beneficial to modify the figures and writing to make the authors' claims clearer to readers. Furthermore, the paper gives an overall impression of being less polished in terms of abbreviations, figure numbering, etc. These aspects should be revised to make the paper easier for readers to understand.

Reviewer #1 (Public Review):

Summary:<br /> The authors are trying to develop a microscopy system that generates data output exceeding the previous systems based on huge objectives.

Strengths:<br /> They have accomplished building such a system, with a field of view of 1.5x1.0 cm2 and a resolution of up to 1.2 um. They have also demonstrated their system performance on samples such as organoids, brain sections, and embryos.

Weaknesses:<br /> To be used as a volumetric imaging technique, the authors only showcase the implementation of multi-focal confocal sectioning. On the other hand, most of the real biological samples were acquired under wide-field illumination, and processed with so-called computational sectioning. Despite the claim that it improves the contrast, sometimes I felt that the images were oversharpened and the quantitative nature of these fluorescence images may be perturbed.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript introduced a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide 3D tissues and embryos. In terms of technique, this paper is just a minor improvement of the authors' previous work, which is a fluorescence imaging system working at visible wavelength region (https://www.nature.com/articles/s41598-021-95930-7).

Strengths:<br /> In this study, the authors enhanced the system's resolution and sensitivity by increasing the numerical aperture (NA) of the lens. Furthermore, they achieved volumetric imaging by integrating optical sectioning and computational sectioning. This study encompasses a broad range of biological applications, including imaging and analysis of organoids, mouse brains, and quail embryos, respectively. Overall, this method is useful and versatile.

Weaknesses:<br /> The unique application that only can be done by this high-throughput system remains vague. Meanwhile, there are also several outstanding issues in this paper, such as the lack of technical advances, unclear method details, and non-standardized figures.

Reviewer #1 (Public Review):

Summary:

In their study, Petersen et al. investigated the relationship between parameters of metabolic syndrome (MetS) and cortical thickness using partial least-squares correlation analysis (PLS) and performed subsequently a group comparison (sensitivity analysis). To do this, they utilized data from two large-scale population-based cohorts: the UK BioBank (UKB) and the Hamburg City Health Study (HCHS). They identified a latent variable that explained 77% of the shared variance, driven by several measures related to MetS, with obesity-related measures having the strongest contribution. Their results highlighted that higher cortical thickness in the orbitofrontal, lateral prefrontal, insular, anterior cingulate, and temporal areas is associated with lower MetS severity. Conversely, the opposite pattern was observed in the superior frontal, parietal, and occipital regions. A similar pattern was then observed in the sensitivity analysis when comparing two groups (MetS vs. matched controls) separately.

Interestingly, after including HbA1c (a blood glycemic marker, which reflects insulin resistance much better than non-fasting glucose) in their revision, the authors identified a second latent variable accounting for 22% of shared variance mostly driven by HbA1c and blood glucose. The authors conclude that the distinct covariance profile of this variable likely indicates a separate pathological mechanistic connection between MetS components and brain morphology.

They then mapped local cellular and network topological attributes to the observed cortical changes associated with MetS. This was achieved using cell-type-specific gene expressions from the Allen Human Brain Atlas and the group consensus functional and structural connectomes of the Human Connectome Project (HCP), respectively. This contextualization analysis allowed them to identify potential cellular contributions in these structures driven by endothelial cells, microglial cells, and excitatory neurons. It also indicated functional and structural interconnectedness of areas experiencing similar MetS effects.

Strengths:

The effects of metabolic syndrome on the brain are still incompletely understood, and such multi-scale analyses are important for the field. Despite the study's sole 'correlation-based' nature, it yields valuable results, including several scales of brain parameters (cortical thickness, cellular, and network-based). The results are robust and benefit from two 'large-scale' datasets, resulting in highly powered statistics

Weaknesses:

The weakness of this study lies mostly on the non-causative approach used here. Nevertheless, the authors are aware of the limitations of the study and carefully frame their language accordingly.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Petersen et al. aimed for a comprehensive assessment of the relationship between cardiometabolic risk factors and cortical thickness. They found that a latent variable reflecting higher obesity, hypertension, LDL cholesterol, triglyerides, non-fasting glucose, HbA1c and lower HDL cholesterol was associated with lower cortical thickness in orbitofrontal, lateral prefrontal, insular, anterior cingulate and temporal areas as well as lower subcortical volumes. In sensitivity analyses they showed that this pattern replicated across cohorts and was also consistent with a clinical definition of the metabolic syndrome.

Further, when including cognition into the multivariate analysis, the pattern remained unchanged and mediation analyses showed that the relationship between the first latent variable and worse cognitive performance across several tests was mediated by the brain morphological differences.

The authors investigated the cell types implicated in the regions associated with cardiometabolic risk using the Allen Brain Atlas and found that the density of excitatory neurons type 8, endothelial cells and microglia reliably co-located with the pattern of cortical thickness. Furthermore, they showed that cortial regions more strongly associated with MetS were more closely structurally & functionally connected than others.

Strengths:

This study performed a comprehensive assessment of the combined association of cardiometabolic risk factors and brain structure and investigated micro-and macroscopic underpinnings. A major strength of the study is the methodological approach of partial least squares which allows one to not single out risk factors but to take them into account simultaneously. The large sample size from two cohorts allowed for different sensitivity analyses and convincing evidence for the stability of the first latent variable. The authors demonstrated that the component was also reliably related to cognitive performance and that the association of the individual cardiometabolic risk on cognition was mediated by brain morphological differences, replicating multiple previous studies which evidenced associations of different components of the MetS with worse cognitive performance.

The novel contribution of the study lies in the virtual histology and brain topology investigation of the cortical pattern related to MetS. The virtual histology provided convincing evidence of the co-localization of endothelial, glial and excitatory neuronal cells with the regions of MetS-associated cortical thinning while the brain topology analysis highlighted the disproportionate structural and functional connectivity between associated regions. This analysis provides insights into the role of inflammatory processes and the intricate link between gray matter morphology and microvasculature, both locally and in relation to long-range connectivity. This information is valuable to inform future mechanistic studies.

Weaknesses:

The study is exclusively cross-sectional which does not allow disentangling potential causes from consequences. While studies indicate that most of the differences seen in middle age are probably consequences of the MetS on the vasculature, blood-brain barrier or inflammatory processes, differences in cortical morphology might also represent a risk factor for weight gain.<br /> The study is exploratory in nature and for the contextualization analyses it is difficult to judge whether those were selected from a larger pool of analyses.

Reviewer #1 (Public Review):

This is a well-designed study that explores the BEF relationships in fragmented landscapes. Although there are massive studies on BEF relationships, most of them were conducted at local scales, few considered the impacts of landscape variables. This study used a large dataset to specifically address this question and found that habitat loss weakened the BEF relationships. Overall, this manuscript is clearly written and has important implications for BEF studies as well as for ecosystem restoration.

I have read the response letter provided by the authors and think they have done a great job in addressing my concerns.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Yan et al. assess the effect of two facets of habitat fragmentation (i.e., habitat loss and habitat fragmentation per se) on biodiversity, ecosystem function, and the biodiversity-ecosystem function (BEF) relationship in grasslands of an agro-pastoral ecotone landscape in northern China. The authors use a stratified random sampling to select 130 study sites located within 500 m - radius landscapes varying along gradients of habitat loss and habitat fragmentation per se. In these study sites, the authors measure grassland specialist and generalist plant richness via field surveys, as well as above-ground biomass by harvesting and dry-weighting the grass communities in each 3 x 1m2 plots of the 130 study sites. The authors find that habitat loss and fragmentation per se have different effects on biodiversity, ecosystem function and the BEF relationship: whereas habitat loss was associated with a decrease in plant richness, fragmentation per se was not; and whereas fragmentation per se was associated with a decrease in above-ground biomass, habitat loss was not. Finally, habitat loss, but not fragmentation per se was linked to a decrease in the magnitude of the positive biodiversity-ecosystem functioning relationship, via reducing the percentage of grassland specialists in the community.

Strengths:<br /> This study by Yan et al. is an exceptionally well-designed, well-written, clear and concise study shedding light on a longstanding, important question in landscape ecology and biodiversity-ecosystem functioning research. Via a stratified random sampling approach (cf. also "quasi-experimental design" Butsic et al. 2017), Yan et al. create an ideal set of study sites, where habitat loss and habitat fragmentation per se (usually highly correlated) are decorrelated and hence, separate effects of each of these facets on biodiversity and ecosystem function can be assessed statistically in "real-world" (and not experimental, cf. Duffy et al. 2017) communities. The authors use adequate and well-described methods to investigate their questions. The findings of this study add important empirical evidence from real-world grassland ecosystems that help to advance our theoretical understanding on landscape-moderation of biodiversity effects and provide important guidelines for conservation management likewise. Also, all figures are well-designed and clear.

Weaknesses:<br /> I found only a few minor issues, mostly unclear descriptions that have now been revised for more clarity.

Reviewer #3 (Public Review):

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

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

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

Reviewer #2 (Public Review):

The authors have used microfluidic channels to study the response of budding yeast to variable environments. Namely, they tested the ability of the cells to divide when the medium was repeatedly switched between two different conditions at various frequencies. They first characterized the response to changes in glucose availability or in the presence of hyper-osmotic stress via the addition of sorbitol to the medium. Subsequently, the two stresses were combined by applying the alternatively or simultaneously (in-phase). Interestingly, they observed that the in-phase stress pattern allowed more divisions and low levels of cell mortality compared to the alternating stresses where cells were dividing slowly and many cells died. A number of mutants in the HOG pathway were tested in these conditions to evaluate their responses. Moreover, the activation of the MAPK Hog1 and the transcriptional induction of the hyper-osmotic stress promoter STL1 were quantified by fluorescence microscopy.

Overall, the manuscript is well structured and data are presented in a clear way. The time-lapse experiments were analyzed with high precision. The experiments confirm the importance of performing dynamic analysis of signal transduction pathways. While the experiments reveal some unexpected behavior, I find that the biological insights gained on this system remain relatively modest.

In the discussion section, the authors mention two important behaviors that their data unveil: resource allocation (between glycolysis and HOG-driven adaptation) and regulation of the HOG-pathway based on the presence of glucose. These types of behaviors had been already observed in other reports (Sharifan et al. 2015 or Shen et al. 2023, for instance). The experimental set-up used in this study provides highlights new aspects of the interplay between hyper-osmotic stress response and glucose availability.

The authors have tested various processes that could explain the slow growth observed in the alternating stress regime. Unfortunately, neither glycogen accumulation, cell-cycle arrest via Sic1 or the inhibition of protein production in starved cells could explain the observed behavior. However, one clear evidence that is presented is the link between glycerol accumulation during the sorbitol treatment and the cell death phenotype upon starvation in alternating stress condition.

One question which remains open is to what extent the findings presented here can be extended to other types of perturbations which for instance would combine Nitrogen limitation and hyper-osmotic stress.

Reviewer #1 (Public Review):

In this study, the authors aimed to investigate how cells respond to dynamic combinations of two stresses compared to dynamic inputs of a single stress. They applied the two stresses - carbon stress and hyperosmotic stress - either in or out of phase, adding and removing glucose and sorbitol.

Both a strength and a weakness is that the cells' hyperosmotic response strongly requires glucose. For in-phase stress, cells are exposed to hyperosmotic shock without glucose, limiting their ability to respond with the well-studied HOG pathway; for anti-phase stress, cells do have glucose when hyperosmotically shocked, but experience a hypo-osmotic shock when both glucose and sorbitol are simultaneously removed. Responding with the HOG pathway and so amassing intracellular glycerol amplifies the impact of this hypo-osmotic shock. Counterintuitively then, it is the presence of glucose rather than the stress of its absence that is deleterious for the cells.

The bulk of the paper supports these conclusions with clean, compelling time-lapse microscopy, including extensive analysis of gene deletions in the HOG network and measurements of both division and death rates. The methodology the authors develop is powerful and widely applicable.

The authors' findings demonstrate the tight links that can exist between metabolism and the ability to respond to stress and the novel insights that can be gained using multiple dynamic inputs.

Reviewer #3 (Public Review):

Summary:

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

Comments for the previous version:

Major comments:

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

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

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

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

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

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

Comments for the revised version:

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

Reviewer #1 (Public Review):

Recently discovered extrachromosomal DNA (ecDNA) provides an alternative non-chromosomal means for oncogene amplification and a potent substrate for selective evolution of tumors. The current work aims to identify key genes whose expression distinguishes ecDNA+ and ecDNA- tumors and the associated processes to shed light on the biological mechanisms underlying ecDNA genesis and their oncogenic effects. This is clearly an important question and through detailed analysis this work points to specific GO processes associated (up and down) with ecDNA+ tumors, namely, specific DNA damage repair processes and specific oncogenic processes.

In the initial submission I had commented on lack of clarity of method, potential biases, and in some cases inappropriate interpretation. In the revised version, the authors have addressed all my comments satisfactorily and I think this is an important work furthering our understanding of mechanisms underlying ecDNA+ tumors.

Reviewer #2 (Public Review):

In their manuscript Lin et al. describe an important study on the transcriptional programs associated with the presence of extrachromosomal DNA in a cohort of 870 cancers of different origins. The authors find that compared to cancers lacking such amplifications, ecDNA+ cancers express higher levels of DNA damage repair-associated genes, but lower levels of immune-related gene programs.

This work is very timely and its findings have the potential to be very impactful, as the transcriptional context differences between ecDNA+ and ecDNA- cancers are currently largely unknown. The observation that immune programs are downregulated in ecDNA+ cancers may initiate new preclinical and translational studies that impact the way ecDNA+ cancers are treated in the future. Thus, this study has important theoretical implications that have the potential to substantially advance our understanding of ecDNA+ cancers.

Strengths:

The authors provide compelling evidence for their conclusions based on large patient datasets. The methods they used and analyses are rigorous.

Weaknesses:

The biological interpretation of the data remains observational. The direct implication of these genes in ecDNA(+) tumors is not tested experimentally.

Reviewer #1 (Public Review):

The authors deploy a combination of their own previously developed computational methods and databases (SIGNOR and CellNOptR) to model the FLT3 signaling landscape in AML and identify synergistic drug combinations that may overcome the resistance AML cells harboring ITD mutations in the TKI domain of FLT3 to FLT3 inhibitors. I did not closely evaluate the details of these computational models since they are outside of my area of expertise and have been previously published. The manuscript has significant issues with data interpretation and clarity, as detailed below, which, in my view, call into question the main conclusions of the paper.

The authors train the model by including perturbation data where TKI-resistant and TKI-sensitive cells are treated with various inhibitors and the activity (i.e. phosphorylation levels) of the key downstream nodes are evaluated. Specifically, in the Results section (p. 6) they state "TKIs sensitive and resistant cells were subjected to 16 experimental conditions, including TNFa and IGF1 stimulation, the presence or absence of the FLT3 inhibitor, midostaurin, and in combination with six small-molecule inhibitors targeting crucial kinases in our PKN (p38, JNK, PI3K, mTOR, MEK1/2 and GSK3)". I would appreciate more details on which specific inhibitors and concentrations were used for this experiment. More importantly, I was very puzzled by the fact that this training dataset appears to contain, among other conditions, the combination of midostaurin with JNK inhibition, i.e. the very combination of drugs that the authors later present as being predicted by their model to have a synergistic effect. Unless my interpretation of this is incorrect, it appears to be a "self-fulfilling prophecy", i.e. an inappropriate use of the same data in training and verification/test datasets.

My most significant criticism is that the proof-of-principle experiment evaluating the combination effects of midostaurin and SP600125 in FLT3-ITD-TKD cell line model does not appear to show any synergism, in my view. The authors' interpretation of the data is that the addition of SP600125 to midostaurin rescues midostaurin resistance and results in increased apoptosis and decreased viability of the midostaurin-resistant cells. Indeed, they write on p.9: "Strikingly, the combined treatment of JNK inhibitor (SP600125) and midostaurin (PKC412) significantly increased the percentage of FLT3ITD-TKD cells in apoptosis (Fig. 4D). Consistently, in these experimental conditions, we observed a significant reduction of proliferating FLT3ITD- TKD cells versus cells treated with midostaurin alone (Fig. 4E)." However, looking at Figs 4D and 4E, it appears that the effects of the midostaurin/SP600125 combination are virtually identical to SP600125 alone, and midostaurin provides no additional benefit. No p-values are provided to compare midostaurin+SP600125 to SP600125 alone but there seems to be no appreciable difference between the two by eye. In addition, the evaluation of synergism (versus additive effects) requires the use of specialized mathematical models (see for example Duarte and Vale, 2022). That said, I do not appreciate even an additive effect of midostaurin combined with SP600125 in the data presented.

In my view, there are significant issues with clarity and detail throughout the manuscript. For example, additional details and improved clarity are needed, in my view, with respect to the design and readouts of the signaling perturbation experiments (Methods, p. 15 and Fig 2B legend). For example, the Fig 2B legend states: "Schematic representation of the experimental design: FLT3 ITD-JMD and FLT3 ITD-JMD cells were cultured in starvation medium (w/o FBS) overnight and treated with selected kinase inhibitors for 90 minutes and IGF1 and TNFa for 10 minutes. Control cells are starved and treated with PKC412 for 90 minutes, while "untreated" cells are treated with IGF1 100ng/ml and TNFa 10ng/ml with PKC412 for 90 minutes.", which does not make sense to me. The "untreated" cells appear to be treated with more agents than the control cells. The logic behind cytokine stimulation is not adequately explained and it is not entirely clear to me whether the cytokines were used alone or in combination. Fig 2B is quite confusing overall, and it is not clear to me what the horizontal axis (i.e. columns of "experimental conditions", as opposed to "treatments") represents. The Method section states "Key cell signaling players were analyzed through the X-Map Luminex technology: we measured the analytes included in the MILLIPLEX assays" but the identities of the evaluated proteins are not given in the Methods. At the same time, the Results section states "TKIs sensitive and resistant cells were subjected to 16 experimental conditions" but these conditions do not appear to be listed (except in Supplementary data; and Fig 2B lists 9 conditions, not 16). In my subjective view, the manuscript would benefit from a clearer explanation and depiction of the experimental details and inhibitors used in the main text of the paper, as opposed to various Supplemental files/figures. The lack of clarity on what exactly were the experimental conditions makes the interpretation of Fig 2 very challenging. In the same vein, in the PCA analysis (Fig 2C) there seems to be no reference to the cytokine stimulation status while the authors claim that PC2 stratifies cells according to IGF1 vs TNFalpha. There are numerous other examples of incomplete or confusing legends and descriptions which, in my view, need to be addressed to make the paper more accessible.

I am not sure that I see significant value in the patient-specific logic models because they are not supported by empirical evidence. Treating primary cells from AML patients with relevant drug combinations would be a feasible and convincing way to validate the computational models and evaluate their potential benefit in the clinical setting.

Reviewer #2 (Public Review):

Summary:

This manuscript by Latini et al describes a methodology to develop Boolean-based predictive logic models that can be applied to uncover altered protein/signalling networks in cancer cells and discover potential new therapeutic targets. As a proof-of-concept, they have implemented their strategy on a hematopoietic cell line engineered to express one of two types of FLT3 internal tandem mutations (FLT3-ITD) found in patients, FLT3-ITD-TKD (which are less sensitive to tyrosine kinase inhibitors/TKIs) and FLT3-ITD-JMD (which are more sensitive to TKIs).

Strengths:

This useful work could potentially represent a step forward towards personalised targeted therapy, by describing a methodology using Boolean-based predictive logic models to uncover altered protein/signalling networks within cancer cells.

Authors have validated their approach by analysing independent, real-world data

Weaknesses:

No weaknesses were observed by this reviewer for the revised version.

Reviewer #3 (Public Review):

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

Strengths:

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

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

Weaknesses:

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

Reviewer #2 (Public Review):

The authors examine the use of metformin in the treatment of hepatic ischemia/reperfusion injury (HIRI) and suggest the mechanism of action is mediated in part by the gut microbiota and changes in hepatic ferroptosis. The concept is intriguing and their results have potential to better understand the pleiotropic functions of metformin. The histological and imaging studies were considered a strength and reveal a significant impact of metformin post-HIRI. The connections with GABA producing bacteria adds to our understanding of the chemical signals exchanged between the host and microbiota. While the authors have characterized these connections in mice, how/if these observations translate to humans remains to be determined.

Reviewer #1 (Public Review):

Many drugs have off-target effects on the gut microbiota but the downstream consequences for drug efficacy and side effect profiles remain unclear. Herein, Wang et al. use a mouse model of liver injury coupled to antibiotic and microbiota transplantation experiments. Their results suggest that metformin-induced shifts in gut microbial community structure and metabolite levels may contribute to drug efficacy. This study provides valuable mechanistic insights that could be dissected further in future studies, including efforts to identify which specific bacterial species, genes, and metabolites play a causal role in drug response. Importantly, although some pilot data from human subjects is shown, the clinical relevance of these findings for liver disease remain to be determined.

Comments on revised version:

The authors have now addressed my original concerns.

Reviewer #3 (Public Review):

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

Reviewer #1 (Public Review):

The work by Ohigashi and colleagues addresses the developmental and lineage relationship of a newly characterized thymus epithelial cell (TEC) progenitor subset. The authors take advantage of an elegant and powerful set of experimental approaches to demonstrate that CCL21-expressing TECs appear early in thymus organogenesis and that these cells, which are centrally located, go on to give rise to medullary (m)TECs. What makes the findings intriguing is that these CCL21-expressing mTECs are a distinct subset, which do not express RANK or AIRE, and transcriptomic and lineage tracing approaches point to these cells as potential mTEC progenitor-like cells. Of note, using in vitro and in vivo precursor-product cell transfer experiments, the authors show that this subset has a developmental potential to give rise to AIRE+ self-antigen-displaying mTECs, revealing that CCL21-expressing mTECs can give rise to distinct mTEC subsets. This functional duality provides an attractive rationale for the necessary function of mTECs, which is to attract CCR7+ thymocytes that have just undergone positive selection in the thymus cortex to enter the medulla to undergo tolerance-induction against self-antigen-displaying mTECs. Overall, the work is well supported and offers new insights into the diverse functions of the medullary compartment, and how two distinct subsets of mTECs can achieve it.

Reviewer #2 (Public Review):

The authors set out to discover a developmental pathway leading to functionally diverse mTEC subsets. They show that Ccl21 is expressed early during thymus ontogeny in the medullary area. Fate-mapping gives evidence for the Ccl21 positive history of Aire positive mTECs as well as of thymic tuft cells and postnatally of a certain percentage of cTECs. Therefore, the differentiation potential of Ccl21+ TECs is tested in reaggregate thymus experiments - using embryonic or postnatal Ccl21+ TECs. From these experiments, the authors conclude that at least embryonic mTECs in large part pass through a Ccl21 positive stage prior to differentiation towards an Aire expressing or tuft cell stage.

The authors are using Ccl21a as a marker for a bipotent progenitor that is detectable in the embryonic thymus and is still present at the adult stage mainly giving rise to mTECs. The choice of this marker gene is very interesting since Ccl21 expression can directly be linked to an important aspect in thymus biology: the expression of Ccl21 by cells in the thymic medulla allows trafficking of T cells into the medulla in order to undergo T cell selection. Making use of the Ccl21 detection, the authors can nicely show that cells actively expressing Ccl21 are localized throughout the medulla at an embryonic stage but also in adult thymus tissue. This suggests, that this progenitor is not accumulating at a specific area inside the medulla. This is a new finding. Moreover, the finding that a Ccl21+ progenitor population plays a functional role in thymocyte trafficking towards the medulla has not been described. Thus, Ccl21 expression may be used to localize a late bipotent progenitor in the thymic lobes. In addition, in Fig.8, the authors provide evidence that these progenitor cells have the potential to self-maintain as well as to differentiate in reaggregate experiments at E17 (not at 4 weeks of age). The first point is of great interest and importance since these cells in theory can be of therapeutic use.

Overall assessment:

The authors highlight a developmental pathway starting from a Ccl21-expressing TEC progenitor that contributes to a functionally diverse mTEC repertoire. This is a welcome addition to current knowledge of TEC differentiation.

Reviewer #1 (Public Review):

This study reports a long-term, multisite study of tropical herbivory on Piper plants. The results are clear that lack of water leads to lower plant survival and altered herbivory. The results varied substantially among sites. The caveats are that ecosystem processes beyond water availability are not investigated although they are brought into play in the title and in the paper, that herbivory beyond leaf damage was not reported (there might be none, reader needs to be shown the evidence for this), that herbivore diversity is defined by leaf damage (authors need to give evidence that this is a valid inference), that the plots were isolated from herbivores beyond their borders, that the effects of extreme climate events were isolated to Peru, that intraspecific variation in the host plants needs to be explained and interpreted in more detail, the results as reported are extremely complicated, the discussion is overly long and diffuse.

Reviewer #2 (Public Review):

This is an important and large experimental study examining the effects of plant species richness, plant genotypic richness, and soil water availability on herbivory patterns on Piper species in tropical forests.

A major strength is the size of the study and the fact that it tackled so many potentially important factors simultaneously. The authors examined both interspecific plant diversity and intraspecific plant diversity. They crossed that with a water availability treatment. And they repeated the experiment across five geographically separated sites.

The authors find that both water availability and plant diversity, intraspecific and interspecific, influence herbivore diversity and herbivory, but that the effects differ in important ways across sites. I found the study to be solid and the results to be very convincing. The results will help the field grapple with the importance of environmental change and biodiversity loss and how they structure communities and alter species interactions.

Reviewer #1 (Public Review):

The authors developed an extension to the pairwise sequentially Markov coalecent model that allows to simultaneously analyze multiple types of polymorphism data. In this paper, they focus on SNPs and DNA methylation data. Since methylation markers mutate at a much faster rate than SNPs, this potentially gives the method better power to infer size history in the recent past. Additionally, they explored a model where there are both local and regional epimutational processes.

Integrating additional types of heritable markers into SMC is a nice idea which I like in principle. However, a major caveat to this approach seems to be a strong dependence on knowing the epimutation rate. In Fig. 6 it is seen that, when the epimutation rate is known, inferences do indeed look better; but this is not necessarily true when the rate is not known. (See also major comment #1 below about the interpretation of these plots.) A roughly similar pattern emerges in Supp. Figs. 4-7; in general, results when the rates have to be estimated don't seem that much better than when focusing on SNPs alone. This carries over to the real data analysis too: the interpretation in Fig. 7 appears to hinge on whether the rates are known or estimated, and the estimated rates differ by a large amount from earlier published ones.

Overall, this is an interesting research direction, and I think the method may hold more promise as we get more and better epigenetic data, and in particular better knowledge of the epigenetic mutational process. At the same time, I would be careful about placing too much emphasis on new findings that emerge solely by switching to SNP+SMP analysis.

Major comments:<br /> - For all of the simulated demographic inference results, only plots are presented. This allows for qualitative but not quantitative comparisons to be made across different methods. It is not easy to tell which result is actually better. For example, in Supp. Fig. 5, eSMC2 seems slightly better in the ancient past, and times the trough more effectively, while SMCm seems a bit better in the very recent past. For a more rigorous approach, it would be useful to have accompanying tables that measure e.g. mean-squared error (along with confidence intervals) for each of the different scenarios, similar to what is already done in Tables 1 and 2 for estimating $r$.

- 434: The discussion downplays the really odd result that inputting the true value of the mutation rate, in some cases, produces much worse estimates than when they are learned from data (SFig. 6)! I can't think of any reason why this should happen other than some sort of mathematical error or software bug. I strongly encourage the authors to pin down the cause of this puzzling behaviour. (Comment addressed in revision. Still, I find the explanation added at 449ff to be somewhat puzzling -- shouldn't the results of the regional HMM scan only improve if the true mutation rate is given?)

- As noted at 580, all of the added power from integrating SMPs/DMRs should come from improved estimation of recent TMRCAs. So, another way to study how much improvement there is would be to look at the true vs. estimated/posterior TMRCAs. Although I agree that demographic inference is ultimately the most relevant task, comparing TMRCA inference would eliminate other sources of differences between the methods (different optimization schemes, algorithmic/numerical quirks, and so forth). This could be a useful addition, and may also give you more insight into why the augmented SMC methods do worse in some cases. (Comment addressed in revision via Supp. Table 7.).

- A general remark on the derivations in Section 2 of the supplement: I checked these formulas as best I could. But a cleaner, less tedious way of calculating these probabilities would be to express the mutation processes as continuous time Markov chains. Then all that is needed is to specify the rate matrices; computing the emission probabilities needed for the SMC methods reduces to manipulating the results of some matrix exponentials. In fact, because the processes are noninteracting, the rate matrix decomposes into a Kronecker sum of the individual rate matrices for each process, which is very easy to code up. And this structure can be exploited when computing the matrix exponential, if speed is an issue.

- Most (all?) of the SNP-only SMC methods allow for binning together consecutive observations to cut down on computation time. I did not see binning mentioned anywhere, did you consider it? If the method really processes every site, how long does it take to run?

- 486: The assumed site and region (de)methylation rates listed here are several OOM different from what your method estimated (Supp. Tables 5-6). Yet, on simulated data your method is usually correct to within an order of magnitude (Supp. Table 4). How are we to interpret this much larger difference between the published estimates and yours? If the published estimates are not reliable, doesn't that call into question your interpretation of the blue line in Fig. 7 at 533? (Comment addressed in revision.)

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 #3 (Public Review):

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

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

Reviewer #1 (Public Review):

Summary: This study examines the role of host blood meal source, temperature, and photoperiod on the reproductive traits of Cx. quinquefasciatus, an important vector of numerous pathogens of medical importance. The host use pattern of Cx. quinquefasciatus is interesting in that it feeds on birds during spring and shifts to feeding on mammals towards fall. Various hypotheses have been proposed to explain the seasonal shift in host use in this species but have provided limited evidence. This study examines whether the shifting of host classes from birds to mammals towards autumn offers any reproductive advantages to Cx. quinquefasciatus in terms of enhanced fecundity, fertility, and hatchability of the offspring. The authors found no evidence of this, suggesting that alternate mechanisms may drive the seasonal shift in host use in Cx. quinquefasciatus.

Strengths: Host blood meal source, temperature, and photoperiod were all examined together.

Weaknesses: The study was conducted in laboratory conditions with a local population of Cx. quinquefasciatus from Argentina. I'm not sure if there is any evidence for a seasonal shift in the host use pattern in Cx. quinquefasciatus populations from the southern latitudes.

Comments on the revision:

Overall, I am not quite convinced about the possible shift in host use in the Argentinian populations of Cx. quinquefasciatus. The evidence from the papers that the authors cite is not strong enough to derive this conclusion. Therefore, I think that the introduction and discussion parts where they talk about host shift in Cx. quinquefasciatus should be removed completely as it misleads the readers. I suggest limiting the manuscript to talking only about the effects of blood meal source and seasonality on the reproductive outcomes of Cx. quinquefasciatus.

Reviewer #2 (Public Review):

Summary:

Conceptually, this study is interesting and is the first attempt to account for the potentially interactive effects of seasonality and blood source on mosquito fitness, which the authors frame as a possible explanation for previously observed host-switching of Culex quinquefasciatus from birds to mammals in the fall. The authors hypothesize that if changes in fitness by blood source change between seasons, higher fitness on birds in the summer and on mammals in the autumn could drive observed host switching. To test this, the authors fed individuals from a colony of Cx. quinquefasciatus on chickens (bird model) and mice (mammal model) and subjected each of these two groups to two different environmental conditions reflecting the high and low temperatures and photoperiod experienced in summer and autumn in Córdoba, Argentina (aka seasonality). They measured fecundity, fertility, and hatchability over two gonotrophic cycles. The authors then used a generalized linear model to evaluate the impact of host species, seasonality, and gonotrophic cycle on fecundity, fertility, and hatchability. The authors were trying to test their hypothesis by determining whether there was an interactive effect of season and host species on mosquito fitness. This is an interesting hypothesis; if it had been supported, it would provide support for a new mechanism driving host switching. While the authors did report an interactive impact of seasonality and host species, the directionality of the effect was the opposite from that hypothesized. The authors have done a very good job of addressing many of the reviewer concerns, with several exception that continue to cause concern about the conclusions of the study.

Strengths:

(1) Using a combination of laboratory feedings and incubators to simulate seasonal environmental conditions is a good, controlled way to assess the potentially interactive impact of host species and seasonality on the fitness of Culex quinquefasciatus in the lab.<br /> (2) The driving hypothesis is an interesting and creative way to think about a potential driver of host switching observed in the field.<br /> (3) The manuscript has become a lot clearer and easier to read with the revisions - thank you to the authors for working hard to make many of the suggested changes.

Weaknesses:

(1) The authors have decided not to follow the suggestion of conducting experimental replicates of the study. This is understandable given the significant investment of resources and time necessary, however, it leaves the study lacking support. Experimental replication is an important feature of a strong study and helps to provide confidence that the observed patterns are real and replicable. Without replication, I continue to lack confidence in the conclusions of the study.<br /> (2) The authors have included some additional discussion about the counterintuitive nature of their results, but the paragraph discussing this in the discussion was confusing. I believe that this should be revised. This is a key point of the paper and needs to be clear to the reader.<br /> (3) There should be more discussion of the host switching observed in the two studies conducted in Argentina referenced by the authors. Since host switching is the foundation for the hypothesis tested in this paper, it is important to fully explain what is currently known in Argentina.<br /> (4) In some cases, the explanations of referenced papers are not entirely accurate. For example, when referencing Erram et al 2022, I think the authors misrepresented the paper's discussion regarding pre-diuresis- Erram et al. are suggesting that pre-diuresis might be the mechanism by which C. furens compensates for the lower nutritional value of avian blood, leading to no significant difference between avian/mammal blood on fecundity/fertility (rather than leading to higher fecundity on birds, as stated in this manuscript). The study performed by Erram et al. also didn't prove this phenomenon, they just suggest it as a possible mechanism to explain their results, so that should be made clear when referencing the paper.<br /> (5) In some cases, the conclusions continue to be too strongly worded for the evidence available. For example, lines 322-324: I don't think the data is sufficient to conclude that a different physiological state is induced, nor that they are required to feed on a blood source that results in higher fitness.<br /> (6) There is limited mention of the caveat that this experiment performed with simulated seasonality that does not perfectly replicate seasonality in the field. I think this caveat should be discussed in the discussion (e.g. that humidity is held constant).

Reviewer #1 (Public Review):

In their manuscript, Laporte et al. analyze the process of formation of the quiescent-cell nuclear microtubule (Q-nMT) bundle, a set of parallel MTs that emanate from the nuclear side of the spindle pole bodies (SPBs) upon the entry of Saccharomyces cerevisiae cells in quiescence. Based on their results, the authors propose that Q-nMT bundle formation is a multistep process that comprises three distinct sequential phases. The authors further evaluate the role of different factors during the growth of the Q-nMT bundle upon quiescence entry, as well as during the disassembly of this structure once the cells resume their proliferation.

The Q-nMT is an interesting cellular structure whose physiological function is still widely unknown. Hence, providing new insights into the dynamics of Q-nMT bundle formation and identifying new factors involved in this process is an interesting topic of relevance in the field. The authors made a substantial effort in order to evaluate Q-nMT bundle establishment and provide a considerable amount of data, mainly obtained from microscopy analyses. Overall, the experiments are mostly well described and properly executed, and the data in the manuscript are clearly presented. Despite the interest of the study, nonetheless, there are several issues that could affect the validity of some conclusions drawn. In this way, regarding the analysis of the dynamics of Q-nMT bundle formation, the described experimental setup raises certain concerns, which mostly derive from the use of the microtubule-depolymerizing agent nocodazole as the only approach to evaluate this process. Also, regarding the factors involved in Q-nMT formation, the differences in microtubule length with the wild type strain, despite being statistically significant, are really subtle for many of the mutants analyzed (e.g., bir1, slk19, etc.). Furthermore, it is also puzzling that an effect on Q-nMT formation is proposed for meiosis-specific factors such as Mam1, which might as well be present during quiescence, but seems to be also detected in proliferating cells. Lastly, the evidence shown are insufficient to provide a direct link between defects in cell viability and aberrant Q-nMT formation.

Reviewer #2 (Public Review):

Summary: The authors investigate the assembly of the Q-nMT, a stable microtubule structure that is assembled during quiescence. Notably, the authors show that the formation of the Q-nMT cannot be solely explained by changes in the physico-chemical properties of quiescent cells. The authors report that Q-nMT assembly occurs in three regulated steps and identify kinesin motor proteins involved in the assembly and disassembly of the structure.

Strengths: The findings provide new insight into the assembly and possible function of the Q-nMT with respect to the response of haploid budding yeast to glucose starvation.

Weaknesses: The manuscript would benefit from more precise language and requires additional clarification regarding how claims are supported by the evidence. Clear definitions are also required, for example "active process" is not defined. Some conclusions are not supported by the results, for example the claim that the Q-nMT functions as a checkpoint effector that inhibits re-entry into the cell cycle.

After reviewing the responses of the authors and the revised manuscript I am now satisfied with the study in its current form.

Reviewer #1 (Public Review):

The study presented in this manuscript presents very convincing evidence that purifying selection is the main force shaping the landscape of TE polymorphisms in B. distachyon, with only a few putatively adaptive variants detected, even though most conclusions are based on the 10% of polymorphisms contributed by retrotransposons. That first conclusion is not novel, however, as it had already been clearly established in natural A. thaliana strains (Baduel et al. Genome Biol 2021) and in experimental D. simulans lines (Langmüller et al. NAR 2023). In contrast to the conclusions reached in A. thaliana, however, Horvath et al. report here a seemingly deleterious effect of TE insertions even very far away from genes (>5kb), a striking observation for a genome of relatively similar size. However, SNPs within these regions show similar allele frequency deviations, suggesting this effect may be due to mapping quality artefacts in gene poor regions of the genome. An additional caveat of this study is the lack of orthogonal benchmarking of the TE polymorphisms calls by a pipeline known for a high rate of false positives (see detailed Private Recommendations #1). The authors note that their conclusions are still valid using only the highest covered samples (>20x), yet this coverage threshold is relatively low and higher coverage would mostly reduce the rate of false negatives.

Nonetheless, this set of observations makes an important addition to the knowledge of TE dynamics in the wild and questions our understanding of the main molecular mechanisms through which TEs can impact fitness.

Reviewer #2 (Public Review):

Transposable elements are known to have a strong potential to generate diversity and impact gene regulation, and they are thought to play an important role in plant adaptation to changing environments. Nevertheless, very few studies have performed genome-wide analyses to understand the global effect of selection on TEs in natural populations. Horvath et al., used available whole-genome re-sequencing data from a representative panel of B. distachyon accessions to detect TE insertion polymorphisms (TIPs) and estimate their time of origin. Using a thorough combination of population genomics approaches, the authors demonstrate that only a small amount of the TE polymorphisms are targeted by positive selection or potentially involved in adaptation. By comparing the age-adjusted population frequencies of TE polymorphisms and neutral SNPs, the authors found that retrotransposons are affected by purifying selection independently of their distance to genes. Finally, using forward simulations they were able to quantify the strength of selection acting on TE polymorphisms, finding that retrotransposons are mainly under moderate purifying selection, with only a minority of the insertions evolving neutrally.

Horvath et al., use a convincing set of strategies and their conclusions are well supported by the data. I think that incorporating polymorphism's age to the analysis of purifying selection is an interesting way to reduce the possible bias introduced by the fact that SNPs and TEs polymorphisms do not occur at the same pace. The fact that TE polymorphisms far from genes are also under purifying selection is an interesting result that reinforces the idea that trans-regulatory effect of TE insertions might not be a rare phenomenon, a matter that may be demonstrated in future studies.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Wu et al. investigated the microbiome in the rhizosphere and roots of plant species along an elevational gradient. They found that: (i) plants with higher root nitrogen ("fast" strategy) were more likely to be associated with saprotrophic fungi, plant pathogenic fungi, and AM fungi, but plants with lower root nitrogen ("slow" strategy) were more likely to be associated with ectomycorrhizal fungi; (ii) bacterial functional guilds were associated with root-zone pH but not root traits.

Strengths:<br /> This study is novel in the sense that it revealed the associations between microbiome and trait dimensions of plants. This has been rarely explored even though we acknowledge the importance of plant-microbe interactions.

Weaknesses:<br /> The authors tried to include the relative abundances of bacterial and fungal guilds into the root economics framework, which I disagree with because they are just associated with the root economics framework. The title also states that the authors' aim is to link microbial functional guilds to root economics. Therefore, I would suggest that the analyses should be redone to elaborate on the relationships between microbiome and root functional traits.

Below I provide some critiques and comments that outline my concerns and provide recommendations to hopefully improve the current manuscript.

-Figures 2 and 3: The authors included soil properties, relative abundances of bacterial or fungal guilds, and root traits in the root economics spectrum. However, soil properties and relative abundances of bacterial or fungal guilds are not root traits, they are just associated with root traits. These bacterial or fungal guilds are the consequence of root traits. Also, the authors did not elaborate on the root trait dimensions of the plants. The only trait dimension they discussed is the "fast-slow" axis. Therefore, I would suggest the authors first analyze the trait dimensions of plants by only using the root traits (PCA), and then explore how the soil properties and relative abundances of bacterial or fungal guilds are associated with the trait dimensions (e.g., envfit in the vegan package).

-When exploring the associations between microbial functional guilds and root traits, it is unnecessary to analyze the bacterial and fungal functional guilds separately. The bacterial and fungal functional guilds can be included in the same models, and their relative importance and patterns can be compared.

-For fungi, the authors used FUNGuild to infer functional guilds from taxonomy. qPCR was also performed to validate the results of AMF. This is fantastic. For bacteria, the authors used FAPROTAX to infer functional guilds from taxonomy. However, archaea are also considered in some functions in FAPROTAX. For example, both bacteria (ammonia-oxidizing bacteria) and archaea (ammonia-oxidizing archaea) play critical roles in nitrification. I would assume the authors have removed archaea from the dataset because they stated that the functions of bacteria are inferred from FAPROTAX. Therefore, the importance of nitrification might be underestimated.

-Key methodological details are missing. First, maps of the sampling site and plots are missing. It would be great if the authors provided maps showing the location of the sampling site and the spatial distribution of the 11 plots. Second, in lines 304-306 the authors claimed that they sampled the most common species in the plots, but they did not provide the coverage or relative abundances of plant species in the plots.

Reviewer #2 (Public Review):

Summary:<br /> The authors aimed to determine to what extent root morphology, chemistry, and soil characteristics explained the relative abundance of functional groups of bacteria and fungi associated with roots. To do so, they sample roots and rhizhospheric soil of trees along an elevation gradient. This type of work is common in the field of microbial ecology. The main novelties I see are two: a) a focus on the functional groups of bacteria and fungi rather than just taxonomic abundance. I think this approach is valuable because it provides information about the potential functions of these microorganisms; b) using the root economic spectrum to frame the findings. The root economic spectrum reflects a gradient along which plant roots can be allocated from 'short-lived that provide fast investment return' to 'long-lived that provide a slow investment return'. It is logical to expect (as the authors did) that variation along this gradient will be an important factor in explaining the variation in functional groups.

Strengths:<br /> The main strength is using the root economic spectrum as a framework to interpret the data. There are countless studies addressing variation in the relative abundance of microbial communities along environmental gradients which tend to be more descriptive. I think using this framework advances the field by suggesting that while the root economic spectrum exists it is not a very important explanatory variable to predict changes in functional diversity. I also think the authors use state-of-the art methods to collect and process the sample (i.e. to obtain the data).

Weaknesses:<br /> The main weakness is with the presentation of statistical methods as it currently stands. The authors use distance-based redundancy analysis as the main statistical method. However, my understanding is that this method is not advised for a relative abundance of communities. At least not with Euclidean distances which is the default option of the functions dbrda in vegan. The use of this distance would group together communities with no species in common as close to each other (which is an incorrect interpretation). I think the authors should specify what distance they use. My guess is that they actually used bray-curtis in which case this weakness does not apply. However, as it stands it is not specified what metric they use and if they indeed use Euclidean distances it may lead to inaccurate conclusions. In addition, they also mention they use PCA on the relative abundance of functional groups. By definition, PCA is also based on Euclidean distances, which gives a similar problem as dbrda. Thus, I encourage the authors to use bray-curtis distance and specify it in the text.

Reviewer #3 (Public Review):

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #4 (Public Review):

Summary:<br /> Recent progress in root economics has revealed global-scale axes of covaried root traits that reflect various root resource acquisition strategies. These covariance patterns are powerful tools for understanding root functional diversity. However, roots do not function in isolation for below-ground resource acquisition. Rather, symbiotic fungi and rhizosphere microorganisms often collaborate with plant roots, forming a root-microbial-soil continuum. This study seeks to provide novel insights into this continuum by extending the existing framework of root economics to include the structures of root-associated microorganisms. I find this topic highly relevant. Considering the role of soil microorganisms is undoubtedly crucial for a more comprehensive understanding of below-ground resource strategies.

Major comments:<br /> A key finding of this study is a relationship between root N and the tendency for roots to associate with particular types of mycorrhizal associations (Line 27, Fig. 2). The authors concluded that this indicates "a linkage from simple root traits to fungal-mediated carbon nutrient cycling" (line 27) and integrates "microbial functions into the root economics framework," (line 32). If substantiated, this correlation could represent a significant discovery about the connection between root functional traits and root-associated fungi. It suggests that low root N, indicative of low metabolic activity within the root economics framework, is linked with forming EcM associations. However, I am not fully convinced this is the case based on the current data presentation and interpretation.

First, there is no biological interpretation of this relationship between root N and mycorrhizal type. It merely noted that root N is indicative of root metabolic activity, and thus by relating root N to fungal composition, "the trait-related root economics and fungal-driven nutrient economics may be integrated into a unified framework" (lines 221-224). Why would roots with low N and low metabolic activity tend to favor EcM associations? What are the potential mechanisms? Biological interpretation is essential for understanding whether a statistical correlation reflects a causal and meaningful relationship or is coincidental.

I am also concerned that this relationship may be spurious, especially when it lacks biological interpretation. EcM is underrepresented in this study (8 EcM species, of which more than half are conifers and oaks vs. 44 AM) and seems to cluster at higher elevations (line 231). Thus, the tree species/individual data points are not independent, but phylogenetically and geographically clustered. The unique properties at higher elevations (e.g., distinct plant community structures, low levels of mineral N) may drive both the lower root N and the prevalence of EcM associations. This scenario aligns with the observation that at higher elevations, AM roots also exhibited low root N (Line 231). In this case, root N may not directly relate to mycorrhizal type but is characteristic of certain locations (or closely related species), and it would be misleading to suggest that low root N/metabolic activity, a proxy in fast-slow root economics, is directly linked to the preference for a particular mycorrhizal type (lines 27-28, 220 - 224). In summary, because the studied tree species appear to be clustered both phylogenetically and geographically, these factors need to be carefully taken into account in the statistical analysis and data interpretation to understand the underlying causes of the apparent relationship and prevent overinterpretation. I also recommend, if possible, providing a visual presentation of the geographical and phylogenetic distribution of the studied tree species.

That being said, this dataset is undoubtedly valuable in revealing the shifts in the compositional structures of root-associated soil microorganisms. However, integrating the traits of microbial composition to root trait economics would require more caution and careful examination of the potential driving causes.

Reviewer #1 (Public Review):

This study used a multi-day learning paradigm combined with fMRI to reveal neural changes reflecting the learning of new (arbitrary) shape-sound associations. In the scanner, the shapes and sounds are presented separately and together, both before and after learning. When they are presented together, they can be either consistent or inconsistent with the learned associations. The analyses focus on auditory and visual cortices, as well as the object-selective cortex (LOC) and anterior temporal lobe regions (temporal pole (TP) and perirhinal cortex (PRC)). Results revealed several learning-induced changes, particularly in the anterior temporal lobe regions. First, the LOC and PRC showed a reduced bias to shapes vs sounds (presented separately) after learning. Second, the TP responded more strongly to incongruent than congruent shape-sound pairs after learning. Third, the similarity of TP activity patterns to sounds and shapes (presented separately) was increased for non-matching shape-sound comparisons after learning. Fourth, when comparing the pattern similarity of individual features to combined shape-sound stimuli, the PRC showed a reduced bias towards visual features after learning. Finally, comparing patterns to combined shape-sound stimuli before and after learning revealed a reduced (and negative) similarity for incongruent combinations in PRC. These results are all interpreted as evidence for an explicit integrative code of newly learned multimodal objects, in which the whole is different from the sum of the parts.

The study has many strengths. It addresses a fundamental question that is of broad interest, the learning paradigm is well-designed and controlled, and the stimuli are real 3D stimuli that participants interact with. The manuscript is well written and the figures are very informative, clearly illustrating the analyses performed.

There are also some weaknesses. The sample size (N=17) is small for detecting the subtle effects of learning. Most of the statistical analyses are not corrected for multiple comparisons (ROIs), and the specificity of the key results to specific regions is also not tested. Furthermore, the evidence for an integrative representation is rather indirect, and alternative interpretations for these results are not considered.

Reviewer #2 (Public Review):

Li et al. used a four-day fMRI design to investigate how unimodal feature information is combined, integrated, or abstracted to form a multimodal object representation. The experimental question is of great interest and understanding how the human brain combines featural information to form complex representations is relevant for a wide range of researchers in neuroscience, cognitive science, and AI. While most fMRI research on object representations is limited to visual information, the authors examined how visual and auditory information is integrated to form a multimodal object representation. The experimental design is elegant and clever. Three visual shapes and three auditory sounds were used as the unimodal features; the visual shapes were used to create 3D-printed objects. On Day 1, the participants interacted with the 3D objects to learn the visual features, but the objects were not paired with the auditory features, which were played separately. On Day 2, participants were scanned with fMRI while they were exposed to the unimodal visual and auditory features as well as pairs of visual-auditory cues. On Day 3, participants again interacted with the 3D objects but now each was paired with one of the three sounds that played from an internal speaker. On Day 4, participants completed the same fMRI scanning runs they completed on Day 2, except now some visual-auditory feature pairs corresponded with Congruent (learned) objects, and some with Incongruent (unlearned) objects. Using the same fMRI design on Days 2 and 4 enables a well-controlled comparison between feature- and object-evoked neural representations before and after learning. The notable results corresponded to findings in the perirhinal cortex and temporal pole. The authors report (1) that a visual bias on Day 2 for unimodal features in the perirhinal cortex was attenuated after learning on Day 4, (2) a decreased univariate response to congruent vs. incongruent visual-auditory objects in the temporal pole on Day 4, (3) decreased pattern similarity between congruent vs. incongruent pairs of visual and auditory unimodal features in the temporal pole on Day 4, (4) in the perirhinal cortex, visual unimodal features on Day 2 do not correlate with their respective visual-auditory objects on Day 4, and (5) in the perirhinal cortex, multimodal object representations across Days 2 and 4 are uncorrelated for congruent objects and anticorrelated for incongruent. The authors claim that each of these results supports the theory that multimodal objects are represented in an "explicit integrative" code separate from feature representations. While these data are valuable and the results are interesting, the authors' claims are not well supported by their findings.

(1) In the introduction, the authors contrast two theories: (a) multimodal objects are represented in the co-activation of unimodal features, and (b) multimodal objects are represented in an explicit integrative code such that the whole is different than the sum of its parts. However, the distinction between these two theories is not straightforward. An explanation of what is precisely meant by "explicit" and "integrative" would clarify the authors' theoretical stance. Perhaps we can assume that an "explicit" representation is a new representation that is created to represent a multimodal object. What is meant by "integrative" is more ambiguous-unimodal features could be integrated within a representation in a manner that preserves the decodability of the unimodal features, or alternatively the multimodal representation could be completely abstracted away from the constituent features such that the features are no longer decodable. Even if the object representation is "explicit" and distinct from the unimodal feature representations, it can in theory still contain featural information, though perhaps warped or transformed. The authors do not clearly commit to a degree of featural abstraction in their theory of "explicit integrative" multimodal object representations which makes it difficult to assess the validity of their claims.

(2) After participants learned the multimodal objects, the authors report a decreased univariate response to congruent visual-auditory objects relative to incongruent objects in the temporal pole. This is claimed to support the existence of an explicit, integrative code for multimodal objects. Given the number of alternative explanations for this finding, this claim seems unwarranted. A simpler interpretation of these results is that the temporal pole is responding to the novelty of the incongruent visual-auditory objects. If there is in fact an explicit, integrative multimodal object representation in the temporal pole, it is unclear why this would manifest in a decreased univariate response.

(3) The authors ran a neural pattern similarity analysis on the unimodal features before and after multimodal object learning. They found that the similarity between visual and auditory features that composed congruent objects decreased in the temporal pole after multimodal object learning. This was interpreted to reflect an explicit integrative code for multimodal objects, though it is not clear why. First, behavioral data show that participants reported increased similarity between the visual and auditory unimodal features within congruent objects after learning, the opposite of what was found in the temporal pole. Second, it is unclear why an analysis of the unimodal features would be interpreted to reflect the nature of the multimodal object representations. Since the same features corresponded with both congruent and incongruent objects, the nature of the feature representations cannot be interpreted to reflect the nature of the object representations per se. Third, using unimodal feature representations to make claims about object representations seems to contradict the theoretical claim that explicit, integrative object representations are distinct from unimodal features. If the learned multimodal object representation exists separately from the unimodal feature representations, there is no reason why the unimodal features themselves would be influenced by the formation of the object representation. Instead, these results seem to more strongly support the theory that multimodal object learning results in a transformation or warping of feature space.

(4) The most compelling evidence the authors provide for their theoretical claims is the finding that, in the perirhinal cortex, the unimodal feature representations on Day 2 do not correlate with the multimodal objects they comprise on Day 4. This suggests that the learned multimodal object representations are not combinations of their unimodal features. If unimodal features are not decodable within the congruent object representations, this would support the authors' explicit integrative hypothesis. However, the analyses provided do not go all the way in convincing the reader of this claim. First, the analyses reported do not differentiate between congruent and incongruent objects. If this result in the perirhinal cortex reflects the formation of new multimodal object representations, it should only be true for congruent objects but not incongruent objects. Since the analyses combine congruent and incongruent objects it is not possible to know whether this was the case. Second, just because feature representations on Day 2 do not correlate with multimodal object patterns on Day 4 does not mean that the object representations on Day 4 do not contain featural information. This could be directly tested by correlating feature representations on Day 4 with congruent vs. incongruent object representations on Day 4. It could be that representations in the perirhinal cortex are not stable over time and all representations-including unimodal feature representations-shift between sessions, which could explain these results yet not entail the existence of abstracted object representations.

In sum, the authors have collected a fantastic dataset that has the potential to answer questions about the formation of multimodal object representations in the brain. A more precise delineation of different theoretical accounts and additional analyses are needed to provide convincing support for the theory that "explicit integrative" multimodal object representations are formed during learning.

Reviewer #3 (Public Review):

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public Review):

The hypothesis that the MA myristyl switch is a trigger for M-PMV maturation is derived from previously published findings, and is well supported by the data presented in this manuscript. The results suggest a new function for the myristyl switch, one that could perhaps be relevant for other proteins. Below are some suggestions for improving the MS.

Reviewer #2 (Public Review):

This manuscript presents measurements of proteolytic digestion and structural studies using both hydrogen-deuterium exchange and NMR. The data test the idea that membrane association leads to a rearrangement of the MA domain of the MPMV Gag protein, as the myristate chain at the N-terminus of the protein is "switched" from a hydrophobic pocket within the protein into lipid layers, finally rendering the protein efficiently digestible by the viral protease. In my opinion, the data are highly convincing, and the underlying hypothesis is a useful contribution to the field, providing for this retrovirus a solution to the long-standing problem of how proteolytic maturation is activated.

Reviewer #3 (Public Review):

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

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

Reviewer #1 (Public Review):

This study demonstrates that Langerhans ADAM17 is lower in nonlesional skin and type I interferons have effects on ADAM17. ADAM17 releases EGFR ligands that preserve epidermal integrity. LC ROS is lower with high type I interferons, accompanied by reduced epidermal EGFR phosphorylation in nonlesional skin in SLE. The authors did an outstanding job with data from 3 animal models and human lupus skin to demonstrate their findings.

Reviewer #2 (Public Review):

Many of the questions about type I interferon and photosensitivity have already been studied in murine lupus models but most importantly in skin biopsies from both lesional and non-lesional cutaneous lupus. The authors should try to link their data to the existing literature and validate their results by using human samples, as not all murine lupus models have a strong interferon-mediated disease. Other important aspects of the work include whether or not the authors have considered knocking out the mice for ADAM17 and reassessing the function of the Langerhans cells? Last but not least, some of the data presented will need to be validated by more in vitro work that will shed more light on the functional properties of ADAM17 in Langerhans cells and inflammatory response in cutaneous lupus.

Reviewer #3 (Public Review):

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

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

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

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

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

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

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

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

Reviewer #1 (Public Review):

The work by Porciello and colleagues provides scientific evidence that the acidic content of the stomach covaries with the experienced level of disgust and fear evoked by disgusting videos. The working of the inside of the gut during cognitive or emotional processes have remained elusive due to the invasiveness of the methods to study it. The major strength of the paper is the use of the non-invasive smart pill technology, which senses changes in Ph, pressure and temperature as it travels through the gut, allowing authors to investigate how different emotions induced with validated video clips modulate the state of the gut. The experimental paradigm used to evoke distinct emotions was also successful, as participants reported the expected emotions after each emotion block. While the reported evidence is correlational in nature, I believe these results open up new avenues for studying brain-body interactions during emotions in cognitive neuroscience, and future causal manipulations will shed more insight on this phenomena. Indeed, this is the first study to provide evidence for a link between gastric acidity and emotional experience beyond single patient studies, and it has major implications for the advancement of our understanding of disorders with psycho-somatic influences, such as stress and it's influence of gastritis.

As for the limitations, little insight is provided on the mechanisms, time scales, and inter-individual variability of the link between gastric Ph and emotional induction. Since this is a novel phenomena, it would be important to further validate and characterize this finding. On this line, one of the most well known influences of disgust on the gut is tachygastria, the acceleration of the gastric rhythm. It would be important to understand how acid secretion by disgusting film is related to tachygastria, but authors only examine the influence of disgusting film on the normogastric frequency range. Additionally, only one channel of the electrogastrogram (EGG) was used to measure the gastric rhythm, and no information is provided on the quality of the recordings. With only one channel of EGG, it is often impossible to identify the gastric rhythm as the position of the stomach varies from person to person, yielding inaccurate estimates of the frequency of the gastric rhythm. Finally, I believe that the results do not show evidence in favor of the discrete nature of emotions theory as they claim in the discussion. Authors chose to use stimuli inducing discrete emotions, and only asked subjective reports of these same discrete emotions, so these results shed no light on whether emotions are represented discretely vs continuously in the brain.

Reviewer #2 (Public Review):

To measure the role of gastric state in emotion, the authors used an ingestible smart pill to measure pH, pressure, and temperature in the gastrointestinal tract (stomach, small bowel, large bowel) while participants watched videos that induced disgust, fear, happiness, sadness, or a control (neutral). The study has a number of strengths, including the novelty of the measurement (very few studies have ever measured these gut properties during emotion processing) and the apparent robustness of their main finding (that during disgusting video clips, participants who experienced more feelings of disgust (and to a lesser degree which might not survive more stringent multiple comparison correction, fear) had more acidic stomach measurements, while participants who experienced more happiness during the disgusting video clips had a less acidic (more basic) stomach pH. Although the study is correlational (which all discussion should carefully reflect) and is restricted to a moderately-sized, homogenous sample, the results support their general conclusion that stomach pH is related to emotion experience during disgust induction. There may be additional analyses to conduct in order for the authors to claim this effect is specific to the stomach. Nevertheless, this work is likely to have a large impact on the field, which currently tends to rely on noninvasive measures of gastric activity such as electrogastrography (which the authors also collect for comparison); the authors' minimally-invasive approach yields new and useful measurements of gastric state. These new measures could have relevance beyond emotion processing in understanding the role of gut pH (and perhaps temperature and pressure) in cognitive processes (e.g. interoception) as well as mental and physical health.

Reviewer #3 (Public Review):

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

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

Reviewer #1 (Public Review):

This manuscript describes the results of closed-loop SWR disruption in rats experiencing a short-term memory task that they previously acquired successfully. The authors aim to show that SWRs are dispensable for STM tasks requiring multiple match-to-sample trial rules, single-trial non-match-to-sample rules, and spatial sequence memory. In all cases, the analysis and intervention were performed at the higher standards, providing a clear proof-of-principle of appropriate detection and the necessary control. I found the experiments well executed and analyzed. Results may help to advance our understanding of the role of awake SWRs in STM. However, since the results consist of a lack of evidence there is a need for some additional positive controls to fully support the main claim of the study.

Reviewer #2 (Public Review):

This study aims to test the role of awake replay in short-term memory, a type of memory that operates on the timescale of seconds and minutes. Replay refers to a time-compressed burst of neuronal population activity during a particular oscillatory local field potential event in the hippocampus, called the sharp-wave ripple (SWR). SWRs are found during sleep and in the awake state and are always associated with the animal being quiescent. The paper compares results from three different behavioral tasks ranging in memory requirements and memory timescales. First, rats were trained on either a spatial match-to-sample task (MTS), a non-match-to-sample task (NMTS), or a task requiring the memorization of sequences (maze arms to be visited in a specific temporal order). In this initial training phase, the animals were allowed to learn the maze structure and the rules governing these tasks for all these behavioral paradigms. Then, awake sharp-SWRs were disrupted as the animal performed these tasks (both during instruction and test phases) via an online detection system combined with closed-loop electrical stimulation of the ventral hippocampal commissure. Notably, this manipulation appeared not to affect performance in all three tasks, as determined using various behavioral parameters. Trials with no stimulation or delayed stimulation serve as controls. Thus, the authors conclude that awake SWRs are not involved in these short-term memory-guided behaviors. I do have a few comments that the authors should discuss or address:

(1) This study adds to a large number of studies investigating the role of awake SWRs in spatial learning and memory tasks. The results of these previous studies are quite contradictory and range from awake SWRs are not crucial in guiding decisions at all to SWRs are only essential during task rule learning to SWRs do guide behavior. Could the authors comment on these seemingly contradictory results? Why are these experiments now the right ones?<br /> (2) None of the experiments presented here test the role of replay. I suggest making this distinction in the paper and the title clear. As the results are presented now, is it possible that the SWR content is not affected sufficiently to have a behavioral effect or that there is a bias towards detecting specific SWRs, e.g., longer SWRs?

Reviewer #3 (Public Review):

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

Major comments:

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public Review):

Summary: Zai et al test if songbirds can recover the capacity to sing auditory targets without singing experience or sensory feedback. Past work showed that after the pitch of targeted song syllables are driven outside of birds' preferred target range with external reinforcement, birds revert to baseline (i.e. restore their song to their target). Here the authors tested the extent to which this restoration occurs in muted or deafened birds. If these birds can restore, this would suggest an internal model that allows for sensory-to-motor mapping. If they cannot, this would suggest that learning relies entirely on feedback dependent mechanisms, e.g. reinforcement learning (RL). The authors find that deafened birds exhibit moderate but significant restoration, consistent with the existence of a previously under-appreciated internal model in songbirds.

Strengths:

The experimental approach of studying vocal plasticity in deafened or muted birds is innovative, technically difficult and perfectly suited for the question of feedback-independent learning. The finding in Figure 4 that deafened birds exhibit subtle but significant plasticity toward restoration of their pre-deafening target is surprising and important for the songbird and vocal learning fields, in general.

In this revision, the authors suitably addressed the confusion about some statistical methods related to Fig. 4, where the main finding of vocal plasticity in deafened birds was presented.

There remain minor issues in the presentation early in the results section and in Fig. 4 that should be straightforward to clarify in revision.

Reviewer #3 (Public Review):

Summary:

Zai et al. test whether birds can modify their vocal behavior in a manner consistent with planning. They point out that while some animals are known to be capable of volitional control of vocalizations, it has been unclear if animals are capable of planning vocalizations-that is, modifying vocalizations towards a desired target without the need to learn this modification by practicing and comparing sensory feedback of practiced behavior to the behavioral target. They study zebra finches that have been trained to shift the pitch of song syllables away from their baseline values. It is known that once this training ends, zebra finches have a drive to modify pitch so that it is restored back to its baseline value. They take advantage of this drive to ask whether birds can implement this targeted pitch modification in a manner that looks like planning, by comparing the time course and magnitude of pitch modification in separate groups of birds who have undergone different manipulations of sensory and motor capabilities. A key finding is that birds who are deafened immediately before the onset of this pitch restoration paradigm, but after they have been shifted away from baseline, are able to shift pitch partially back towards their baseline target. In other words, this targeted pitch shift occurs even when birds don't have access to auditory feedback, which argues that this shift is not due to reinforcement-learning-guided practice, but is instead planned based on the difference between an internal representation of the target (baseline pitch) and current behavior (pitch the bird was singing immediately before deafening).

The authors present additional behavioral studies arguing that this pitch shift requires auditory experience of song in its state after it has been shifted away from baseline (birds deafened early on, before the initial pitch shift away from baseline, do not exhibit any shift back towards baseline), and that a full shift back to baseline requires auditory feedback. The authors synthesize these results to argue that different mechanisms operate for small shifts (planning, which does not need auditory feedback) and large shifts (through a mechanism that requires auditory feedback).

The authors also make a distinction between two kinds of planning: covert-not requiring any motor practice-and overt-requiring motor practice, but without access to auditory experience from which target mismatch could be computed. They argue that birds plan overtly, based on these deafening experiments as well as an analogous experiment involving temporary muting, which suggest that indeed motor practice is required for pitch shifts.

Strengths:

The primary finding (that partially restorative pitch shift occurs even after deafening) rests on strong behavioral evidence. It is less clear to what extent this shift requires practice, since their analysis of pitch after deafening takes the average over within the first two hours of singing. If this shift is already evident in the first few renditions then this would be evidence for covert planning. Technical hurdles, such as limited sample sizes and unstable song after surgical deafening, make this difficult to test. (Similarly, the authors could test whether the first few renditions after recovery from muting already exhibit a shift back towards baseline.)

This work will be a valuable addition to others studying birdsong learning and its neural mechanisms. It documents features of birdsong plasticity that are unexpected in standard models of birdsong learning based on reinforcement and are consistent with an additional, perhaps more cognitive, mechanism involving planning. As the authors point out, perhaps this framework offers a reinterpretation of the neural mechanisms underlying a prior finding of covert pitch learning in songbirds (Charlesworth et al., 2012).

A strength of this work is the variety and detail in its behavioral studies, combined with sensory and motor manipulations, which on their own form a rich set of observations that are useful behavioral constraints on future studies.

Weaknesses:

The argument that pitch modification in deafened birds requires some experience hearing their song in its shifted state prior to deafening (Fig. 4) is solid, but has an important caveat. Their argument rests on comparing two experimental conditions: one with and one without auditory experience of shifted pitch. However, these conditions also differ in the pitch training paradigm: the "with experience" condition was performed using white noise training, while the "without experience" condition used "lights off" training (Fig. 4A). It is possible that the differences in ability for these two groups to restore pitch to baseline reflects the training paradigm, not whether subjects had auditory experience of the pitch shift. Ideally, a control study would use one of the training paradigms for both conditions, which would be "lights off" or electrical stimulation (McGregor et al. 2022), since WN training cannot be performed in deafened birds. In the Discussion, in response to this point the authors point out that birds are known to recover their pitch shift if those shifts are driven using electrical stimulation as reinforcement (McGregor et al. 2022); however, it is arguably still relevant to know whether a similar recovery occurs for the "lights off" paradigm used here.

Reviewer #1 (Public Review):

Summary:<br /> By examining the prevalence of interactions with ancient amino acids of coenzymes in ancient versus recent folds, the authors noticed an increased interaction propensity for ancient interactions. They infer from this that coenzymes might have played an important role in prebiotic proteins.

Strengths:<br /> (1) The analysis, which is very straightforward, is technically correct. However, the conclusions might not be as strong as presented.

(2) This paper presents an excellent summary of contemporary thought on what might have constituted prebiotic proteins and their properties.

(3) The paper is clearly written.

Weaknesses:<br /> (1) The conclusions might not be as strong as presented. First of all, while ancient amino acids interact less frequently in late with a given coenzyme, maybe this just reflects the fact that proteins that evolved later might be using residues that have a more favorable binding free energy.

(2) What about other small molecules that existed in the probiotic soup? Do they also prefer such ancient amino acids? If so, this might reflect the interaction propensity of specific amino acids rather than the inferred important role of coenzymes.

(3) Perhaps the conclusions just reflect the types of active sites that evolved first and nothing more.

Reviewer #2 (Public Review):

I enjoyed reading this paper and appreciate the careful analysis performed by the investigators examining whether 'ancient' cofactors are preferentially bound by the first-available amino acids, and whether later 'LUCA' cofactors are bound by the late-arriving amino acids. I've always found this question fascinating as there is a contradiction in inorganic metal-protein complexes (not what is focused on here). Metal coordination of Fe, Ni heavily relies on softer ligands like His and Cys - which are by most models latecomer amino acids. There are no traces of thiols or imidazoles in meteorites - although work by Dvorkin has indicated that could very well be due to acid degradation during extraction. Chris Dupont (PNAS 2005) showed that metal speciation in the early earth (such as proposed by Anbar and prior RJP Williams) matched the purported order of fold emergence.

As such, cofactor-protein interactions as a driving force for evolution has always made sense to me and I admittedly read this paper biased in its favor. But to make sure, I started to play around with the data that the authors kindly and importantly shared in the supplementary files. Here's what I found:

Point 1: The correlation between abundance of amino acids and protein age is dominated by glycine.

There is a small, but visible difference in old vs new amino acid fractional abundance between Ancient and LUCA proteins (Figure 3, Supplementary Table 3). However, the bias is not evenly distributed among the amino acids - which Figure 4A shows but is hard to digest as presented. So instead I used the spreadsheet in Supplement 3 to calculate the fractional difference FDaa = F(old aa)-F(new aa). As expected from Figure 3, the mean FD for Ancient is greater than the mean FD for LUCA. But when you look at the same table for each amino acid FDcofactor = F(ancient cofactor) - F(LUCA cofactor), you now see that the bias is not evenly distributed between older and newer amino acids at all. In fact, most of the difference can be explained by glycine (FDcofactor = 3.8) and the rest by also including tryptophan (FDcofactor = -3.8). If you remove these two amino acids from the analysis, the trend seen in Figure 3 all but disappears.

Troubling - so you might argue that Gly is the oldest of the old and Trp is the newest of the new so the argument still stands. Unfortunately, Gly is a lot of things - flexible, small, polar - so what is the real correlation, age, or chemistry? This leads to point 2.

Point 2 - The correlation is dominated by phosphate.

In the ancient cofactor list, all but 4 comprise at least one phosphate (SAM, tetrahydrofolic acid, biopterin, and heme). Except for SAM, the rest have very low Gly abundance. The overall high Gly abundance in the ancient enzymes is due to the chemical property of glycine that can occupy the right-hand side of the Ramachandran plot. This allows it to make the alternating alphaleft-alpharight conformation of the P-loop forming Milner-White's anionic nest. If you remove phosphate binding folds from the analysis the trend in Figure 3 vanishes.

Likewise, Trp is an important functional residue for binding quinones and tuning its redox potential. The LUCA cofactor set is dominated by quinone and derivatives, which likely drives up the new amino acid score for this class of cofactors.

In summary, while I still believe the premise that cofactors drove the shape of peptides and the folds that came from them - and that Rossmann folds are ancient phosphate-binding proteins, this analysis does not really bring anything new to these ideas that have already been stated by Tawfik/Longo, Milner-White/Russell, and many others.

I did this analysis ad hoc on a slice of the data the authors provided and could easily have missed something and I encourage the authors to check my work. If it holds up it should be noted that negative results can often be as informative as strong positive ones. I think the signal here is too weak to see in the noise using the current approach.

Reviewer #1 (Public Review):

In this study, Hunt et al investigated the role of the ubiquitin-conjugating enzyme UBE2D/effete (eff) in maintaining proteostasis during aging. Utilizing Drosophila as a model, the researchers observed diverse roles of E2 ubiquitin-conjugating enzymes in handling the aggregation-prone protein huntingtin-polyQ in the retina. While some E2s facilitated aggregate assembly, UBE2D/eff and other E2s were crucial for degradation of htt-polyQ. The study also highlights the significance of UBE2D/eff in skeletal muscle, showing that declining levels of eff during aging correlate with proteostasis disruptions. Knockdown of eff in muscle led to accelerated accumulation of poly-ubiquitinated proteins, shortened lifespan, and mirrored proteomic changes observed in aged muscles. The introduction of human UBE2D2, analogous to eff, partially rescued the deficits in lifespan and proteostasis caused by eff-RNAi expression in muscles.

The conclusions of this paper are mostly well supported by data, although a more precise mechanistic explanation of phenotypes associated with UBE2D/eff deficiency would have strengthened the study. Additionally, some aspects of image quantification and data analysis need to be clarified and/or extended.

Reviewer #2 (Public Review):

Important findings:

• Knockdown of UBE2D increases HTT aggregation.

• Knockdown of UBE2D leads to an accumulation of ubiquitinated proteins and reduces the lifespan of Drosophila, which is rescued by an ectopic expression of the human homolog.

• UBE2D protein levels decline with aging.

• UBE2D knockdown is associated with an up- and downregulation of several different cellular pathways, including proteostasis components.

Caveats:

• The readout of HTT aggregation (with methods that are not suitable) as a proxy for the role of UBE2D in proteostasis is not convincing. It would probably improve the manuscript to start with the proteomic analysis of UBE2D to demonstrate that its protein levels decrease with aging. The authors could then induce UBE2D in aged animals to assess the role of UBE2D in the proteome with aging.

• UBE2D knockdown increases the number of HTT foci (Figure 1A), but the quantification is less convincing as depicted in Figure 1B, and other E2 enzymes show a stronger effect (e.g. Ubc6 that is only studied in Figures 1 and 2 without an explanation and Ubc84D). The graph is hard to interpret. What is the sample size and which genetic conditions show a significant change? P values and statistical analyses are missing.

• The quantification of the HTT fluorescence cannot be used as a proxy for HTT aggregation. The authors should assess HTT aggregation by e.g. SDD-AGE, FRAP, filter retardation, etc. The quantification of the higher MW species of HTT in the SDS-PAGE is not ideal either as this simply reflects material that is stuck in the wells that could not enter the gel. Aggregation and hence high MW size could be one reason, but it can also be HTT trapped in cell debris, etc.

• Does UBE2D ubiquitinate HTT? And thus, is HTT accumulation a suitable readout for the functional assessment of the E2 enzyme UBE2D?

• The proteomic analyses could help to identify potential substrates for UBE2D.

• Are there mutants available for UBE2D or conditional mutants? One caveat of RNAi is: first not complete knockdown and second, variable knockdown efficiencies that increase variability.

• The analysis of the E3 enzymes does not add anything to this manuscript.

• Figure 2B: the fluorescence intensities in images 2 and 4 are rather similar, yet the quantification shows significant differences.

• The proteomic analyses could provide insights into the functional spectrum of UBE2D or even the identification of substrates. Yet apart from a DAVID analysis, none of the hits were followed up. In addition, only a few hits were labelled in the volcano plots (Figure 5). On what basis did the authors select those?

• The manuscript remains at this stage rather descriptive.

Reviewer #3 (Public Review):

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

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

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

Reviewer #1 (Public Review):

This study explores the relationship between neurodegeneration's most common spatial patterns and the density of different cell types in the cerebral cortex. The authors present data showing that atrophy patterns in Alzheimer's disease and Frontotemporal dementia (FTD) strongly associated with the abundance of astrocytes and microglia. This work (the original manuscript and the revision) takes a step in the right direction by emphasizing the critical role that cells other than neurons play in the degeneration patterns observable with neuroimaging.

Comments on revised version:

I appreciate the revisions the authors made to address my main comments:<br /> - adding whole-brain maps showing cellular abundance and atrophy<br /> - stratifying the FTD group into the three clinically defined categories bvFTD (behavior-variant), nfvPPA (nonfluent/agrammatic-variant primary progressive aphasia), and svPPA (semantic-variant primary progressive aphasia).

I reiterate my agreement with the authors that this work demonstrates the need to "surpass the current neuro-centric view of brain diseases and the imperative for identifying cell-specific therapeutic targets in neurodegeneration".

Reviewer #3 (Public Review):

This study is a fine example of a recent productive trend in the integration of neuroimaging and molecular biology of the brain: in brief, overlaying some neuroimaging data (usually from a large cohort) onto the high spatial resolution gene expression in the Allen Human Brain Atlas data, derived from 6 individuals. By projecting structural MRI images over cell type proportions identified in the Allen data, the authors can represent various diseases in terms of their spatially-associated cell types. The result has implications for prioritizing the contributions of various cell types to each disease and creates an even-handed cell type profile through which the 11 diseases can be compared.

Reviewer #1 (Public Review):

Summary:

In this paper, Ruggiero, Leite and colleagues assess the effects of early life seizures on a large number of anatomical, physiological, behavioral and neurochemical measures. They find that prolonged early life seizures do not lead to obvious cell loss, but lead to astrogliosis, working memory deficits on the radial arm maze, increased startle response, decreased paired pulse inhibition, and increased hippocampal-PFC LTP. There was a U-shaped relationship between LTP and cognitive deficits. There is increased theta power during the awake state in ELS animals but reduced PFC theta-gamma coupling and reduced theta HPC-PFC coherence. Theta coherence seems to be similar in ACT and REM states in ELS animals while in decreases in active relative REM in controls.

Strengths:

The main strength of the paper is the number of convergent techniques used to understand how hippocampal PFC neural dynamics and behavior change after early life seizures. The sheer scale, breadth and reach of the experiments are praiseworthy. It is clear that the paper is a major contribution to the field as far as understanding the impact of early life seizures. The LTP findings are robust and provide an important avenue for future study. The experiments are performed carefully and the analysis is appropriate. The paper is well-written and the figures are clear.

Weaknesses:

The main weakness of the paper remains the lack of causal manipulations to determine whether prevention or augmentation of any of the findings have any impact on behavior or cognition. Alternatively, if other manipulations would enhance working memory in ELS animals, it would have been interesting to see the effects on any of these parameters measured in the paper. The authors now discuss the lack of causal manipulations in the discussion but have not performed new experiments to address this weakness. Also, I find the sections where correlations and dimensionality reduction techniques are used to compare all possible variables to each other less compelling than the rest of the paper (with the exception of the findings of U shaped relationship of cognition to LTP). In fact, I think these sections take away from the impact of the actual findings. The rationale for the apomorphine experiments are now explained more fully.

Reviewer #3 (Public Review):

Summary:

The manuscript examines an important question, namely how the brain associates events spaced in time. It uses a variety of neural methods including fiber photometry as well as area-specific and pathway-silencing methods with the exquisite dissociation of norepinephrine and dopamine. The data show that neurons in the locus coeruleus (LC) respond to auditory cue onset, offset, and shock. These responses are stronger if the cue is paired with shock in a trace procedure. Optogenetic stimulation similar to the neural response captured by fiber photometry enhances associative learning. LC terminals in the dorsal hippocampus also showed phasic responses during fear conditioning and drove dopamine and norepinephrine responses. Pharmacological methods revealed that dopamine and not norepinephrine are critical for fear learning.

Strengths:

The examination of the neural signal to different tone intensities, different shock intensities, repeated tone presentation (habituation), and conditioning, offers an unprecedented account of the neural signal to non-associative and associative processes. This kind of deconstruction of the elements of conditioning offers a strong account of how the brain processes the stimuli used and their interaction during learning.

Excellent use of data acquired with fiber photometry in the optogenetic interrogation study.

The use of pharmacology to disentangle dopamine and norepinephrine was excellent.

Comments on revised version:

The authors have thoroughly and thoughtfully addressed my prior concerns.

Reviewer #1 (Public Review):

Summary:

The authors conducted two tasks at 300 days separation. First, a social perception task, where Ps responded whether a pictured person either deserved or needed help. Second, an altruism task, where Ps are offered monetary allocations for themselves and a partner. Ps decide whether to accept, or a default allocation of 20 dollars each. The partners differed in perceived merit, such that they were highly deserving, undeserving or unknown. This categorisation was decided on the basis of a prisoners dilemma game the partner played beforehand. "Need" was also manipulated, by altering the probability that the partner must have their hand in cold water at the end of the experiment and this partner can use the money to buy themselves out. These two tasks were conducted to assess the perception of need/merit in the first instance, and how this relates to social behaviour in the second. fMRI data were collected alongside behavioural.

The authors present many analyses of behaviour (including DDM results) and fMRI. E.g., they demonstrate that they could decode across the mentalising network whether someone was making a need or deserving judgement vs control judgements but couldn't decode need vs deserving. And that brain responses during merit inferences (merit - control) systematically covaried with participants' merit sensitivity scores in the rTPJ. They also found relationships between behaviour and rTPJ in the altruism task. And that merit sensitivity in the perception task predicted influence of merit on social behaviour in the altruism task.

Strengths:

This manuscript represents a sensible model to predict social perceptions and behaviours, and a tidy study design with interesting findings. The introduction introduced the field especially brilliantly for a general audience.

Weaknesses:

These are small samples. This is especially the case for the correlational questions. The limitation is acknowledged, but does mean that we cannot conclude much from absent relationships, where the likelihood of Type II error is high.

Decoding analyses. The authors decode need vs merit, and need+merit vs control, not the content of these inferences. The logic of these analyses implies that there is a distributed representation of merit that does not relate to its content but is an abstracted version that applies to all merit judgements. However, these analyses are not central to the authors' aims and conclusions, so this is just a minor point.

Reviewer #2 (Public Review):

When people help others is an important psychological and neuroscientific question. It has received much attention from the psychological side, but comparatively less from neuroscience. The paper translates some ideas from a social Psychology domain to neuroscience using a neuroeconomically oriented computational approach. In particular, the paper is concerned with the idea that people help others based on perceptions of merit/deservingness, but also because they require/need help. To this end, the authors conduct two experiments with an overlapping participant pool:

(1) A social perception task in which people see images of people that have previously been rated on merit and need scales by other participants. In a blockwise fashion, people decide to whether the depicted person a) deserves help, b) needs help, and c) whether the person uses both hands (== control condition)<br /> (2) In an altruism task, people make costly helping decisions by deciding between giving a certain amount of money to themselves or another person. It is manipulated how much the other person needs and deserves the money.<br /> The authors use sound and robust computational modelling approach for both tasks using evidence accumulation models. They analyse behavioural data for both tasks, showing that the behaviour is indeed influenced, as expected, by the deservingness and the need of the shown people. Neurally, the authors use a block-wise analysis approach to find differences in activity levels across conditions of the social perception task. The authors do find large activation clusters in areas related to theory of mind. Interestingly, they also find that activity in TPJ that relates to the deservingness condition correlates with people's deservingness ratings while they do the task, but also with computational parameters related to helping others in the second task, the one that was conducted many months later. Also some behavioural parameters correlate across the two tasks, suggesting that how deserving of help others are perceived reflects a relatively stable feature that translates into concrete helping decisions later-on.

The conclusions of the paper are overall well supported by the data.

(1) I found that the modelling was done very thoroughly for both tasks. Overall, I had the impression that the methods are very solid with many supplementary analyses. The computational modelling is done very well.<br /> (2) A slight caveat, however, regarding this aspect, is that, in my view, the tasks are relatively simplistic, so that even the complex computational models do not as much as they can in the case of more complex paradigms. For example, the bias term in the model seems to correspond to the mean response rate in a very direct way (please correct me if I am wrong).<br /> (3) Related to the simple tasks: The fMRI data is analysed in a simple block-fashion. This is in my view not appropriate to discern the more subtle neural substrates of merit/need-based decision making or person perception. Correspondingly, the neural activation patterns (merit > control, need > control) are relatively broad and unspecific. They do not seem to differ in the classic theory of mind regions, that are the focus of the analyses.<br /> (4) However, the relationship between neural signal and behavioural merit sensitivity in TPJ is noteworthy.<br /> (5) The latter is even more the case, as the neural signal and aspects of the behaviour are correlated across subjects with the second task that is conducted much later. Such a correlation is very impressive and suggests that the tasks are sensitive for important individual differences in helping perception/behaviour.<br /> (6) That being said, the number of participants in the latter analyses are at the lower end of the number of participants that are these days used for across-participant correlations.

Reviewer #3 (Public Review):

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

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

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

Reviewer #1 (Public Review):

Summary:<br /> A long literature in cognitive neuroscience studies how humans and animals adjudicate between conflicting goals. However, despite decades of research on the topic, a clear computational account of control has been difficult to pin down. In this project, Petri, Musslick, & Cohen attempt to formalize and quantify the problem of control in the context of toy neural networks performing conflicting tasks.

This manuscript builds on the formalism introduced in Petri et al (2021), "Topological limits to the parallel processing capability of network architectures", which describes a set of tasks as a graph in which input nodes (stimuli) are connected to output nodes (responses). Each edge in this graph links an input node to an output node, representing a "task"; i.e. a word reading task connects the input node "word" to the output node "read". Cleverly, patterns of interference and conflict between tasks can be quantified from this graph. In the current manuscript, the authors extend this framework by converting these graphs into neural networks and a) allowing edges to be continuous rather than binary; b) introducing "hidden layers" of units between input and output nodes; and c) introducing a "control" signal that modulates edge weights. The authors then examine how, in such a network, optimal behavior may involve serial versus parallel execution of different sets of tasks.

Strengths:<br /> There is a longstanding belief in cognitive neuroscience that "control" manages conflicts by scheduling tasks to be executed in parallel versus serially; I applaud the efforts of the authors to give these intuitions a more concrete computational grounding.

My main scientific concern is that the authors focus on what seems like an arbitrary set of network architectures. The networks considered here are derived by converting task graphs, which represent a multitasking problem, into networks for _performing_ that multitasking problem. Frankly, these networks do not look like any neural network a computer scientist would use to actually solve a problem, nor do they seem biologically realistic. Furthermore, adding hidden layers to these networks only ever seems to make performance worse (Figures 4, 11), introducing unnecessary noise and interference; it would seem more useful to study a network architecture in which hidden layers fulfilled some useful purpose (as they do in the brain and machine learning).

However, this scientific concern is secondary to the major problem with this paper, which is clarity.

Major problem: A lack of clarity

I found this paper extremely difficult to read. To illustrate my difficulty, I will describe a subset of my confusion.

The authors define the "entropy" of an action in equation 1, but the content of the equation gives what is sometimes referred to as the "surprisal" of the action. Conventionally (as per Wikipedia and any introductory textbook I am familiar with), entropy is the "expected surprisal" of a random variable, not the surprisal of a single action. This creates immediate confusion going into the results. Furthermore, defining "entropy" this way means that "information" is functionally equivalent to accuracy for the purposes of this paper, in which case I do not know what has been gained by this excursion into (non-standard) information-theoretic terminology.

They next assert that equation 1 is the information _cost_ of an action. No motivation is given for this statement and I do not know what it means. In what sense is a "cost" associated with the negative logarithm of a probability?

In the next section II.B, the authors introduce a new formalism in which responses are represented by task graph nodes _R_. What is the relationship between an action _a_ and the responses _R_? Later, in section II.C, edges _f_ in the task graph are used as seemingly drop-in replacements for actions _a_.

I simply have no idea what is going on in equations 31 through 33. Where are the functions _R_ (not to be confused with the response nodes _R_) and _S_ defined? Or how are they approximated? What does the variable _t_ mean and why does it appear and disappear from equations seemingly at random?

Response times seem to be important, but as far as I can tell, nowhere do the authors actually describe how response times are calculated for the simulated networks.

Similar issues persist through the rest of the paper: unconventional formalism is regularly introduced using under-explained notation and without a clear relationship to the scientific questions at hand. As a result, the content and significance of the findings are largely inscrutable to me, and I suspect also to the vast majority of readers.

Reviewer #2 (Public Review):

Summary:<br /> The authors develop a normative account of automaticity-control trade-offs using the mathematics of information theory, which they apply to abstract neural networks. They use this framework to derive optimal trade-off solutions under particular task conditions.

Strengths:<br /> On the positive side, I appreciate the effort to rigorously synthesize ideas about multi-tasking within an information-theoretic framework. There is potentially a lot of promise in this approach. The analyis is quite comprehensive and careful.

Weaknesses:<br /> Generally speaking, the paper is very long and dense. I don't in principle mind reading long and dense papers (though conciseness is a virtue); it becomes more of a slog when it's not clear what new insights are being gained from laboring through the math. For example, after reading the Stroop section, I wasn't sure what new insight was provided by the information-theoretic formalism which goes beyond earlier models. Is this just an elegant formalism for expressing previously conceived ideas, or is there something fundamentally new here that's not predicted by other frameworks? The authors cite multiple related frameworks addressing the same kinds of data, but there is no systematic comparison of predictions or theoretical interpretations. Even in the Discussion, where related work is directly addressed, I didn't see much in terms of explaining how different models made different predictions, or even what predictions any of them make.

After a discussion of the Stroop task early in the paper, the analysis quickly becomes disconnected from any empirical data. The analysis could be much more impactful if it was more tightly integrated with relevant empirical data.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript describes a series of experiments using human intracranial neural recordings designed to evaluate the processing of self-generated speech in the setting of feedback delays. Specifically, the authors aim to address the question about the relationship between speech-induced suppression and feedback sensitivity in the auditory cortex, whose relationship has been conflicting in the literature. They found a correlation between speech suppression and feedback delay sensitivity, suggesting a common process. Additional controls were done for possible forward suppression/adaptation, as well as controlling for other confounds due to amplification, etc.

Strengths:<br /> The primary strength of the manuscript is the use of human intracranial recording, which is a valuable resource and gives better spatial and temporal resolution than many other approaches. The use of delayed auditory feedback is also novel and has seen less attention than other forms of shifted feedback during vocalization. Analyses are robust, and include demonstrating a scaling of neural activity with the degree of feedback delay, and more robust evidence for error encoding than simply using a single feedback perturbation.

Weaknesses:<br /> Some of the analyses performed differ from those used in past work, which limits the ability to directly compare the results. Notably, past work has compared feedback effects between production and listening, which was not done here. There were also some unusual effects in the data, such as increased activity with no feedback delay when wearing headphones, that the authors attempted to control for with additional experiments, but remain unclear. Confounds by behavioral results of delayed feedback are also unclear.

Overall the work is well done and clearly explained. The manuscript addresses an area of some controversy and does so in a rigorous fashion, namely the correlation between speech-induced suppression and feedback sensitivity (or lack thereof). While the data presented overlaps that collected and used for a previous paper, this is expected given the rare commodity these neural recordings represent. Contrasting these results to previous ones using pitch-shifted feedback should spawn additional discussion and research, including verification of the previous finding, looking at how the brain encodes feedback during speech over multiple acoustic dimensions, and how this information can be used in speech motor control.

Reviewer #2 (Public Review):

Summary:<br /> In "Speech-induced suppression and vocal feedback sensitivity in human cortex", Ozker and colleagues use intracranial EEG to understand audiomotor feedback during speech production using a speech production and delayed auditory feedback task. The purpose of the paper is to understand where and how speaker-induced suppression occurs, and whether this suppression might be related to feedback monitoring. First, they identified sites that showed auditory suppression during speech production using a single-word auditory repetition task and a visual reading task, then observed whether and how these electrodes show sensitivity to auditory feedback using a DAF paradigm. The stimuli were single words played auditorily or shown visually and repeated or read aloud by the participant. Neural data were recorded from regular- and high-density grids from the left and right hemispheres. The main findings were:<br /> • Speaker-induced suppression is strongest in the STG and MTG, and enhancement is generally seen in frontal/motor areas except for small regions of interest in the dorsal sensorimotor cortex and IFG, which can also show suppression.<br /> • Delayed auditory feedback, even when simultaneous, induces larger response amplitudes compared to the typical auditory word repetition and visual reading tasks. The authors presume this may be due to the effort and attention required to perform the DAF task.<br /> • The degree of speaker-induced suppression is correlated with sensitivity to delayed auditory feedback.<br /> • pSTG (behind TTS) is more strongly modulated by DAF than mid-anterior STG

Strengths:<br /> Overall, I found the manuscript to be clear, the methodology and statistics to be solid, and the findings mostly quite robust. The large number of participants with high-density coverage over both the left and right lateral hemispheres allows for a greater dissection of the topography of speaker-induced suppression and changes due to audiomotor feedback. The tasks were well-designed and controlled for repetition suppression and other potential caveats.

Weaknesses:<br /> (1) In Figure 1D, it would make more sense to align the results to the onset of articulation rather than the onset of the auditory or visual cue, since the point is to show that the responses during articulation are relatively similar. In this form, the more obvious difference is that there is an auditory response to the auditory stimulus, and none to the visual, which is expected, but not what I think the authors want to convey.<br /> (2) The DAF paradigm includes playing auditory feedback at 0, 50, 100, and 200 ms lag, and it is expected that some of these lags are more likely to induce dysfluencies than others. It would be helpful to include some analysis of whether the degree of suppression or enhancement varies by performance on the task, since some participants may find some lags more interfering than others.<br /> (3) Figure 3 shows data from only two electrodes from one patient. An analysis of how amplitude changes as a function of the lag across all of the participants who performed this task would be helpful to see how replicable these patterns of activity are across patients. Is sensitivity to DAF always seen as a change in amplitude, or are there ever changes in latency as well? The analysis in Figure 4 gets at which electrodes are sensitive to DAF but does not give a sense of whether the temporal profile is similar to those shown in Figure 3.<br /> (4) While the sensitivity index helps to show whether increasing amounts of feedback delay are correlated with increased response enhancement, it is not sensitive to nonlinear changes as a function of feedback delay, and it is not clear from Figure 3 or 4 whether such relationships exist. A deeper investigation into the response types observed during DAF would help to clarify whether this is truly a linear relationship, dependent on behavioral errors, or something else.

Reviewer #1 (Public Review):

Summary:<br /> I really enjoyed this manuscript from Torsekar et al on "Contrasting responses to aridity by different-sized decomposers cause similar decomposition rates across a precipitation gradient". The authors aimed to examine how climate interacts with decomposers of different size categories to influence litter decomposition. They proposed a new hypothesis: "The opposing climatic dependencies of macrofauna and that of microorganisms and mesofauna should lead to similar overall decomposition rates across precipitation gradients".

This study emphasizes the importance as well as the contribution of different groups of organisms (micro, meso, macro, and whole community) across different seasons (summer with the following characteristics: hot with no precipitation, and winter with the following characteristics: cooler and wetter winter) along a precipitation gradient. The authors made use of 1050 litter baskets with different mesh sizes to capture decomposers contribution. They proposed a new hypothesis that was aiming to understand the "dryland decomposition conundrum". They combined their decomposition experiment with the sampling of decomposers by using pittfall traps across both experiment seasons. This study was carried out in Israel and based on a single litter species that is native to all seven sites. The authors found that microorganism contribution dominated in winter while macrofauna decomposition dominated the overall decomposition in summer. These seasonality differences combined with the differences in different decomposers groups fluctuation along precipitation resulted in similar overall decomposition rates across sites.<br /> I believe this manuscript has a potential to advance our knowledge on litter decomposition.

Strengths:<br /> Well design study with combination of different approaches (methods) and consideration of seasonality to generalize pattern.<br /> The study expands to current understanding of litter decomposition and interaction between factors affecting the process (here climate and decomposers).

Weaknesses:<br /> The study was only based on a single litter species.

Reviewer #2 (Public Review):

Summary: Torsekar et al. use a leaf litter decomposition experiment across seasons, and in an aridity gradient, to provide a careful test of the role of different-sized soil invertebrates in shaping the rates of leaf litter decomposition. The authors found that large-sized invertebrates are more active in the summer and small-sized invertebrates in the winter. The summed effects of all invets then translated into similar levels of decomposition across seasons. The system breaks down in hyper-arid sites.

Strengths: This is a well-written manuscript that provides a complete statistical analysis of a nice dataset. The authors provide a complete discussion of their results in the current literature.

Weaknesses: I have only three minor comments. Please standardize the color across ALL figures (use the same color always for the same thing, and be friendly to color-blind people). Fig 1 may benefit from separating the orange line (micro and meso) into two lines that reflect your experimental setup and results. I would mention the dryland decomposition conundrum earlier in the Introduction. And the manuscript is full of minor grammatical errors. Some careful reading and fixing of all these minor mistakes here and there would be needed.